The Interactive Fly
Genes involved in tissue and organ development
How stem cells generate both differentiating and self-renewing daughter cells is unclear. This study shows that Drosophila larval neuroblasts - stem cell-like precursors of the adult brain - regulate proliferation by segregating the growth inhibitor Brat and the transcription factor Prospero into only one daughter cell. Like Prospero, Brat binds and cosegregates with the adaptor protein Miranda. In larval neuroblasts, both Brat and Prospero are required to inhibit self-renewal in one of the two daughter cells. While Prospero regulates cell-cycle gene transcription, Brat acts as a posttranscriptional inhibitor of dMyc. In brat or prospero mutants, both daughter cells grow and behave like neuroblasts leading to the formation of larval brain tumors. Similar defects are seen in lethal giant larvae (lgl) mutants where Brat and Prospero are not asymmetric. This study has identified a molecular mechanism that may control self-renewal and prevent tumor formation in other stem cells as well (Betschinger, 2006).
These data reveal a molecular mechanism that controls self-renewal in Drosophila larval neuroblasts. The growth regulator Brat segregates asymmetrically during neuroblast division and inhibits self-renewal in one of the two daughter cells. Together with the asymmetrically segregating transcription factor Prospero, Brat ensures that this daughter cell will stop growing, exit the cell cycle, and differentiate into neurons. In brat or prospero mutants, or in lgl mutants, where Brat and Prospero are not asymmetrically segregated, both daughter cells proliferate leading to the formation of a brain tumor and death of the animal. These tumors are neoplastic and can be transplanted into the abdomen of wild-type flies where they overgrow, invade other tissues, and eventually kill the host (Betschinger, 2006).
Asymmetric cell division has been studied in the Drosophila central and peripheral nervous systems. In the peripheral nervous system, the determinants Numb and Neuralized segregate into one of the two daughter cells, and in their absence, this cell is transformed into its sister cell. In the embryonic central nervous system, Prospero acts as a segregating determinant, but in prospero mutants, many GMCs are still correctly specified. The data suggest that this is because Prospero acts partially redundant with Brat. In embryos double mutant for prospero and brat, most GMCs expressing the marker Eve are missing and neuronal differentiation in the embryonic CNS is greatly impaired. These observations suggest that Brat and Prospero act together to specify GMC fate in Drosophila embryos (Betschinger, 2006).
Although cell-cycle markers are expressed longer and stronger in prospero and brat, prospero mutant embryos, uncontrolled overproliferation has not been described in Drosophila embryos so far. In larvae, however, both brat and prospero mutant neuroblasts can initiate tumor formation. It is proposed that this difference is due to distinct mechanisms of cell growth during the two stages. During embryogenesis, cell number increases dramatically but the total volume of the embryo remains constant. Embryonic neuroblasts therefore shrink with each division and they might exit the cell cycle simply because they become too small. Support for this model comes from mutations affecting cell size asymmetry during neuroblast divisions, like Gβ13F (Fuse, 2003) or Ric-8 (Hampoelz, 2005): in these mutants, GMCs are larger, neuroblasts shrink faster and, as a consequence, divide less often. In larval neuroblasts, the situation is quite different. Several results indicate that larval neuroblasts grow significantly while cell growth is inhibited in GMCs. First, the total volume of clones generated from individual neuroblasts is several times more than the initial volume of the neuroblast. Second, the size of 'old' and 'young' (earlier and more recently generated) GMCs is approximately the same, indicating that GMCs do not grow significantly during clone formation. Third, larval neuroblasts do not become progressively smaller during development indicating that the loss of cytoplasm from each division must be compensated for by growth. Taken together, these results suggest that larval neuroblasts might be more appropriate as a model for the control of self-renewal in stem cells (Betschinger, 2006).
These experiments show that the restriction of cell growth in the GMC requires the genes lgl, brat, and prospero. While lgl seems to be required indirectly due to its role in asymmetric protein segregation, Prospero and Brat act in the GMC to regulate several important events: They repress neuroblast fate, inhibit cell-cycle progression, and prevent cell growth. Prospero is a homeodomain transcription factor, and the cell-cycle genes Cyclin A, Cyclin E, and Dacapo (the fly homolog of the CDK inhibitor p21) were shown to be among its transcriptional targets. Similar to Drosophila Prospero, its vertebrate homolog Prox-1 has been shown to regulate cell-cycle genes, and loss of prox-1 leads to increased proliferation of retinal progenitor cells (Betschinger, 2006).
For Brat, two different functions have been described: First, it acts as a translational regulator of the gap-gene hunchback. Hunchback is expressed in the embryonic nervous system but is not present in wild-type or brat mutant larval neuroblasts and is unlikely to be relevant for the growth regulatory activity of Brat. More likely, Brat acts through its well-described inhibitory activity on ribosomal RNA synthesis. Cells mutant for brat or its C. elegans homolog ncl-1 have larger nucleoli, more ribosomal RNA, and higher rates of protein synthesis, and these activities have been made responsible for the cell size increase that is observed in C. elegans and Drosophila brat mutant cells. These data suggest that this second function of Brat is also linked to posttranscriptional gene regulation. It is proposed that Brat downregulates dMyc in one of the two daughter cells and thereby inhibits protein synthesis and cell growth. Whether Brat controls dMyc translation, protein stability, or RNA stability is unclear. Interestingly, the C. elegans Brat homolog ncl-1 has been identified as one of the genes required for RNAi (Kim, 2005). Since the microRNA pathway was shown to be involved in regulation of Drosophila stem cell proliferation (Hatfield, 2005), differential regulation of this pathway in neuroblasts and GMCs by Brat could provide another explanation for its mutant phenotype (Betschinger, 2006).
Brat is part of a protein family that is characterized by a C-terminal NHL domain, several zinc-finger like B boxes, and a coiled-coil region. While the vertebrate members of this family (TRIM-2, TRIM-3, and TRIM-32) are not well characterized, the mutant phenotype of the two other Drosophila members (Dappled/Wech and Mei-P26) suggests a common function as tumor suppressors. Mutations in dappled cause melanomic tumors of the fat body, and mei-P26 mutations lead to ovarian tumors. While dappled tumors have not been well characterized, the mei-P26 phenotype has been attributed to overproliferation of undifferentiated germ cells. It is similar to-and genetically interacts with-bag of marbles, a well-characterized repressor of proliferation in the daughter cells of germline stem cells. Thus, it is conceivable that proliferation control in stem cells is a common activity of NHL domain proteins (Betschinger, 2006).
Recent evidence suggests that some human brain tumors contain stem cell-like neural progenitors that are responsible for tumor formation. Together with the identification of stem cell-like subpopulations in leukaemia, multiple myeloma, and breast cancer, this has led to the so-called cancer stem cell hypothesis which proposes that only a small population of cells in a tumor have the ability to proliferate and self-renew. This discovery suggests mechanisms for tumorigenesis other than the simple loss of proliferation control, in particular dedifferentiation of cells into additional stem cells and symmetric division of stem cells. Animal models for tumor stem cells are essential for developing new therapeutic approaches that target these mechanisms. Although Drosophila can only mimic some aspects of tumorigenesis, it might contribute to the identification of the molecular pathways operating in tumor stem cells. Human Lgl has already been implicated in tumor progression, and the characterization of Brat homologs will verify the relevance of Drosophila as a tumor stem cell model (Betschinger, 2006).
Brain development in Drosophila is characterized by two neurogenic
periods, one during embryogenesis and a second during larval life. Although
much is known about embryonic neurogenesis, little is known about the genetic
control of postembryonic brain development. This study used mosaic analysis with a
repressible cell marker (MARCM) to study the role of the brain tumor
(brat) gene in neural proliferation control and tumour suppression in
postembryonic brain development of Drosophila. The findings indicate
that overproliferation in brat mutants is due to loss of
proliferation control in the larval central brain and not in the optic lobe.
Clonal analysis indicates that the brat mutation affects cell
proliferation in a cell-autonomous manner and cell cycle marker expression
shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes
cell cycle exit and differentiation of brat mutant cells, thereby
abrogating brain tumour formation. Taken together, these results provide
evidence that the tumour suppressor brat negatively regulates cell
proliferation during larval central brain development of Drosophila,
and suggest that Prospero acts as a key downstream effector of brat
in cell fate specification and proliferation control (Bello, 2006).
Previous studies suggested that brat loss-of-function mutants lead
to massive cellular overgrowth and tumour formation in larval optic lobes of
Drosophila. These studies also indicated an embryonic requirement for
brat to suppress tumour formation. By contrast, the current analysis showed that the brat overproliferation phenotype is due to loss of proliferation control in the larval central brain; the optic lobes initially appear wild-type-like but subsequently are overgrown by neoplastic central brain brat mutant tissue. This conclusion is further supported by MARCM clonal analysis which demonstrated that loss of brat function causes overproliferation in the larval central brain only (Bello, 2006).
In vivo mosaic analysis reveals a cell-autonomous, larval requirement
for brat to limit cell proliferation in the brain. Although
brat is expressed in all parts of the nervous system both in the
embryo, induction of brat mutant clones in the first larval instar is
sufficient to cause massive overproliferation in the central brain but not the
ventral ganglia. This may suggest that either unknown compensatory mechanisms
actively suppress a brat mutant phenotype in the larval ventral
ganglia, or that this reflects region-specific differences in cell cycle
control. Indeed, transcriptional activity of the mitotic regulator
string/Cdc25 is regulated by a plethora of cis-acting elements, most
of which are devoted to differential control of cell proliferation during
embryonic and larval neurogenesis (Bello, 2006).
During postembryonic neurogenesis, intense proliferation takes place in the
brain. This analysis shows that central brain brat mutant clones display
sustained cell cycle marker expression, indicating that mutant cells are
unable to withdraw from the cell cycle. This is further supported by the
presence of enormous brat mutant clones with pronounced proliferative
activity even in 3-week-old adult brains, an observation that contrasts with
the postmitotic adult wild-type brain. Previous studies have shown that
cessation of proliferation in the developing Drosophila brain occurs
during metamorphosis, although the underlying genetic mechanisms are currently
unknown. The elevated and aberrant cell cycle activity of central brain
brat mutant cells suggests that these cells are either able to escape
or that they lack cell cycle termination signals (Bello, 2006).
Mosaic analysis demonstrates that enlarged brat mutant clones
comprise cells that display sustained expression of neural progenitor cell
markers, and simultaneously lack marker gene expression specific for
differentiating ganglion cells. Indeed, lack of axonal processes suggests that
brat mutant clones comprise an excessive number of mutant cells that
are unable to exit the cell cycle and hence do not differentiate into ganglion
cells but rather continue to proliferate. These data indicate that
brat mutation impairs proliferation control of neural progenitor
cells, namely either neuroblasts and GMCs or only one of these progenitors,
since in the wild-type central brain only these two cell types are actively
engaged in the cell cycle. Based on this analysis it is not possible to distinguish
unambiguously between the two possibilities and the underlying mechanisms. The possibility that differentiating ganglion cells
de-differentiate due to brat mutation was excluded, because lack of differentiation was consistently observed right after clone induction and also at any later stages of mutant clone development. This was especially exemplified by
the lack of nuclear Pros expression, which in the wild type is unambiguously
detectable in differentiating progeny of larval neuroblast lineages, namely
GMCs as well as ganglion cells (Bello, 2006).
Moreover, loss-of-function analysis indicates that brat mutant
MARCM clones lack Pros and also phenocopy pros mutant clones. Thus,
enlarged pros mutant clones consist of cells that are devoid of Elav
expression, that lack axonal processes but display sustained expression of Grh
and Mira as well as cell cycle markers such as CycE, CycB and PH3. These data
suggest that mutant clones are essentially devoid of terminally
differentiating postmitotic ganglion cells, indicating that Pros functions
like Brat in terminating neural progenitor cell proliferation and inducing
ganglion cell differentiation. In the embryonic CNS, Pros functions to
terminate cell proliferation by repression of cell-cycle activators and
simultaneously to induce a differentiation program, effectively coupling the
two events. This Pros function appears to be warranted by its localization in the basal cortex of asymmetrically dividing neuroblasts and hence its distribution to only one daughter cell, the GMC. Upon completion of mitosis, Pros translocates from cytoplasm into the nucleus where it executes its transcriptional program ensuring both terminal division of the GMC and cell differentiation of its progeny. In the larval CNS nuclear localisation of Pros is observed in GMCs and ganglion cells but not in the neuroblast, suggesting that Pros has comparable functional features in larval central brain neurogenesis (Bello, 2006).
In addition, the results provide evidence that Pros acts downstream of Brat
in neural proliferation control. The following points support this notion: (1)
brat mutant clones lack nuclear Pros; (2) brat and
pros mutant clones are indistinguishable both at the morphological
and at the molecular level; (3) Brat expression is unaltered in pros
mutant clones, which together with point no. 1 strongly suggests that Brat is
epistatic over Pros; and (4) trans-activation of wild-type pros in
brat mutant clones is sufficient to promote both cell cycle exit and
differentiation. The experiments, however, do not provide any evidence about
the direct or indirect nature of their interaction. Since overexpressed Pros
is detected specifically in brat mutant clones in a wild-type-like
pattern, the possibility that brat acts as a translational repressor
of Pros, comparable to its role in hunchback repression
during embryonic abdominal segmentation, is excluded. In
addition, brat mutation apparently does not affect pros
transcription, since pros RNA in situ hybridization in zygotic
brat mutants produced a pattern indistinguishable from wild-type
controls. Thus, Brat and Pros may act indirectly in the same pathway, regulating progenitor cell proliferation control in the brain. Alternatively, Brat may act in a process required to cargo Pros, comparable to the function of its mammalian homolog BERP (Bello, 2006).
In vivo mosaic analysis demonstrates that a single mutation in either
brat or pros is sufficient to cause brain tumour formation
in a cell-autonomous manner, suggesting that indefinite proliferation of
brat and pros mutant cells is a cell intrinsic property.
GFP-labelled MARCM cells each derive from a common precursor cell, implying
that brat and pros mutant cells all descend from individual
tumour cells of origin and hence lead to brain tumour formation in a clonally
related manner. Moreover, the data indicate that pros and
brat mutant clones in the larval central brain are composed of an
excessive number of mutant progenitor cells that are unable to differentiate
into ganglion cells but rather continue to proliferate. In this sense the
results provide in vivo support for the notion that the initiating event in
the formation of a malignant tumour is an error in the process of normal
differentiation (Bello, 2006).
In addition, the unlimited capacity to generate undifferentiated,
proliferating progeny suggests that cells mutant for brat or
pros retain self-renewing capacities. In human, brain cancers are
thought to arise either from normal stem cells or from progenitor cells in
which self-renewal pathways have become activated, however the underlying mechanisms are elusive. The results in Drosophila may therefore provide a rationale and genetic model for the origin of brain cancer stem cells. Although parallels to human tumour formation are speculative, it is noteworthy that TRIM3, a human
homolog of brat is located on chromosome 11p15, a region frequently deleted in brain tumours. Moreover, functional studies have shown that the pros homologue Prox1 regulates proliferation and differentiation of neural
progenitor cells in the mammalian retina. These data may indicate that brat and pros function in cell differentiation and tumour suppression in an evolutionarily conserved manner (Bello, 2006).
The Drosophila central nervous system is produced by two rounds of neurogenesis: one during embryogenesis to form the larval brain and one during larval stages to form the adult central nervous system. Neurogenesis caused by the activation of neural stem division in the larval brain is essential for the proper patterning and functionality of the adult central nervous system. Initiation of neuroblast proliferation requires signaling by the Fibroblast Growth Factor homolog Branchless and by the Hedgehog growth factor. The Branchless and Hedgehog pathways form a positive feedback loop to regulate the onset of neuroblast division. This feedback loop is initiated during embryogenesis. Genetic and molecular studies demonstrate that the absolute level of Branchless and Hedgehog signaling is critical to fully activate stem cell division. Furthermore, over-expression and mutant studies establish that signaling by Branchless is the crucial output of the feedback loop that stimulates neuroblast division and that Branchless signaling is necessary for initiating the division of all mitotically regulated neuroblasts in the brain lobes. These studies establish the molecular mechanism through which Branchless and Hedgehog signaling interface to regulate the activation of neural stem cell division (Barretta, 2008).
These studies have demonstrated that Hh and Bnl act in a positive feedback loop in the larval brain to control the onset of neuroblast proliferation. The feedback loop acts at the transcriptional level, such that Hh signaling activity is essential to control the level of bnl expression and vice versa. Double mutant analyses showed that an absolute level of signaling by both Bnl and Hh are required to maintain normal neuroblast activation, rather than other possible models that would suggest a certain balance of signaling activity (for example more Bnl than Hh) is sufficient regardless of the exact magnitude of signaling activity. The discovery that Bnl signaling is the critical output of the feedback loop suggests that the main function of Hh signaling is to maintain the proper level of Bnl production and signaling. Furthermore, the observation that only the mushroom body and ventral lateral neuroblasts continue to divide in bnl null mutants regardless of the level of Hh signaling indicates that all the regulated neuroblasts, both optic lobe and central brain sets, require the input of the Bnl pathway to enter S phase. Thus the Hh-Bnl feedback loop appears to control cell cycle progression in all the mitotically arrested neuroblasts that begin cell division in first instar (Barretta, 2008).
Other developmental events that require Hedgehog and FGF signaling have used those pathways in different manners to achieve their goals. For example, in the mouse ventral telencephalon, Hedgehog and FGF/MAPK signaling operate as two independent pathways. FGF signaling is independent of Sonic Hedgehog (SHH) and does not affect expression of either SHH itself or its target gene and effector GLI1. Other systems have shown a linear dependence of FGF expression on SHH signaling and vice versa. During budding morphogenesis in the mouse lung Hedgehog signaling inhibits expression of FGF10 but up-regulates FGF7. In the Xenopus eye, expression of Banded Hedgehog increases expression of FGF8. In the zebrafish forebrain inhibition of Hh signaling decreases expression of FGF3, FGF8 and FGF19. Hedgehog also regulates FGF expression in the zebrafish mid/hindbrain. However, in the zebrafish forebrain HH expression requires FGF signaling. Inhibition of both FGF3 and FGF8 expression resulted in a downregulation of SHH. Alternatively, the HH and FGF pathways can integrate at the level of intracellular components. FGF has been shown to induce expression of GLI2, a transcription factor and HH signaling effector in ventroposterior development in zebrafish (Barretta, 2008).
Of course the classic example of FGF and SHH interplay is the development of the chick limb bud. In this system, several FGFs set up a signaling center at the tip of the bud that turns on expression of SHH in the posterior limb mesenchyme. In turn, SHH signaling is required for maintenance of FGF4, FGF9 and FGF17 expression in the bud tip. This function of SHH occurs through the expression of Gremlin, an inhibitor of Bone Morphogenetic Protein signaling. Gremlin inhibition of Bone Morphogenetic Protein signaling prevents down-regulation of the FGFs. Thus a positive feedback loop exists between SHH and FGFs, mediated by Gremlin (Barretta, 2008).
The model of the Hh-Bnl feedback loop proposed in this study is most similar to the classic SHH-FGF feedback loop described in the vertebrate limb bud. In is not yet known whether the regulation of bnl expression by Hh signaling is direct or if it is mediated by another signaling pathway such as the Gremlin/Bone Morphogenetic Protein connection that operates in the limb bud. However, like the distinct domains of FGF and SHH in the limb bud, bnl and hh expression also occur in distinct regions of the brain lobe. The fact that the Hh-Bnl feedback loop is activated during embryogenesis, but that the first regulated neuroblasts do not enter S phase until 8-10 h after larval hatching also suggests that additional events must take place downstream of Bnl signaling to permit mitotically arrested stem cells to transit through G1 to S phase. One such possibility is exposure to the steroid hormone ecdysone, which is necessary during first larval instar for the initiation of neuroblast division a few hours later. Both SHH and FGF2 have been shown to be necessary for the division of different subsets of neural stem cells in many different vertebrate and mammalian models and in multiple contexts. This is the first time that the interactions between these two pathways necessary to stimulate the reactivation of stem cell division in quiescent neural stem cells have been elucidated. The next challenge will be to determine whether different molecular mechanisms tying these two signaling pathways are used for different developmental decisions such as progeny cell fate, initiation of cell division and maintenance of stem cell identity (Barretta, 2008).
In postembryonic neuroblasts, transition in gene expression programs of a cascade of transcription factors (also known as the temporal series) acts together with the asymmetric division machinery to generate diverse neurons with distinct identities and regulate the end of neuroblast proliferation. However, the underlying mechanism of how this 'temporal series' acts during development remains unclear. This study shows that Hh signaling in the postembryonic brain is temporally regulated; excess (earlier onset of) Hh signaling causes premature neuroblast cell cycle exit and under-proliferation, whereas loss of Hh signaling causes delayed cell cycle exit and excess proliferation. Moreover, the Hh pathway functions downstream of Castor but upstream of Grainyhead, two components of the temporal series, to schedule neuroblast cell cycle exit. Interestingly, Hh is likely a target of Castor. Hence, Hh signaling provides a link between the temporal series and the asymmetric division machinery in scheduling the end of neurogenesis (Chai, 2013).
This study shows that Hh signaling functions during later postembryonic development and acts together with the NB temporal transcription factor cascade to regulate NB cell cycle exit. It was further demonstrated that hh is a downstream target of Cas, a member of temporal series that determines the time at which NBs terminate proliferation via down-regulation of Grh. While increased Hh signaling results in increased cell cycle length and premature NB cell cycle exit, loss of Hh signaling decreases NB cell cycle length and also prolongs the duration of NB proliferation (Chai, 2013).
Hh family proteins can act as short- or long-range morphogens covering distances as few as ten cell diameters (20 µm), or as far as a field containing many more cell diameters (200 µm). In the postembryonic brain, hh is expressed predominantly in the NBs and the newborn GMCs, whereas the expression of its target gene reporter, ptc-lacZ is observed in a narrow area covering the adjacent NB and the sibling GMCs, indicating a limited response to and suggesting a limited spread of Hh ligand. In addition, Hh protein is always found to be enriched and clustering around the NBs in a punctuated form rather than forming a gradient. These data, together with the lineage autonomous phenotype of hh mutant NB clones, strongly suggest that Hh acts locally at short range in the larval brain. This is consistent with the structural arrangement of the larval brain, where each NB lineage comprising of the NB itself, GMCs, and neurons, is encapsulated by a meshwork of glial processes that form a three-dimensional scaffold that potentially acts as a stem cell niche. Such a spatial arrangement may serve as a barrier to restrict spread of the ligand and confine signaling events within a particular lineage so that an individual NB lineage can development with considerable independence from its neighbouring lineages. Indeed, a NB clone derived from a hh null allele exhibits the GMC pool expansion phenotype even though GMCs from its neighbouring lineages are competent in producing Hh ligand (Chai, 2013).
While it is tempting to assume that Hh can also act on the GMCs in an autocrine mode of action judging from the presence of ptc-lacZ expression, no noticeable GMC fate transformation or change in their proliferative capability was seen in ptcS2 and smoIA3 clones. The higher mitotic rate in hh loss-of-function NBs could largely explain the amplification of the GMC pool and enlarged clone-size; however, a possible delay in GMC differentiation cannot be ruled out. The proposition that Hh ligand, which is produced by the NB and daughter GMCs, feeds back on the NB to control its own proliferative capacity and the timing of cell cycle exit is interesting but not totally unfamiliar. Similar feedback signalling mechanism has been demonstrated in the mouse brain in which post-mitotic neurons signal back to the progenitor to control cell fate decisions, as well as the number of neurons and glia produced during corticogenesis (Chai, 2013).
Hh signal reception is detectable in NBs as early as in L2 and persists throughout larval life and in early pupae when NBs undergo Pros-dependent cell cycle exit. This delay of approximately 96 h between the start of Hh reception and the ultimate outcome of cell cycle exit may be due to a requirement for cumulative exposure of NBs to increasing local concentrations of Hh. Such a graded response will enable the wt postembryonic NBs to progress from high to low proliferative stages before ceasing division, in line with the development of the larva. Evidence supporting this notion includes gradual accumulation of Hh on the NBs, lengthening of NB cell cycle time, as well as the necessity of high levels of Hh signaling to trigger cell cycle exit. It is worthwhile to note that even at pre-pupal stage during which most NBs are starting to undergo cell cycle exit, fewer than 20% of them are associated with Hh puncta at any point of time. One likely explanation is that not all the NB lineages within the larval central brain respond synchronously to Hh-mediated temporal transition. However, unlike the embryonic central nervous system in which hh expression is localized to rows 6-7 of the neuroectoderm, this study found it difficult to pinpoint a specific expression pattern in the postembryonic central brain due to the disorganized array of NB lineages. It is equally possible that different NBs exit cell cycle progression at different time points. This is also consistent with the structural organization of individual NB into different 'trophospongium' or stem cell niches (Hoyle, 1986). Nevertheless, the possiblility cannot be ruled out that Hh signal activation primes another yet-to-be-identified developmentally regulated signal/event to schedule NB cell cycle exit (Chai, 2013).
Interestingly, a recently proposed 'cell cycle length hypothesis' postulates that cell cycle length, particularly the length of G1 phase in neural stem cells acts as a switch to trigger the transition from proliferative to neurogenesis mode (Salomoni, 2010). In fact, experiments have shown that manipulation of cdk4/cyclinD1 expression and cdk2/cyclinE activity that result in the lengthening of G1 is sufficient to induce precocious neurogenesis; while inhibition of physiological lengthening of G1 delays neurogenesis and promotes expansion of intermediate progenitors. The curren results show that Drosophila postembryonic NBs in the central brain exhibit a comparable trend of cell cycle lengthening from young to old larval stages. Interestingly, NBs with excess Hh signaling have an extended cell cycle time, consistent with the idea that there is a forward shift of the 'perceived' age, leading to premature cell cycle exit. In contrast, Hh loss-of-function NBs have a shorter cell cycle time compared to their wt counterparts of the same actual age; hence, they have a younger 'perceived' age and are able to maintain their proliferative phase over a longer period of time. Consistent with this, it was shown that persistent NB proliferation in smoIA3 clones as well as the early termination of ptcS2 NBs proliferation, are always associated with the presence and absence of CycE expression, respectively. However, loss of Hh signaling in NBs merely extends their proliferative phase but is not sufficient to ensure perpetual proliferation as no mitotic NB is observed in the adult brain. It is also noted that a previous report suggested that the cell cycle time of the larval NBs reduced during their growth and reached a peak at late third instar with a minimum cell cycle time of 55 min. However, this study was conducted on thoracic NBs from the neuromeres T1 to T3, which have a very distinctive proliferative profile to the central brain NBs assayed in the current study. Indeed, has been shown in that abdominal NBs exhibit significantly different cell cycle times compared to their thoracic counterparts (Chai, 2013).
In Drosophila, the precise timing of NB cell cycle exit is governed by a highly regulated process that involves sequential expression of a series of transcription factors: Hb->Kr->Pdm1->Cas, known as the temporal series. It is known that the temporal series probably utilizes Grh in the postembryonic NBs to regulate Pros localization or apoptotic gene activity, thus determining the time at which proliferation ends. In addition, the temporal series also regulates postembryonic Chinmo->Br-C neuronal switch, which specifies the size and the identity of the neurons. The current data show that Hh signaling does not regulate early to late neuronal transition as Chinmo and Br-C expression timings appear unaffected in both ptc and smo mutant clones. In contrast, excess Hh signaling leads to a variety of features associated with NB cell cycle exit: (1) premature down-regulation of Grh, (2) nuclear localization of Pros (in NBs), and (3) reduction of NB size. Taken together with the extended proliferative duration of Hh loss-of-function NBs, it is apparent that Hh signaling is a potent effector of the temporal series and functions late to promote NB cell cycle exit (Chai, 2013).
The results from the current genetic interaction assays with Hh pathway components and grh reaffirmed the conclusions from previous studies that Grh is necessary to maintain the mitotic activity of the postembryonic NBs. The loss of Hh signaling keeps the central brain type I NBs in their proliferative state and this is largely contributed by persistent grh expression past their normal developmental timing at around 24 h APF. Even though Grh is necessary to extend the proliferative phase of these NBs, it is not sufficient to rescue all aspects of the premature cell cycle exit phenotype seen in ptc mutant NBs. Hence, down-regulation of grh by over-activating Hh signaling is not solely responsible for NB proliferative defects, and this implies that Hh signaling may terminate NB cell cycle via other mechanisms in addition to Grh (Chai, 2013).
The expression of hh appears to be dependent on the pulse of Cas expression at the transition between L1 and L2, as induction of cas mutant clones after that stage does not significantly affect hh expression. Moreover, ChIP assays suggest that Cas binds the hh genomic region, thereby placing Hh as a direct downstream target of the temporal series. However, it is intriguing to speculate on how the early pulse of Cas can mediate hh expression, which only comes on later during larval development. One possible explanation involves a relay mechanism in which that pulse of Cas activates an (or a cascade of) unknown components, which persist and eventually turns on the later hh expression. Yet, in such a model, Cas need not interact directly with the hh locus as the ChIP assay clearly suggests. Moreover, there are at least two pulses of hh expression during larval brain development, and the earlier, shorter pulse that is required for the activation of quiescent NBs appear to be independent from Cas regulation as Cas is only switched on in the larval NBs upon reactivation. Most importantly, the data show that mis-expression of cas abolishes, rather than triggers ectopic hh expression. Thus, the findings do not favour the continuous expression of a hh activator downstream of Cas. Alternatively, Cas may be involve in the epigenetic modifications of the hh locus such that it is primed for expression at a much later stage. This may also explain why saturating the system with Cas for prolonged period of time via mis-expression can negatively affect subsequent hh expression because of to its potential aberrant association with the chromatin. Although such a function has not been reported for Cas, previous studies have postulated that components of the temporal series, such as Hb (or mammalian homolog Ikaros) and Svp (or mammalian homolog COUP-TFI/II), play a role in modulating chromatin structure, hence modifying the competency of downstream gene expression subsequently (Chai, 2013).
The relationship between svp and Hh signaling within the postembryonic temporal series cascade is interesting yet unexpected. svp was thought to be a downstream component of cas on the basis of studies in postembryonic NBs in the thoracic segment of the ventral nerve. This is supported by the observations that the pulse Svp occurs at 40-60 h ALH following the pulse of Cas at 30-50 h ALH. Moreover, both svp and cas mutant clones affect Chinmo/Br-C neuronal target transition, apart from causing NBs' failure to exit the cell cycle at early pupal stage. However, examinations of Svp and Cas expression patterns in the central brain region in this study reveal that the Cas expression window overlaps with the peak of the Svp expression window, even though the latter has a much wider expression window in which low expression levels can still be detected in the NBs at 96 h ALH. Moreover, the data show that abolishment of cas function starting from the embryonic stage does not reduce Svp expression in the NBs at 24 h ALH. Hence, previous interpretation that svp functions downstream of cas in the thoracic postembryonic NBs may not be easily extrapolated to NBs in other brain regions. On the basis of the current results, it is tempting to postulate that Cas and Svp constitute two parallel pathways within the temporal series and Hh signaling is regulated by Cas but not Svp. Nevertheless, such a hypothesis warrants more in depth studies (Chai, 2013).
The precise generation of diverse cell types with distinct function from a single progenitor is important for the formation of a functional nervous system during animal development. It has been shown that, in Drosophila, the developmental timing mechanism (the temporal series) is tightly coupled with the asymmetric machinery. However, the underlying mechanism of this coordination remains elusive. The current data suggest that on the one hand, Hh signaling is under the control of the temporal series (hh expression is directly regulated by Cas), while on the other hand, Hh signaling participates in asymmetric segregation of Mira/Pros during NB division. Introduction of ectopic/premature Hh signaling (in ptc mutant clones) during developmental stages in which NBs are proliferating results in cytoplasmic localization of Mira/Pros during mitosis, reduction of NB size, and slow-down of NB cell cycle progression, reminiscent of the final division of NBs in early pupa just before cessation of proliferation. Consequently, these NBs exit the cell cycle prematurely. It is speculated that Pros may be a direct or indirect target of Hh signaling as elevated pathway activity invariantly leads to increased pros expression in the NBs. Furthermore, reducing the level of Pros protein by removing one copy of function pros is able to rescue the Mira delocalization phenotype seen in ptc mutant NBs. Thus, it is plausible that Hh signaling impinges on the asymmetric division apparatus, likely through Pros, to diminish NB fate gradually (as seen with the absence of Dpn and Mira delocalization) prior to the final cell cycle exit. Despite the results indicating a tight correlation between nuclear entry of Pros into the NBs and the eventual cell cycle exit of these NBs during pupal stage, it should be considered that Pros may not be the direct causative agent in controlling NB cell cycle exit. Therefore the actual role of Pros in this process is purely speculative as far as this study is concerned (Chai, 2013).
In contrast, loss of Hh signaling (e.g., in Smo mutant clones) maintains NBs in their 'younger' proliferating stage far beyond the time when they normally exit the cell cycle. Thus, Hh signaling couples the developmental timing mechanism (the temporal series) with the NB intrinsic asymmetric machinery for the generation of a functional nervous system (Chai, 2013).
In vertebrates, constitutive activation of the Sonic hedgehog (SHH, a homologue of Drosophila Hh), signaling pathway through inactivation mutations in PTCH1, activating mutations in SMO, as well as other mutations involving SHH, IHH, GLI1, GLI2, GLI3, and SUFU, has been implicated in a vast array of malignancies. The proven association of Hh signaling pathway with tumourigenesis and tumour cell growth fuel the view that Hh constitutes a mitogenic signal that promotes pro-proliferative responses of the target cells. Moreover, Hh acts as a stem cell factor in somatic stem cells in the Drosophila ovary, human hematopoietic stem cells, and mouse embryonic stem cells, possibly by exerting its effects on the cell cycle machinery (Chai, 2013).
This report provides an opposing facet of Hh signaling where it is required for timely NB cell cycle exit in the postembryonic pupal brain. This may sound astonishing, but the essential roles of Hh signaling as a negative regulator of the cell cycle has been eclipsed by the common bias that it stimulates proliferation, given the many examples of malignancies with the Hh pathway dysregulation. Indeed, studies have indicated that cell cycle exit and differentiation of a number of cell types, such as absorptive colonocytes of the mammalian gut, zebrafish, and Drosophila retina, require Hh activities. SHH signaling pathway is highly activated in human embryonic stem cell (hESC) and such activity is crucial for hESC differentiation as embryoid bodies. The opposing functions of Hh signaling pathway in different cell types reveal that the ultimate effect of this pathway is likely to be tissue specific, depending on its interaction with other regulatory pathways. The current data indicate that in Drosophila postembryonic NBs of the brain this does indeed appear to be the case, because in this system, Hh signaling pathway interacts with NB-specific temporal series and likely the asymmetric cell division machinery to promote pros nuclear localization to trigger cell cycle exit (Chai, 2013).
The intrinsic neurons of mushroom bodies (MBs), centers of olfactory learning in the Drosophila brain, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and exhibit uninterrupted proliferation till the end of the pupal stage. Whereas MB provides a unique model to study proliferation of neural progenitors, the underlying mechanism that controls persistent activity of MB-Nbs is poorly understood. This study shows that Tailless (Tll), a conserved orphan nuclear receptor, is required for optimum proliferation activity and prolonged maintenance of MB-Nbs and ganglion mother cells (GMCs). Mutations of tll progressively impair cell cycle in MB-Nbs and cause premature loss of MB-Nbs in the early pupal stage. Tll is also expressed in MB-GMCs to prevent apoptosis and promote cell cycling. In addition, it was shown that ectopic expression of tll leads to brain tumors, in which Prospero, a key regulator of progenitor proliferation and differentiation, is suppressed whereas localization of molecular components involved in asymmetric Nb division is unaffected. These results as a whole uncover a distinct regulatory mechanism of self-renewal and differentiation of the MB progenitors that is different from the mechanisms found in other progenitors (Kurusu, 2009).
tll was expressed in the dividing MB-Nbs and GMCs, but not in the postmitotic neurons, through the stages of MB development. Tll expression is initially found in almost all procephalic neuroblasts, but became largely restricted to anterior cells by stage 16. Double immunostaining with an anti-Dac antibody, which labels MB neurons, confirmed that they were MB-Nbs and GMCs. In the larval stages, Tll is expressed in the MB-Nbs and GMCs as well as in lamina precursor cells. While the expression in lamina precursor cells disappears by the end of the larval stage, Tll expression in the MB progenitors is maintained during the pupal stages. In newly eclosed flies, Tll expression was found in a few GMC-like cells in the middle of the MB cell clusters, although their exact identity is unknown (Kurusu, 2009).
Several lines of evidence indicate that Tll is cell autonomously required for efficient proliferation activity MB-Nbs. BrdU labeling experiments
demonstrate that DNA synthesis is partially suppressed in tll mutant Nbs in both the larval and the pupal stages. Cell cycle defects in the mutant MB-Nbs are not evident in the larval stage but confirmed by marked suppression of PH3 and Cyc B activity at 20 h APF before the disappearance of mutant Nbs. As a whole, these data suggest that Tll is required to maintain efficient
cell cycle progression in MB-Nbs, particularly in the pupal stage. In contrast, although the premature loss of the mutant Nbs might be a consequence of cell cycle exit as has been suggested with other Nbs,
the exact mechanism of the disappearance of mutant MB-Nbs in the early
pupal stage is unknown. It is also plausible that mutant Nbs are
removed by apoptosis, as is the case with mutant GMCs, although TUNEL signals for MB-Nbs were not detected at 20 h APF, shortly
before their disappearance whereas cell death signals in GMCs are
evident at both the larval and pupal stages (Kurusu, 2009).
Despite marginal reduction in cell division activity of MB-Nbs at the larval stage, loss of tll activity results in significant reduction of the larval MB clones. Instead, the results demonstrate that cell cycle progression is impaired in larval MB-GMCs. Moreover, the majority of the MB-GMCs are lost by cell death. The molecular mechanism underlying these GMC defects is yet to be investigated, but it is unlikely that they are mediated by altered Pros
expression since Pros is co-expressed with Tll in wild-type MB-GMCs, and its expression is unaltered in mutant GMCs. In addition, the results demonstrating that neither p35 nor Diap1 rescues GMC death suggest that Tll might be involved in suppression of an unconventional cell death pathway (Kurusu, 2009).
What is the molecular function of Tll in the regulation of MB progenitors?
The fact that Tll is a transcription factor localized in the nucleus
suggests that Tll might specify neuronal identity of MB progenitors by
regulating cell-type specific genes. However, unlike other regulatory
factors that confer either spatial or temporal identity, Tll is
expressed only in Nbs and GMCs, and mutant neurons exhibit wild-type
like dendritic and axonal wiring patterns even in the adult stage, in which perdurance of wild-type tll activity in the mutant clones is unlikely. Rather, Tll might provide MB progenitors with cellular identity that specify a distinctive proliferation pattern, either by promoting cell cycle or by preventing apoptosis or by both in parallel. In any case, such identity cannot be determined by Tll on its own because Tll is expressed in other neuronal
progenitors such as lamina precursor cells in the optic lobes.
Instead, it is presumed that the proliferation identity of MB progenitors
may be specified in combination with other regulatory factors such as
Eyeless, which is expressed in MB-Nbs, GMCs and postmitotic neurons to
control MB development (Kurusu, 2009).
In the course of MB proliferation, Tll might downregulate key regulatory
genes involved in cell-cycle exit and differentiation, particularly
given the fact that Tll functions mostly as a repressor in the early
embryogenesis. One such candidate gene is pros. Pros is detected in MB-GMCs, but not MB-Nbs. However, loss of pros causes neither tumorous transformation of MB progenitors nor suppression of tll phenotype in pros tll double mutant clones. Moreover, Pros is not upregulated in tll mutant clones. Thus, these data argue against the involvement of pros in the regulation of MB progenitors although they do not exclude a
redundant mechanism involving Pros cooperating with other factors.
Alternatively, Tll could indirectly control cell cycle progression by
downregulating genes that suppress progenitor division. In support of
this, it is noteworthy that the mammalian homolog Tlx (NR2E1) represses a tumor suppressor gene, Pten, via consensus Tll/TLX binding sites located in the pten promoter, and thereby indirectly stimulates the expression of various cell cycle genes including Cyclin D1, p57 kip2, and p27 kip1 (Kurusu, 2009).
Studies on Drosophila neural progenitors reveal heterogeneity among the brain Nbs in terms of temporal windows of cell division, patterns of self-renewal, and susceptibility to mutations that regulate proliferation and termination of progenitors. Among the Nbs in the Drosophila
brain, MB-Nbs exhibit a highly unique proliferation pattern. Most Nbs
pause cell division between the late embryonic and the early first instar stages, and cease proliferation by the early pupal stage. By contrast, MB-Nbs divide continuously from the embryonic stage till the end of pupal stage, generating diverse identities of neurons by temporal order. In house cricket and moth, proliferation activity of MB-Nbs further extends beyond the pupal stage to exhibit persistent neurogenesis during adult life (Kurusu, 2009).
Although the data clearly indicate a pivotal function of Tll for persistent
proliferation and maintenance of MB-Nbs, the mechanism that determines
the exit from cell cycling at the end of pupal stage remains elusive.
Neither extension of Tll expression beyond the end of the pupal period
nor blocking cell death program, by p35 or Diap1, prolonged MB-Nb
proliferation beyond the pupal stage, suggesting existence of other mechanisms that schedule the end of MB-Nb activity. In most brain Nbs, a burst of Pros in the nucleus at around 120 h after larval hatching (24 h APF) induces cell cycle exit to regulate generation of postmitotic progeny in the brain.
However, no burst of nuclear Pros is detected for MB-Nbs at the end of
the pupal stage when they finally exit from cell cycling,
although the data demonstrate that, as is the case with other Nbs in
the brain, Pros indeed has such regulatory potential in larval MBs that
its overexpression results in partial loss of the MB-Nbs. Moreover, MB clones lacking pros activity, which exhibit normal growth, cease cell division by the end of the pupal stage (Kurusu, 2009).
During asymmetric cell division of Drosophila Nbs, Pros is kept inactive by tethering to the cell cortex by MIRA.
At telophase of Nb cell cycle, Pros is segregated into GMC, where it
enters the nucleus to trigger cell cycle exit and promote
differentiation of post mitotic progeny that are generated by the
division of GMC. Accordingly, nuclear Pros is expressed at high levels
in postmitotic neurons and at moderate levels in GMCs. However, whereas this partition pattern of Pros in the post-embryonic brain is shared between MB and non-MB progenies, Pros seems dispensable for cell-cycle control of MB-GMCs. In non-MB lineages, loss of pros activity in GMCs leads to failure of cell-cycle exit and transforms of GMCs into Nbs. However, loss of pros activity never causes transformation of MB-GMCs although mutant MB neurons exhibit considerable dendritic defects. In contrast, Tll is expressed
and required for MB-GMCs to suppress apoptosis and maintain active cell
cycling. Intriguingly, whereas Pros is suppressed by Tll in non-MB
progenitors, both proteins are coexpressed in MB-GMCs, clearly suggesting that, as compared to the progenitors of non-MB lineages, a different mechanism may operate in MB progenitors to control the expression of regulatory factors that are important for cell division and differentiation (Kurusu, 2009).
The brain hyperplasia produced by Tll overexpression is reminiscent of
brain tumors caused by mislocalization of asymmetric determinants.
Aberrant Nb divisions that disrupt the positioning of such factors
generate brain tumors. Brain tissues from pins, mira, numb, or pros mutants generate tumors when transplanted in the wild-type abdomen. In double mutants of pins and lgl,
mislocalization of aPKC in the basal cortex results in the generation
of supernumerary Nbs at the expense of GMCs, and thus, neurons.
BRAT is required for the asymmetric positioning of Pros, which in turn
suppresses self-renewal of GMC and promotes cell differentiation by
transcriptional control. Mutant clones of either brat or pros are highly tumorigenic, forming a large number of MIRA-positive Nbs (Kurusu, 2009).
While recapitulating the tumor phenotype, ectopic expression of Tll does not
affect asymmetric localization of aPKC, PINS, and BRAT. Instead, Tll downregulates Pros in hyperplasic brains and in overexpression clones, suggesting that the tumorigenesis phenotype caused by Tll
expression is mediated by Pros downregulation in GMCs. This notion is
further supported by the fact that coexpression of Pros with Tll
suppresses brain hyperplasia. Notably, the cis-regulatory region of pros harbors a consensus Tll binding site within 500 base pairs from the
transcriptional initiation site, consistent with the idea that Tll might repress transcription of pros via direct DNA binding (Kurusu, 2009).
Recently, atypical large Nb lineages in the dorsomedial part of the larval brain have been described and designated as Posterior Asense-Negative (PAN)
Nbs. Nbs of such lineages divide asymmetrically to self renew, but, unlike
other Nbs, generate smaller intermediate progenitors that express Nb
markers. The fact that these atypical Nbs are MIRA-positive and Pros
negative raises a possibility that tumor clones induced by Tll could
either correspond to or originate from them. As with other Nbs, clones
of the PAN-Nb lineages accompany only a single large Nb, with their
progeny arranged regularly in a columnar order. By contrast, clones generated by Tll overexpression harbor several large to intermediate-sized Nbs, exhibiting irregular morphology, which is typical of tumors. PAN-Nbs are the Nb subpopulation that exhibits overgrowth in brat mutants. However, it is also unlikely that Tll induced overgrowth originates from overgrowth of PAN Nbs, which correspond to eight Nbs in the DPM group among the ~90 Nbs per hemisphere. On the contrary, Tll induces clonal tumors not only in DPM but also in CM and BLP lineages. Indeed, Tll overgrowth phenotype is not localized to a specific location of the hemisphere, but broadly detectable in the brain
including the optic lobe. Moreover, Tll overgrowth phenotype is also induced in the embryonic CNS, arguing against the involvement of larval PAN-Nbs (Kurusu, 2009).
The Drosophila Tll and the vertebrate homolog TLX (NR2E1) share high sequence similarity in the DNA binding domain. Tlx mutant mice exhibit a reduction of rhinencephalon and limbic structures with emotional and learning defects. Notably, Tlx mutant mice exhibit reduction of neuron numbers in cortical upper layers. Postnatally, TLX is localized to the adult neurogenic regions including the subgranular layer of the dentate gyrus to maintain stem cells in a proliferative and undifferentiated state.
Recent behavioral studies have shown that such TLX-positive neural stem
cells actually contribute to animal's spatial learning. Thus, combined with the current results, these studies highlight a functional commonality of the tll/Tlx homologs between flies and mammals, and imply an intriguing evolutionary conservation of the genetic programs underlying neural
progenitor controls in crucial brain structures involved in memory and
other cognitive functions (Kurusu, 2009).
Intriguingly, the mammalian pros homolog Prox1 promotes cell cycle exit and differentiation of the neural progenitors in the developing subventricular zone and the retina, the neural tissues in which Tlx functions antagonistically to control progenitor proliferation. Based on the tll GOF phenotypes in Drosophila, it is predicted that deregulation of Tlx in the developing brain may cause suppression of Prox1 and could result in severe neurological tumors in humans. On the other hand, consistent with the loss-of-function phenotypes in flies, several
mutations have been identified in the regulatory regions of Tlx in humans with microcephary. Given the commonality in progenitor control, further studies of the Drosophila MB-Nbs may shed light on the molecular basis of the proliferation and differentiation of neural progenitors, and would provide important cues for understanding progenitor disorders in the human brain (Kurusu, 2009).
Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts, the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) was identified as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). These findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization (Sousa-Nunes, 2009).
Drosophila neuroblasts (NBs) are stem-cell-like neural progenitors, which undergo repeated asymmetric divisions to self-renew and generate neurons and/or glia. During each round of division the cell fate determinants Pros (a homeodomain-containing transcription regulator), Numb (a negative regulator of Notch signaling), as well as Brain Tumor (Brat, whose mechanism of action in cell fate specification is unclear) are asymmetrically localized as protein crescents on the NB cortex. In the embryo, the NB mitotic spindle is oriented along the apicobasal axis, the cell fate determinants and their adapter proteins localize to the NB basal cortex and segregate exclusively to the smaller basal daughter, called ganglion mother cell (GMC). The GMC divides terminally to produce two neurons or glial cells. The coordination between the basal localization of the cell fate determinants and the apicobasal orientation of the spindle during mitosis is mediated by several evolutionarily conserved proteins that localize to the apical NB cortex during the G2 stage of the cell cycle. These comprise [1] the Drosophila homologs of the Par3/Par6/aPKC protein cassette, [2] several proteins involved in heterotrimeric G protein signaling—Gαi/Partner of Inscuteable (Pins)/Locomotion defects (Loco), [3] as well as Inscuteable (Insc). In contrast to the embryo, NBs in the larval central brain divide without an apparent fixed orientation. Nevertheless the majority of central brain NBs appear to utilize the same molecular machinery as embryonic NBs, with the apical and basal molecules sharing similar hierarchical relationships and localizing to opposite sides of the NB cortex (Sousa-Nunes, 2009).
Asymmetric localization of Pros and Brat on the one hand and Numb on the other, is mediated through direct interactions with their respective adapters, the coil-coil proteins Miranda (Mira) and Partner of Numb (Pon). Although mutations affecting any of the apical proteins compromise asymmetric localization of basal proteins to varying extents, only in the case of aPKC has any mechanistic insight emerged. aPKC facilitates basal localization of cell fate determinants either through phosphorylation of the cytoskeletal protein Lgl and/or through direct phosphorylation of the determinant. Lgl is uniformly localized throughout the NB cortex, and is essential for cortical association and asymmetric localization of the cell fate determinants and their adapters. aPKC phosphorylates Lgl on three conserved serine residues and the triphosphorylated form appears to be inactive due to a conformational change. The proposed model is that unphosphorylated, active Lgl is restricted to the basal cortex because of apically localized aPKC. Consistent with this model, a nonphosphorylatable version of Lgl, Lgl3A, in which the three target serines have been mutated to alanines, appears to be constitutively active and its expression leads to uniform cortical localization of the normally basally restricted cell fate determinants. Numb is a second protein that can be phosphorylated by aPKC and phosphorylation of three N-terminal serines causes it to become cytoplasmic (Sousa-Nunes, 2009).
How Lgl acts to facilitate the localization of cell fate determinants is less clear. Lgl can bind nonmuscle Myosin II (Zipper) and genetic experiments suggest that Myosin II and Lgl have antagonistic activities. Hence, one possible scenario would be that Myosin II is active at the apical cortex due to the presence of phosphorylated Lgl, which is incapable of binding to Myosin II. Myosin II can then act to exclude basal proteins from the apical cortex. Alternatively, since yeast Lgl orthologs function in exocytosis, it has been suggested that Lgl might act by regulating this process. It is possible that Lgl positively promotes delivery and cortical association of the basal molecules, and that this is antagonized by Myosin II apically. In this scenario, Lgl is inhibited apically both by aPKC and Myosin II, and only basal Lgl is active and able to promote cortical association of the basal proteins (Sousa-Nunes, 2009).
The unconventional Myosin VI (Jaguar, Jar) and Myosin II bind in a mutually exclusive manner to the basal adapter protein Mira. However, in contrast to Myosin II, which acts antagonistically to Lgl, Jar acts in a synergistical manner with Lgl to effect Mira basal localization. In mitotic NBs devoid of Jar, Mira is mislocalized to the cytoplasm. Jar possibly mediates association of Mira with the basal actin cytoskeleton (Sousa-Nunes, 2009).
In addition to aPKC, a few other serine/threonine protein kinases have been shown to play a role in facilitating asymmetric protein localization in NBs. These include Cdk1, required for the asymmetric localization of both apical and basal components during mitosis, Aurora A (AurA), and Polo, both of which mediate Numb and Pon asymmetric localization. With the exception of Polo kinase, which phosphorylates a serine residue within the Pon asymmetric localization domain, substrates for the other kinases have not been identified. The involvement of protein kinases in NB asymmetric divisions implies the involvement of protein phosphatases; however, to date, none have been implicated in the process (Sousa-Nunes, 2009).
In a clonal genetic screen designed to identify genes that mediate NB asymmetric divisions, multiple loss-of-function alleles of flfl. Falafel (Flfl) were identified as a regulatory subunit of the evolutionarily conserved Protein Phosphatase 4 (PP4) Phosphatase complex. PP4 belongs to the best-studied family of cellular protein serine/threonine phosphatases, PP2A (the other major families being PP1, PP2B, and PP2C). Similarly to other PP2A-like phosphatases, PP4 functions as a heterotrimeric complex comprising of a catalytic subunit, PP4C, associated with two regulatory subunits, PP4R2 and PP4R3. PP4, or specifically PP4R3/Flfl, has been implicated in a variety of molecular and cellular processes including regulation of MEK/Erk, insulin receptor substrate 4, Hematopoietic progenitor kinase 1, and Histone deacetylase 3 activities, centrosome maturation, cell cycle progression, apoptosis, DNA repair, cell morphology, and lifespan control (Sousa-Nunes, 2009 and references therein).
This study shows that loss of flfl, as well as attenuation of PP4C/Pp4-19C or PPR2/PPp4R2r function by RNAi specifically results in delocalization of Mira and its associated proteins throughout the cytoplasm in metaphase/anaphase NBs; in addition, both Mira and Pros localize to the NB nucleus prior to nuclear envelope breakdown. Excessive nuclear Mira is dependent on the presence of Pros. These results suggest that whereas cytoplasmic or membrane-associated PP4 is required for asymmetric cortical localization of Mira (and its associated proteins) during metaphase and anaphase, nuclear PP4 is required to exclude Pros (and as a consequence, Mira) from the NB nucleus prior to nuclear envelope breakdown. Moreover, Flfl can complex with Mira in vivo and directly interact with Mira, suggesting that Flfl targets PP4 activity to the Mira complex to facilitate its correct localization (Sousa-Nunes, 2009).
In a clonal screen on third-instar larval (L3) brains, designed to identify novel genes on chromosome arm 3R required for NB asymmetric division, a novel allele of flfl, flfl795 was isolated. In metaphase and anaphase flfl795 clone NBs, Mira displays weak cortical crescents but also a pronounced mislocalization throughout the cytoplasm, whereas in surrounding heterozygous NBs Mira is localized to a robust crescent like in wild type with little cytoplasmic accumulation. As with many mutations that disrupt NB asymmetry during metaphase and anaphase, flfl795 NBs display telophase rescue: The majority of the cytoplasmic Mira relocalizes asymmetrically to the NB cortex at telophase, resulting in asymmetric segregation of Mira into the GMC. Using the flfl795 allele, two additional alleles [flfl795(2), flfl795(3)] were identified via complementation screening of an independent collection of ethylmethane sulfonate (EMS) mutant stocks. Sequencing of these three EMS-induced flfl alleles revealed single point mutations resulting in premature stop codons at positions 324 (flfl795) and 630 [flfl795(2)] of the longest isoform (980 amino acids) and a disruption to the splice acceptor site at the 3′ end of the fourth intron [flfl795(3)]. All three alleles display a mislocalization of Mira to the cytoplasm of metaphase NBs and form an allelic series in terms of phenotypic severity: flfl795 > flfl795(3) > flfl795(2) (Sousa-Nunes, 2009).
Homozygous flfl795 animals survive to pharate adults whereas hemizygous flfl795 animals [using Df(3R)Exel6170 to remove one copy of the flfl coding region] only survive until L3. Furthermore, although the cytoplasmic Mira phenotype of flfl795 homozygotes is highly penetrant, the majority of metaphase NBs still display weak Mira crescents, whereas the majority of metaphase NBs of flfl795 hemizygotes display no crescents. These results suggest that the strongest EMS allele (flfl795) is nevertheless a hypomorph. Therefore a flfl-null allele (flflN42) was generated by imprecise excision of the P-element P{EPgy2}flflEY03585, located ∼1 kb upstream of the flfl translational start site. This allele was confirmed to be a genetic null by the similar expressivities of NB phenotypes in flflN42 homozygotes and flflN42 hemizygotes, as well as in flfl795/flflN42 and flfl795/Df(3R)Exel6170. Consistently, flflN42 NBs are antigen-minus (see below) and molecular analysis indicates that it is a deletion extending into the coding region, deleting the first 1075 base pairs of the coding sequence. Subsequent analyses of the phenotype were carried out using the flflN42 allele, hereafter referred to simply as flfl (Sousa-Nunes, 2009).
In addition to the mislocalization of Mira, the Mira-associated proteins Pros, Brat and Staufen (Stau), are similarly mislocalized to the cytoplasm of metaphase/anaphase flfl NBs. Pros mislocalization occurs in Asense (Ase)-positive NB lineages which comprise the majority of lineages in the central brain; Ase-negative NBs are Pros-negative in flfl as well as in wild-type brains. In contrast, the localization of members of the other basal complex, Pon and Numb, and of apical complexes is unaffected. Hence, during NB division, flfl loss of function specifically affects the localization of the Mira complex (Sousa-Nunes, 2009).
Flfl homologs have been identified in several species, from yeast to humans. They all possess the same domain architecture: a Ran-binding domain (RanBD) at the N terminus, similar in three-dimensional structure to the Ena/VASP homology domain 1 (EVH1, which derives its name from the founding members Enabled and Vasodilator-stimulated phosphoprotein) and to the pleckstrin homology domain; followed by a conserved domain of unknown function (DUF625), a region containing armadillo/HEAT repeats, and a region of low complexity. Within the DUF625 domain, Flfl contains two putative NLSs (NLS1 and NLS2) as well as a nuclear export signal (NES); close to the C terminus Flfl contains a short conserved stretch of acidic and basic amino acid residues that has been shown to be required for nuclear localization of the Dictyostelium discoideum homolog, SMEK (NLS3). Flfl contains many putative target sites for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in its C-terminal 300 amino acids and numerous putative phosphorylation sites throughout, some of which are predicted to be PKC targets (Sousa-Nunes, 2009).
In conclusion, loss of function or RNAi knockdown of the regulatory subunits flfl/PP4R3 or PPP4R2r/PP4R2 as well as knockdown of the catalytic subunit Pp4C-19C/PP4C of PP4 causes mislocalization of Mira/Pros/Brat/Stau to the cytoplasm of metaphase and anaphase NBs (Sousa-Nunes, 2009).
Attenuation of PP4 function above also causes increased frequency of nuclear Mira/Pros prior to nuclear envelope breakdown. The observation that depletion of the catalytic subunit of PP4 results in identical phenotypes to the depletion of its regulatory subunits, suggests that phosphatase activity plays a role in the localization of Mira/Pros throughout the NB cell cycle (Sousa-Nunes, 2009).
Nuclear mislocalization of Mira seen in flfl, jar, or mira2L150 single-mutant NBs requires pros function. This suggests that, when transport of Mira toward or its tethering to the cortex is defective, Pros can take Mira into the nucleus. In this context, the normal relationship between Mira and Pros is reversed, with Pros instructing Mira localization rather than the converse. In the absence of pros, Mira is not localized to the nucleus, even when PP4 function is attenuated. Thus, the role of PP4 on these two temporally distinct localizations of Mira/Pros appears to involve distinct targets since one is a Mira-dependent localization and the other is Pros-dependent (Sousa-Nunes, 2009).
In contrast to serine-threonine kinases, substrate specificity for serine/threonine protein phosphatases is thought to be conferred not primarily by sequences adjacent to the target residues but rather by interaction between the substrate and regulatory subunits of the phosphatase complex. This is the case for the founding family member PP2A, whose variable subunit composition can also target the complex to distinct subcellular domains and is thought to be the case also for PP4. Flfl, a regulatory subunit of PP4, is able to bind Mira and Flfl and Mira are found in a complex in vivo. No binding was detected between Flfl and Pros but since Mira and Pros still colocalize when PP4 function is attenuated, these results also suggest that PP4 function is not required for the Mira-Pros interaction. Therefore, Pros could be recruited to PP4 by its association with Mira, which in turn binds Flfl (Sousa-Nunes, 2009).
Flfl is nuclear before and cytoplasmic after nuclear envelope breakdown. The results from nuclear excluded and membrane targeted versions of Flfl suggest that nuclear Flfl is required to exclude Mira/Pros from the nucleus when inefficiently bound to the cytoskeleton/cortex, whereas cytosolic or membrane-associated Flfl is required for the cortical association and asymmetric localization of Mira/Pros/Brat/Stau at metaphase and anaphase. The localization of Mira/Pros prior to and after nuclear envelope breakdown by PP4 may involve different phosphatase substrates. It is tempting to entertain the possibility that Mira dephosphorylation by PP4 in the cytoplasm is required for its asymmetric cortical localization during mitosis, and that Pros dephosphorylation by PP4 in the nucleus is required for its nuclear exclusion/progression through prometaphase. Indeed, a previous study has shown that cortical Pros is highly phosphorylated relative to nuclear Pros. To test this hypothesis, attempts were made to detect enrichment of a lower mobility band of Mira::3GFP in flfl larval extracts compared with wild type but this it could not be detected, working at the limits of detectability (Sousa-Nunes, 2009).
Asymmetric cortical localization of proteins during NB asymmetric division is dependent on an intact actin cytoskeleton. Although flfl is required for Mira cortical association, at no point in the NB cell cycle does Flfl exhibit cortical enrichment. However, modified versions of Flfl that are either uniformly cytoplasmic or cortically enriched can both drive asymmetric cortical localization of Mira and its associated proteins. Moreover, the Mira mislocalization phenotypes of flfl are strikingly similar to those of Myo VI/jar. Both mutants exhibit nuclear Mira/Pros prior to and cytoplasmic Mira and associated proteins following NB nuclear envelope breakdown; both Flfl and Jar are cytoplasmic at metaphase/anaphase; and genetically, both Jar and Flfl act parallel to or downstream from Lgl. Further propelled by the presence of a putative actin-binding domain in Flfl (the RanBD domain, which is an EVH1-like domain), it was asked whether Flfl too might facilitate association of Miranda with the actin cytoskeleton either separately from or in association with Jar. However, in vitro assays clearly showed that Flfl does not bind F-actin, although Mira alone does, with comparable strength to that of α-Actinin and Jar, used as controls. Furthermore, Jar could not be detected in Flfl containing protein immunoprecipitates. Therefore, it seems unlikely that Flfl acts either directly or in a complex with Jar to facilitate Mira transport along or tethering to the actin cytoskeleton. Still, Flfl could act indirectly; for example, by stabilization of the Mira-Jar association. It is speculated that Flfl may act by targeting PP4 to the Mira complex and that the consequent dephosphorylation of a component of this complex facilitates Jar-Mira association (Sousa-Nunes, 2009).
In Dictyostelium, mutants in the flfl homolog, smkA, exhibit phenotypes similar to strains defective in Myo II assembly, suggesting that smkA may regulate Myo II function. However, in flfl NBs the Mira mislocalization phenotype does not resemble that of Myo II loss of function, which has been described to lead to Mira mislocalization to the mitotic spindle in embryonic NBs (Sousa-Nunes, 2009).
The reduced proliferation seen in flfl NBs correlates with nuclear localization of Pros/Mira. Nuclear Pros negatively regulates transcription of cell cycle genes and positively regulates differentiation genes, and has been shown to limit NB proliferation. Therefore, ectopic nuclear Pros is likely to be at least one cause of the NB underproliferation observed in flfl brains. Still, it is possible that flfl has additional functions in promoting proliferation, independent of its role in excluding Pros/Mira from the NB nucleus. Indeed, an excessive proportion of phospho-histone H3-positive flfl NBs was detected relative to wild type. These NBs typically had a nucleus but the cell morphology was not spheroid, as would be expected in prophase cells. This suggests that flfl NBs either have a block or delay in prometaphase or that PP4 may be required for dephosphorylation of Histone H3; in either case, it seems to be required for dephosphorylation of other proteins involved in cell cycle progression. Nonetheless, pros,flfl double-mutant NB clones are indistinguishable from those of pros single mutants, both showing extensive overproliferation, suggesting that the loss of flfl is unable to override the overproliferation induced by loss of pros (Sousa-Nunes, 2009).
Numb can antagonize Notch signaling to diversify the fates of sister cells. Paired sister cells acquire different fates in all three Drosophila neuronal lineages that make diverse types of antennal lobe projection neurons (PNs). Only one in each pair of postmitotic neurons survives into the adult stage in both anterodorsal (ad) and ventral (v) PN lineages. Notably, Notch signaling specifies the PN fate in the vPN lineage but promotes programmed cell death in the missing siblings in the adPN lineage. In addition, Notch/Numb-mediated binary sibling fates underlie the production of PNs and local interneurons from common precursors in the lAL lineage. Furthermore, Numb is needed in the lateral but not adPN or vPN lineages to prevent the appearance of ectopic neuroblasts and to ensure proper self-renewal of neural progenitors. These lineage-specific outputs of Notch/Numb signaling show that a universal mechanism of binary fate decision can be utilized to govern diverse neural sibling differentiations (Lin, 2010).
In contrast to MB lineages, in which GMCs divide to make two indistinguishable neurons, the three AL neuronal lineages examined produce GMCs that consistently undergo asymmetric cell division and yield daughter cells with distinct fates. This mechanism allows doubling of neuron types, as in the lAL lineage. However, in the adPN and vPN lineages, only one from each pair of daughter cells persists into the adult stage. They are both present as hemilineages. Notably, about 50% of central brain lineages exist as hemilineages, as revealed by clonal analysis with twin-spot MARCM using a pan-neuronal driver. Recovery of the missing hemilineages in the Drosophila VNC has implicated the Notch/Numb-mediated asymmetric cell division as a mechanism for divergent configuration of distinct insect brains. In sum, asymmetric cell division is broadly utilized; depending on the lineages, a GMC may divide to make two identical neurons, two distinct neurons, or only one mature neuron (Lin, 2010).
Notch and Numb underlie asymmetric cell division in diverse contexts, including the asymmetric cell divisions of diverse AL PN precursors. Notably, the output of Notch signaling is grossly opposite in the adPN versus vPN lineage. Each GMC in both lineages makes one PN and one mysterious sibling. Interestingly, Notch-on specifies the PN fate in the vPN lineage but antagonizes the PN fate in the adPN lineage. The cell-fate determinants for PNs of different lineages could be more distinct than their gross phenotypes suggest. In addition, the mysterious siblings of adPNs versus vPNs, upon rescued, might acquire very different fates. These lineage-dependent outputs of Notch signaling support the argument for its involvement in modulating cell differentiation, rather than specifying any de novo cell fate. It appears that two, possibly mutually exclusive, cell fates pre-exist in each precursor, and that Notch signaling, which occurs only in Numb-negative daughter cells, triggers cell differentiation along one rather than the other pre-programmed path (Lin, 2010).
Notch/Numb-dependent asymmetric cell division underlies the derivation of two complex lAL hemilineages that both persist into the adult stage. Distinct PN types are made along the Notch-off hemilineage, whereas diverse types of non-PNs, including various AL LNs and most Acj6-positive progeny, differentiate from Numb-negative daughter cells. As in other neuronal lineages, specific neuron types of the lAL lineage are made at specific times of development. However, it remains uncertain whether specific PN types consistently pair with specific non-PN types through the production of the sister hemilineages. Superficially, there exist many more non-PN types than the recognizable PN types in the lAL lineage, raising the possibility that neuronal temporal identity is altered in distinct paces between the two lAL hemilineages. Determining individual lAL GMCs and their derivatives is essential for resolving the detail and further elucidating how two parallel sets of temporal cell fates can be generated by a common progenitor through repeated self-renewal (Lin, 2010).
Besides governing neuronal cell fates following asymmetric cell division of GMCs, Numb, together with other basal complex proteins, including Brat and Prospero, is selectively segregated into GMCs during self-renewal of Nbs. However, in contrast with its essential role for preventing the transit-amplifying precursors from undergoing tumor-like overproliferation in PAN lineages, the function of Numb in restraining the basally situated Nb offspring from adopting Nb fate varies among non-PAN lineages and depends on the stage of development. Notably, Numb is required in certain non-PAN neuronal lineages, including the lAL lineage, for preventing production of ectopic Nbs. Although Notch is dispensable for maintaining the stem cell fate in lAL Nbs, it remains likely that loss of Numb leads to ectopic Notch signaling, which in turn promotes stem cell fate in otherwise GMCs. The differential requirement of Numb for proper specification of GMCs of different origins could be due to lineage- and/or stage-dependent variations in the abundance of Notch signaling components. Interestingly, the ectopic Nbs apparently maintain proper temporal identity and could make diverse neuron types as the endogenous progenitor. These raise the possibility that dynamic Notch signaling might be utilized in vivo to promote self-renewal versus amplification of Nbs (Lin, 2010).
Taken together, most neuron types in the Drosophila central brain are specified not only according to their lineage origin as well as birth order, but also depending on whether Numb exists to suppress Notch signaling in newly derived postmitotic neurons. It appears that postmitotic neurons are born with two opposing cell fates that were pre-determined in their immediate precursors based on their lineage and temporal origin. Notch signaling then suppresses the otherwise dominating fate. In addition, in certain neuronal lineages, Numb plays a subtle role in ensuring production of GMCs while Nbs undergo self-renewal. A conserved Notch/Numb-dependent mechanism probably governs diverse neural developmental processes through evolution (Lin, 2010).
Neural progenitors of the Drosophila larval brain, called neuroblasts, can be divided into distinct populations based on patterns of proliferation and differentiation. Type I neuroblasts produce ganglion mother cells (GMCs) that divide once to produce differentiated progeny, while type II neuroblasts produce self-renewing intermediate neural progenitors (INPs) and thus generate lineages containing many more progeny. This study identified Taranis (Tara) as an important determinant of type I lineage-specific neural progenitor proliferation patterns. Tara is an ortholog of mammalian SERTAD proteins that are known to regulate cell cycle progression. Tara is differentially-expressed in neural progenitors, with high levels of expression in proliferating type I neuroblasts but no detectable expression in type II lineage INPs. Tara is necessary for cell cycle reactivation in quiescent neuroblasts and for cell cycle progression in type I lineages. Cell cycle defects in tara mutant neuroblasts are due to decreased activation of the E2F1/Dp transcription factor complex and delayed progression through S-phase. Mis-expression of tara in type II lineages delays INP cell cycle progression and induces premature differentiation of INPs into GMCs. Premature INP differentiation can also be induced by loss of E2F1/Dp function and elevated E2F1/Dp expression suppresses Tara-induced INP differentiation. These results show that lineage-specific Tara expression is necessary for proper brain development and suggest that distinct cell cycle regulatory mechanisms exist in type I versus type II neural progenitors (Manansala, 2013).
Brain development requires precise control of neural progenitor proliferation and differentiation. Attempts were made to identify regulators of these processes starting with a TU-tagging approach (Miller, 2009) to identify mRNAs that are enriched in neural progenitors. From the TU-tagging data, tara as a candidate regulator of neurogenesis. Analysis of tara-lacZ expression in larval brains revealed interesting temporal and spatial expression patterns. tara is not expressed in quiescent neuroblasts of newly hatched larvae but is expressed in the persistently proliferating mushroom body neuroblasts. Similarly, tara expression ceases at the end of larval neurogenesis when neuroblasts exit the cell cycle but tara expression continues through pupal stages in the proliferating mushroom body neuroblasts. These temporal patterns suggest that tara expression is regulated by signals that control cell cycle entry and exit. In developing larvae, nutrient status controls secretion of insulin/IGF-like peptides from glial cells and this signaling system is necessary for neuroblast reactivation. Importantly, tara-lacZ expression is only detected after quiescent neuroblasts receive these reactivation signals. Tara is therefore not likely to function in the transduction of these signals but is a likely downstream target of reactivation pathways. This is similar to serum-induced expression of the mammalian Tara orthologs SERTAD1 and SERTAD2 (Sim, 2006). tara-lacZ expression is also detected in post-mitotic neurons of late-stage larval brains, particularly in early-born neurons that surround the central neuropil. This expression pattern suggests that Tara has an additional role in neural function, separate from the cell cycle roles described in this study (Manansala, 2013).
In addition to the temporal patterns of tara-lacZ expression, spatial patterns of tara-lacZ expression were identified that correspond to distinct progenitor populations. tara-lacZ is not expressed in optic lobe neuroepithelia but is expressed in neuroblasts derived from these progenitors. tara is also differentially-expressed in central brain neuroblasts, with high levels of tara-lacZ expression in type I neuroblasts, low or undetectable levels in type II neuroblasts (following reactivation), and no detectable expression in type II lineage INPs. Compared to previously described lineage-specific proteins (Ase, Erm, PntP1), Tara is unique in that it is only expressed in type I neuroblast lineages following reactivation. Importantly, the tara-lacZ expression patterns in type I versus type II lineages correlate with tara loss-of-function and mis-expression phenotypes. Type I lineage tara loss-of-function clones had dramatic proliferation defects while type II lineage loss-of-function clones were not significantly different from wildtype, as predicted based on the absence of tara-lacZ expression in type II lineages. Type II lineages were more dramatically affected by tara mis-expression, compared to the effect of tara over-expression in type I lineages, providing evidence that the absence of Tara is important for normal type II lineage development (Manansala, 2013).
The timing of tara expression during neuroblast reactivation suggested that Tara is necessary for cell cycle re-entry. This role was confirmed in tara1 mutants, in which quiescent neuroblasts fail to reactivate. A neuroblast-autonomous role for Tara in reactivation was demonstrated using the wor-GAL4 driver to express a tara transgene in the tara1 mutant background. Wor-GAL4 driven tara expression was sufficient to rescue quiescent neuroblast reactivation in tara1 mutants, although there were fewer M phase neuroblasts relative to wildtype. Wor-GAL4 driven E2F1/Dp expression in tara1 mutants also rescued quiescent neuroblast reactivation, placing E2F1/Dp downstream of Tara in the reactivation pathway. Given the requirement for Tara during neuroblast reactivation, precocious expression of tara in quiescent neuroblasts might be expected to cause early cell cycle re-entry. Expression of tara in quiescent neuroblasts was sufficient to activate CycE expression but was not sufficient for progression to S phase. Similarly, previous work has shown that precocious expression of CycE fails to induce premature S phase in quiescent neuroblasts. These results are interpreted as evidence that Tara regulates the initial transition from G0 to G1, but temporally-regulated growth signals are required for transition past the G1/S checkpoint. It is worth noting that ectopic expression of tara or E2F1/Dp in neuroblasts using wor-GAL4 did not rescue the lethality of tara1 mutants. This suggests that endogenous regulation of Tara in the nervous system is essential or that Tara is required in other tissues such as mesoderm or muscles (Manansala, 2013).
MARCM analysis showed that tara loss-of-function in type I lineages caused a delay in neuroblast and GMC cell cycle progression, primarily during S-phase. In Drosophila embryos, decreased E2F1/DP activity has been shown to inhibit the G1→S transition (which requires the E2F1/DP target gene CycE) and to delay S phase progression (which requires the E2F1/DP target gene PCNA). Following reactivation, the delayed S phase progression observed in tara1 clones may be due to decreased transcription of E2F1/DP targets, particularly PCNA. tara1 neuroblasts express low levels of PCNA and this would be expected to limit the processivity of DNA polymerase delta and prolong S phase, as previously described (Manansala, 2013).
The absence of tara expression in INPs led to a hypothesis that tara mis-expression might interfere with type II lineage development. tara mis-expression in type II lineages was found to delay INP cell cycle progression and reduced the number of INPs in type II neuroblast MARCM clones. The decreased numbers of INPs appeared to be due to premature INP differentiation, particularly based on the analysis of INP MARCM clones. If tara mis-expression only decreased the rate of INP self-renewing divisions, the frequency of clones that still contain an INP would not differ between wildtype and tara mis-expressing clones. However, there is a significant decrease in the number of clones containing an INP when tara is mis-expressed, indicating premature INP differentiation into a GMC. Delayed progression through G1 and S-phases in INPs could be sufficient to cause premature differentiation, as has been demonstrated in mammalian cortical progenitors. However, experiments using Dap to delay the G1/S transition in INPs demonstrate that this delay is not sufficient to cause differentiation. These results suggest that there is something unique about the cell cycle delay induced by Tara or that Tara causes INP differentiation independent of its effects on cell cycle timing (Manansala, 2013).
Mammalian SERTAD proteins regulate cell cycle entry and progression via interaction with Cdk4 and link cell cycle progression with transcription via interactions with E2F/Dp. SERTAD1 regulates Cdk4 kinase activity in a dose-dependent manner, stimulating Cdk4 at low concentrations and inhibiting Cdk4 at high concentrations (Li, 2004). It was hypothesized that Tara might inhibit Cdk4 in INPs but found that Cdk4 is dispensable for type II lineage development. Similarly, elevated Cdk4 expression does not suppress Tara-induced INP differentiation. These results agree with previous work showing that Cdk4 does not significantly affect cell proliferation rates in Drosophila and instead regulates cell growth in certain tissues. In contrast to Cdk4, loss of E2F1/Dp-dependent transcription causes type II lineage defects that are identical to those observed in tara mis-expressing INPs. Similarly, E2F1/Dp over-expression suppresses Tara-induced INP differentiation. These results support a model in which Tara represses transcription of E2F1/Dp target genes that are necessary for cell cycle progression and self-renewal in INPs. In contrast to mammals, the Drosophila genome encodes a single Dp protein, and only two E2F proteins, E2F1 and E2F2. E2F1/Dp activates transcription of genes necessary for cell cycle progression and E2F2/Dp can repress transcription of these same genes in addition to repressing transcription of a distinct set of genes involved in differentiation. This study did not investigate the potential contribution of E2F2/Dp-Tara complexes in INP differentiation, but the fact that E2F1i2 mutants mimic tara mis-expression phenotypes and elevated E2F1/Dp suppresses tara-mis-expression phenotypes suggests that E2F1 target genes (as opposed to E2F2-specific target genes) are relevant to INP differentiation (Manansala, 2013).
The current findings support a model in which Tara stimulates E2F1/Dp activity in type I lineages but inhibits E2F1/Dp activity when mis-expressed in type II lineages. A likely explanation for these opposing lineage-specific roles of Tara is provided by the fact that mammalian SERTAD1 can interact with transcription factors that either stimulate or inhibit E2F1/Dp1 activity. This interaction occurs via PHD and bromodomain binding motifs that are conserved in Tara (Calgaro, 2002). It is proposed that distinct E2F/Dp-Tara complexes form in type I versus type II lineages and that the absence of Tara from INPs is necessary to avoid formation of complexes that inhibit E2F1/Dp activity and favor differentiation. Elevated Pros expression in INPs has been shown to induce premature differentiation and this study observed an increased number of Proshigh cells in tara mis-expressing clones. Elevated Pros may be the cause of differentiation in tara mis-expressing INPs. Tara is not required for induction of Pros expression in type I lineages, since tara loss-of-function does not result in any detectable changes in Pros levels, but tara mis-expression may increase Pros transcription in INPs. The current findings suggest that Tara establishes lineage-specific E2F1/Dp-dependent transcription programs, and identification of relevant target genes and Tara-interacting transcription factors will be important areas of future investigation. The discovery that a Drosophila SERTAD protein differentially regulates the development of neural lineages also raises the possibility that mammalian SERTAD proteins control cell cycle and cell fate decisions in a lineage-specific or tissue-specific manner (Manansala, 2013).
Developing animals survive periods of starvation by protecting the growth of critical organs at the expense of other tissues. This study used Drosophila to explore the as yet unknown mechanisms regulating this privileged tissue growth. As in mammals, it was observed in Drosophila that the CNS is more highly spared than other tissues during nutrient restriction (NR). Anaplastic lymphoma kinase (Alk) efficiently protects neural progenitor (neuroblast) growth against reductions in amino acids and insulin-like peptides during NR via two mechanisms. First, Alk suppresses the growth requirement for amino acid sensing via Slimfast/Rheb/TOR complex 1. And second, Alk, rather than insulin-like receptor, primarily activates PI3-kinase. Alk maintains PI3-kinase signaling during NR as its ligand, Jelly belly (Jeb), is constitutively expressed from a glial cell niche surrounding neuroblasts. Together, these findings identify a brain-sparing mechanism that shares some regulatory features with the starvation-resistant growth programs of mammalian tumors (Cheng, 2011).
This study found that CNS progenitors are able to continue
growing at their normal rate under fasting conditions severe
enough to shut down all net body growth. Jeb/Alk signaling was
identified as a central regulator of this brain sparing, promoting
tissue-specific modifications in TOR/PI3K signaling that protect
growth against reduced amino acid and Ilp concentrations.
These findings highlight that a 'one size fits all' wiring diagram
of the TOR/PI3K network should not be extrapolated between
different cell types without experimental evidence. The two molecular mechanisms by which Jeb/Alk
signaling confers brain sparing is discussed, and how they may be integrated
into an overall model for starvation-resistant CNS growth (Cheng, 2011).
One mechanism by which Alk spares the CNS is by suppressing
the growth requirement for amino acid sensing via Slif, Rheb,
and TORC1 components in neuroblast lineages. An important
finding of this study is that in the presence of Alk signaling Tor has no
detectable growth input (evidence from Tor clones), but in its
absence (evidence from UAS-AlkDN; Tor clones) Tor reverts to
its typical role as a positive regulator of both growth and proliferation.
The growth requirement for Slif/TORC1 is clearly much
less in the CNS than in other tissues such as the wing disc but
a low-level input cannot be ruled out due to possible perdurance
inherent in any clonal analysis. Although Slif, Rheb, Tor, and
Raptor mutant neuroblast clones attain normal volume, this
reflects increased cell numbers offset by reduced average cell
size. Atypical suppression of proliferation by TORC1 has also
been observed in wing discs, where partial inhibition with rapamycin
advances G2/M progression (Cheng, 2011).
Alk signaling in neuroblast lineages does not override the
growth requirements for all TOR pathway components. The
downstream effectors S6k and 4E-BP retain functions as
positive and negative growth regulators, respectively. 4E-BP
appears to be particularly critical in the CNS as mutant animals
have normal mass, but mutant neuroblast
clones are twice their normal volume. In many tissues, 4E-BP is
phosphorylated by nutrient-dependent TORC1 activity. In CNS
progenitors, however, 4E-BP phosphorylation is regulated in
an NR-resistant manner by Alk, not by TORC1. Hence, although
the pathway linking Alk to 4E-BP is not yet clear, it makes an
important contribution toward protecting CNS growth during
fasting (Cheng, 2011).
A second mechanism by which Alk spares CNS growth is by
maintaining PI3K signaling during NR. Alk suppresses or overrides
the genetic requirement for InR in PI3K signaling, which
may or may not involve the direct binding of intracellular domains
as reported for human ALK and IGF-IR (Shi, 2009). Either
way, in the CNS, glial Jeb expression stimulates Alk-dependent
PI3K signaling and thus neural growth at similar levels during
feeding and NR. In contrast, in tissues such as the salivary gland,
where PI3K signaling is primarily dependent upon InR, falling insulin-like peptides
concentrations during NR strongly reduce growth (Cheng, 2011).
The finding that Alk signals via PI3K during CNS growth differs
from the Ras/MAPK transduction pathway described in
Drosophila visceral muscle. However, a link between ALK and PI3K/Akt/Foxo signaling
during growth is well documented in humans, both in glioblastomas
and in non-Hodgkin lymphoma. Similarities with mammals are less obvious with
regard to Alk ligands, as there is no clear Jeb ortholog and
human ALK can be activated, directly or indirectly, by the
secreted factors Pleiotrophin and Midkine (Cheng, 2011).
A comparison of these results with those of previous studies indicates
that CNS super sparing only becomes fully active at late larval
stages. Earlier in larval life, dietary amino acids are essential
for neuroblasts to re-enter the cell cycle after a period of
quiescence. This nutrient-dependent
reactivation involves a relay whereby Slif-dependent amino
acid sensing in the fat body stimulates Ilp production from a glial
cell niche (Sousa-Nunes, 2011). In turn, glial-derived Ilps
activate InR and PI3K/TOR signaling in neuroblasts thus stimulating
cell cycle re-entry. Hence, the relative importance of Ilps versus Jeb
from the glial cell niche may change in line with the developmental
transition of neuroblast growth from high to low nutrient sensitivity (Cheng, 2011).
The results of this study suggest a working model for super
sparing in the late-larval CNS.
Central to the model is that Jeb/Alk signaling suppresses Slif/
RagA/Rheb/TORC1, InR, and 4E-BP functions and maintains
S6k and PI3K activation, thus freeing CNS growth from the high
dependence upon amino acid sensing and Ilps that exists in
other organs. The CNS also contrasts with other spared diploid
tissues such as the wing disc, in which PI3K-dependent growth
requires a positive Tor input but is kept in check by negative
feedback from TORC1 and S6K. Alk is both necessary (in the CNS) and sufficient
(in the salivary gland) to promote organ growth during fasting.
However, both Alk manipulations produce organ-sparing
percentages intermediate between the 2% salivary gland and
the 96% neuroblast values, arguing that other processes may
also contribute. For example, some Drosophila tissues synthesize
local sources of Ilps that could be more NR resistant than
the systemic supply from the IPCs. In mammals, this type of mechanism
may contribute to brain sparing as it has been observed
that IGF-I messenger RNA (mRNA) levels in the postnatal CNS
are highly buffered against NR. It will
also be worthwhile exploring whether mammalian neural growth
and brain sparing involve Alk and/or atypical TOR signaling.
In this regard, it is intriguing that several studies show that activating
mutations within the kinase domain of human ALK are
associated with childhood neuroblastomas. In addition, fetal growth of the mouse
brain was recently reported to be resistant to loss of function of
TORC1. Finally, a comparison between the current
findings and those of a cancer study, highlights that insulin/IGF independence and constitutive
PI3K activity are features of NR-resistant growth in contexts as
diverse as insect CNS development and human tumorigenesis (Cheng, 2011).
Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor Prospero is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that Prospero is not expressed in postmitotic neuronal progeny. Thus, Prospero is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. This study focused on postembryonic neurogenesis and shows that the expression of Prospero is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, evidence is provided that this expression of Prospero in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) Dacapo. These findings imply that Prospero, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development (Colonques, 2011).
During development, cell cycle progression must be coordinated with the regulation of cell specification and differentiation. The underlying mechanisms of coordination are likely to be particularly complex during neural development due to the enormous cell diversity in the brain. In Drosophila, these mechanisms have been well studied during embryonic CNS development. In embryonic neurogenesis, the homeodomain transcription factor Pros is expressed in the NB but it does not enter the nucleus due to its binding to the carrier protein Mira, which localizes to the cell cortex. This interaction facilitates the segregation of Pros from the parent NB to the GMC during asymmetric NB division. In the GMC, Pros is released from its carrier and translocates to the nucleus where it plays a binary role as a cell fate determinant, and as a promoter of terminal differentiation (Colonques, 2011).
It has been reported that Pros is similarly expressed and asymmetrically segregated during the proliferative activity of (type I) NBs in the larval CB although it does not seem to be expressed in CB dorso-medial lineages (type II) NBs. However, as this study shows, during postembryonic neurogenesis, in the majority of larval CB and OPC (outer proliferation center) neuronal lineages, pros expression is transiently upregulated in new born prospective neurons (GCs), in addition to its earlier expression and asymmetric segregation in some larval NBs. This is clearly different from the situation in embryonic lineages where pros is only transcribed in NBs, and Pros protein is downregulated in GCs after the division of their parent GMC (see Summary of cellular expression pattern of PROS in the larval CNS and lineage alterations in pros mutant clones) (Colonques, 2011).
This transient expression in most newborn postembryonic neurons shortly after the division of the GMC implies a novel role of Pros in postmitotic cells. It is postulated that this role is to inhibit cell cycle progression and promote cell cycle exit. The Pros GoF and LoF experiments support this notion. Pros GoF induces proliferation arrest and Pros LoF results in supernumerary cells with sustained expression of cell cycle markers, indicating an inability to withdraw from the cell cycle (Colonques, 2011).
In addition to the marked difference in Pros expression in postmitotic GCs during embryogenesis versus postembryonic neurogenesis (Pros is undetectable in embryonic GCs and high in postembryonic GCs), there are other functional differences in Pros action during embryonic versus postembryonic CNS development. For example, in pros mutant embryos, overproliferation is followed by abundant apoptotic cell death among the supernumerary cells. By contrast, no increas cell death was found in the larval OL of pros mutants. Moreover, while Pros and Dap seem to act in parallel to end the cell cycle in the embryonic CNS, Dap appears to act downstream of Pros in larval CNS neurons. These initial findings suggest that further differences between the functions of Pros during embryonic and postembryonic CNS neurogenesis may exist and should be considered (Colonques, 2011).
The fact that Pros protein is present in embryonic GMCs (intermediate progenitors) but not in embryonic GCs (prospective neurons), suggests that in the embryonic CNS, Pros initiates the end of mitotic activity in the GMC rather than in the GC. Accordingly, it has been proposed that the GMC is a transition state between the proliferating NB and the differentiating neuron that provides a window in which Pros represses stem cell-specific genes and activates differentiation genes. Nevertheless, it is not well understood how the GMC can go through its terminal cell cycle in spite of the repressive action of Pros on cell cycle regulators (Colonques, 2011).
The results strongly suggest that in postembryonic neurogenesis Pros acts not only in the GMC progenitor but also in the postmitotic GCs produced by the GMC. Thus, this analysis indicates that there are two main pros expression pattern subclasses among CB type I and OPC NB lineages. For the shake of simplicity they have been called them A and B. In type A, Pros is expressed in GCs after the division of GMCs while in type B, Pros is first expressed at low level in the NB and asymmetrically segregated to the GMC, and afterwards, upregulated in new born GCs. These two subsets of expression patterns correlate well with the two main phenotypes found in pros mutant clones. Thus, the LOF of pros in NBs with type A Pros expression appears to preclude cell cycle exit of GCs which, consequently, continue dividing and do not differentiate, yielding a type A clone composed of a single NB, a GMC and several small mitotic cells. By contrast, in lineages with type B Pros expression, the LOF of pros seems to cause primarily a change in the fate of the putative GMC that behaves like a NB maintaining the expression of asymmetric division genes (such as Mira) and overproliferating, to yield a type B clone composed of multiple large NB like cells (Colonques, 2011).
Hence, it is postulated that during postembryonic neurogenesis Pros functions in two sequential phases in type I NB lineages, first as cell fate determinant in some GMCs and later as cell cycle repressor in most GCs. Furthermore, the idea is favored that the different roles of Pros in postembryonic GMCs versus postembryonic GCs might be related to the higher level of expression observed in GCs compared to GMCs. Thus, high levels of Pros might be required to definitively withdraw the GCs from the cell cycle, while low levels might be sufficient to specify GMCs and modulate their cell cycle. The higher level of Dap protein in postembryonic GCs in relation to their parent GMCs and NBs is consistent with this hypothesis. The strong burst of Pros at the end of NB proliferation in ventral ganglia of early pupae is also in agreement with the idea that high levels of Pros are required to stop proliferation. Furthermore, it has been recently shown that the missexpression of Pros at high level suppresses proliferation in type II larval brain NBs lineages without apparent change in their identity (Colonques, 2011).
Taken together, all of these findings imply that different developmental strategies have been selected to couple cell fate decisions and cell cycle regulation during embryonic and postembryonic neurogenesis through the same effector, Pros. It is possible that this change in strategy is a consequence of the evolutionary adaptation to regulate the production of a large number of equivalent neurons in postembryonic lineages in contrast to embryonic neurogenesis where a much more limited set of specific neurons are generated in each lineage through GMC divisions (Colonques, 2011).
This study has shown that Pros is coexpressed with Dap in new born prospective neurons and, moreover, it was found that pros is sufficient and it is required for the expression of dap in these larval brain neuronal precursors. The dap gene encodes a member of the Cip/Kip family of CKIs with homology to mammalian p27kip1. This family of CKIs has been implicated in mediating cell cycle exit prior to terminal differentiation. They function by binding and inhibiting G1/S cyclin dependent kinase complexes. There is compelling data supporting a role of Dap in cell cycle exit during Drosophila embryogenesis. In Drosophila embryonic NB lineages, dap expression becomes apparent just before the terminal neurogenic division of the GMC. In contrast, this study has shown that dap is upregulated in new born postembryonic neurons. Consistent with a role in the termination of cell proliferation, dap expression in the larval OL has been tightly correlated with cells ending proliferation. Interestingly, Pros is required to terminate cell proliferation during embryonic neurogenesis and it has been shown to be involved in the regulation of dap expression in the embryonic nervous system. Thus, the results provide support to the idea that Pros promotes the cell cycle exit of post-embryonic GCs by upregulating the expression of dap. The data also suggest that this upregulation of dap is mediated by inhibiting the expression of Dpn. Dpn is an essential panneural bHLH transcription factor, which has been previously shown to be a suppressor of dap expression in the larval OL. Indeed, the dpn gene contains consensus Pros binding sites and Pros has been shown to be required to terminate the expression of dpn in the embryo (Colonques, 2011).
Quiescent stem cells in adult tissues can be activated for homeostasis or repair. Neural stem cells (NSCs) in Drosophila are reactivated from quiescence in response to nutrition by the insulin signaling pathway. It is widely accepted that quiescent stem cells are arrested in G0. This study, however, demonstrates that quiescent NSCs (qNSCs) are arrested in either G2 or G0. G2-G0 heterogeneity directs NSC behavior: G2 qNSCs reactivate before G0 qNSCs. In addition, this study shows that the evolutionarily conserved pseudokinase Tribbles (Trbl) induces G2 NSCs to enter quiescence by promoting degradation of Cdc25String and that it subsequently maintains quiescence by inhibiting Akt activation. Insulin signaling overrides repression of Akt and silences trbl transcription, allowing NSCs to exit quiescence. These results have implications for identifying and manipulating quiescent stem cells for regenerative purposes (Otsuki, 2018).
Neural stem cells (NSCs) in Drosophila, like those in mammals, proliferate during embryogenesis, become quiescent in the late embryo, and then proliferate again (reactivate) postembryonically to produce neurons and glia. A nutritional stimulus induces reactivation; specifically, dietary amino acids induce glial cells in the blood- brain barrier to secrete Drosophila insulin-like peptides (dILPs). dILPs activate the insulin signaling pathway in neighboring quiescent NSCs (qNSCs), prompting the NSCs to exit quiescence (Otsuki, 2018).
Quiescent stem cells are widely accepted to be arrested in G0, a poorly understood state characterized by a 2n DNA content and a lack of expression of cell cycle progression factors. This study assessed whether Drosophila qNSCs are arrested in G0. As expected, the M phase marker phospho-histone H3 (pH3) was not detected in qNSCs. Previous studies demonstrated that qNSCs do not express the G1 marker cyclin E or incorporate the S phase marker 5-bromo-2'-deoxyuridine (BrdU) or 5-ethynyl-2'-deoxyuridine (EdU). However, it was found that 73% of qNSCs expressed the G2 markers cyclin A (CycA) and cyclin B (CycB). This finding suggests that most qNSCs are arrested in G2 and that qNSCs are arrested heterogeneously in the cell cycle (Otsuki, 2018).
It was verified that ~75% of qNSCs were arrested in G2 by comparing the fluorescent ubiquitination- based cell cycle indicator (FUCCI)-pH3 profiles of qNSCs and proliferating NSCs. CycA-positive (CycA+) qNSCs had twice the DNA content of CycA-negative (CycA-) qNSCs and larger nuclei than CycA- qNSCs. Thus, qNSCs exhibited two types of stem cell quiescence: The majority were arrested in G2, and a minority were arrested in G0. G2 quiescence has not been reported previously for stem cells in mammals or Drosophila (Otsuki, 2018).
The choice of G2 or G0 arrest could be stochastic or preprogrammed. Seven G0 qNSCs were found in the first thoracic hemisegment, T1, and eight G0 qNSCs each in T2 and T3. A consistent subset of qNSCs were always arrested in G0, namely, NB2-2, NB2-4, NB2-5, NB3-4, NB5-3, and NB7-4 (cells are named according to their spatial origin in the neuroectoderm). Of these qNSCs, NB2-4 disappears from T1 during embryogenesis, explaining why fewer qNSCs are arrested in G0 in T1 than in T2 and T3. NB5-4 and NB5-7 were arrested in G2 in 50% of hemisegments but were not always arrested in the same cell cycle phase on either side of the midline. It is concluded that, with the exception of NB5-4 and NB5-7, the choice of G2 or G0 quiescence is entirely invariant (Otsuki, 2018).
Is G2-G0 heterogeneity in qNSCs significant? The reactivation of G2 and G0 qNSCs
by tracking the expression of the reactivation
marker worniu (wor). More than 86% of
G qNSCs reactivated by 20 hours after larval 2
hatching (ALH), compared with 20% of G0 qNSCs. For example, NB3-4, a G0 qNSC, reactivated in fewer than 7% of hemisegments (n = 10 tVNCs, six hemisegments each) . All NSCs reactivated by 48 hours ALH. Thus, G2 qNSCs are faster-reactivating stem cells than G0 qNSCs (Otsuki, 2018).
Next, gene expression was profiled in qNSCs using targeted DamID (TaDa), identifying 1656 genes. Corresponding Gene Ontology (GO) terms included 'nervous system development' (35 genes) and 'neuroblast [NSC] development' (10 genes). To identify quiescence-specific genes, genes common to quiescent and proliferating NSCs, such as deadpan (dpn), were eliminated. tribbles (trbl) is one of the most significantly expressed protein-encoding genes specific to qNSCs. trbl encodes an evolutionarily conserved pseudokinase with three human homologs that have been implicated in insulin and mitogen-
activated protein kinase signaling. It was confirmed that trbl labels quiescent but not proliferating NSCs in vivo. To date, no other gene that labels qNSCs specifically has been identified (Otsuki, 2018).
trbl is necessary for quiescence entry, as NSCs continued to divide during late embryogenesis in trbl hypomorphic mutants or when trbl was knocked down specifically in NSCs. trbl regulates quiescence entry specifically, without affecting division mode or cell viability. The ectopically dividing NSCs in the trblEP3519 mutant were G2,
not G0, qNSCs. G2 but not G0 qNSCs also became significantly smaller in trblEP3519 mutants. As embryonic NSCs do not regrow between cell divisions, the size reduction is consistent with excessive divisions. Consistent with a function in G2 quiescence, Trbl was expressed primarily in G2 qNSCs (Otsuki, 2018).
Trbl is also required to maintain quiescence. RNA interference-mediated knockdown of trbl in qNSCs caused NSCs to leave quiescence and divide. Transgenic flies were generated carrying upstream activation sequence-green fluorescent protein (GFP)-Trbl and drove expression with grainyhead (grh)-GAL4 (4) to assess whether Trbl is sufficient to maintain G2 quiescence. grh-GAL4 expression is initiated at quiescence entry and occurs in ~67% of NSCs, allowing comparison between neighboring GFP-Trbl-expressing and nonexpressing NSCs. Almost all GFP-Trbl-expressing NSCs remained in G2 quiescence and expressed CycA. GFP-Trbl-expressing NSCs retained the primary process that is extended specifically by quiescent NSCs, unlike control NSCs, which had begun to divide. Thus, Trbl is sufficient to maintain G2 quiescence (Otsuki, 2018).
In the embryonic mesoderm, trbl induces G2 arrest by promoting Cdc25String protein degradation. This study found that Cdc25String protein was reduced in NSCs at quiescence entry but that Cdc25String mRNA was maintained (Fig. 4A). Therefore, Cdc25String is regulated posttranscriptionally at quiescence entry. Significantly more NSCs were positive for Cdc25String protein in trblEP3519 mutants than in controls. This increase in Cdc25String is sufficient to explain the excessive NSC proliferation in trbl mutants. Thus, Trbl initiates quiescence entry by promoting Cdc25String protein degradation during late embryogenesis (Otsuki, 2018).
Trbl also maintains NSC quiescence postembryonically; however, it must act through another mechanism, as Cdc25String is no longer expressed in postembryonic qNSCs. Trbl is known to inhibit insulin signaling by binding Akt and preventing its phosphorylation. Consistent with this, Trbl-expressing NSCs had less phosphorylated translation initiation factor 4E-binding protein (p4E-BP) than control NSCs. If Trbl inhibits Akt to maintain quiescence, constitutively active Akt (myr-Akt; here-
after AktACT) should counteract Trbl-induced quiescence. AktACT fully rescued NSC reactivation. In contrast, as Trbl is thought to act downstream of phosphatidylinositol 3-kinase (PI3K), constitutively active PI3K (dp110CAAX; hereafter PI3KACT)should not rescue reactivation, which it did not. Thus, Trbl maintains quiescence by
blocking activation of Akt. This role is specific to postembryonic NSCs, as embryonic NSCs do not depend on insulin signaling to proliferate (Otsuki, 2018).
trbl expression must be repressed to allow NSC reactivation. It was found that insulin signaling is necessary and sufficient to repress trbl transcription. NSCs misexpressing phosphatase and tensin homolog (PTEN), an insulin pathway inhibitor, failed to down-regulate trbl transcription . In contrast, activating the insulin pathway by expressing AktACT in NSCs was sufficient to switch off trbl transcription (Otsuki, 2018).
This study has discovered the mechanisms by which Drosophila NSCs enter, remain in, and exit quiescence in response to nutrition. First, Trbl pseudokinase promotes degradation of Cdc25String protein to induce quiescence; second, it blocks insulin signaling by inhibiting Akt in the same NSCs to maintain quiescence; and third, it is overridden by nutrition-dependent secretion of dILPs from blood-brain barrier glia, which activate insulin signaling in qNSCs, repress trbl expression, and enable reactivation (Otsuki, 2018).
qNSCs were found to be preprogrammed for arrest in G2 or G0, contrary to accepted doctrine. G2 qNSCs are the first to reactivate and generate neurons; this is followed by reactivation of G0 qNSCs. This pattern may ensure that neurons form the correct circuits in the appropriate order during brain development. G2 arrest also enables high-fidelity homologous recombination- mediated repair in response to DNA damage, preserving genomic integrity during quiescence. Quiescent stem cells in mammals may also arrest in G2, with implications for isolating and manipulating quiescent stem cells for therapeutic purposes (Otsuki, 2018).
Spinal muscular atrophy is a severe neurogenic disease that is caused by mutations in the human survival motor neuron 1 (SMN1) gene. SMN protein is required for the assembly of small nuclear ribonucleoproteins and a dramatic reduction of the protein leads to cell death. It is currently unknown how the reduction of this ubiquitously essential protein can lead to tissue-specific abnormalities. In addition, it is still not known whether the disease is caused by developmental or degenerative defects. Using the Drosophila system, this study shows that SMN is enriched in postembryonic neuroblasts and forms a concentration gradient in the differentiating progeny. In addition to the developing Drosophila larval CNS, Drosophila larval and adult testes have a striking SMN gradient. When SMN is reduced in postembryonic neuroblasts using MARCM clonal analysis, cell proliferation and clone formation defects occur. These SMN mutant neuroblasts fail to correctly localise Miranda and have reduced levels of snRNAs. When SMN is removed, germline stem cells are lost more frequently. It was also shown that changes in SMN levels can disrupt the correct timing of cell differentiation. It is concluded that highly regulated SMN levels are essential to drive timely cell proliferation and cell differentiation (Grice, 2011).
This study shows a high demand for SMN in Drosophila stem cells. In addition, striking SMN concentration gradient, inversely proportional to the state of differentiation, has been identified in Drosophila larval CNS and testis. In Drosophila SMN mutant larvae, both the CNS and testis display growth defects which precede the previously reported motor defects and death. These larvae also fail to localise Miranda protein correctly at the basal membrane of the neuroblast. Clonal analysis indicates that SMN deficient stem cells have a reduced number of divisions and also generate cells with lower levels of U2 and U5 snRNPs. Overexpression of SMN alters the timing of CNS growth and disrupts the onset of pupariation and pupation. Using the male germline system, it was shown that prolonged SMN reduction leads to stem cell loss. Finally it was found that ectopic SMN expression in cells along the SMN gradient leads to changes in the timing of cell differentiation. It is therefore suggested that the fine-tuning of SMN levels throughout development can lead to complex developmental defects and reduce the capacity of stem cells to generate new cells in development (Grice, 2011).
SMN levels have been reported to be extremely high in early development. This study shows that SMN up-regulation occurs in neuroblasts prior to the initiation of their cell division, suggesting a distinct increase of SMN levels is required for new rounds of neurogenesis and local proliferation. Fewer immature neurons are generated in the thoracic ganglion of smn mutant MARCM clones. Provisional data has suggested there may be proliferation defects in the spinal cord of severe mouse models. In addition, a recent study using the severe SMA mouse model has shown proliferation defects in the mouse hippocampus, a region associated with higher SMN levels (Wishart, 2010). Together these data suggest that, in part, the pathology observed in more severe forms of SMA may be caused by defects in tissue growth (Grice, 2011).
Proteins involved in processes such as chromatin remodelling, histone generation and cell signalling have been identified as intrinsic factors for the maintenance of Drosophila stem cells. This is the first report of stem cell defects caused by the reduction of a protein involved in snRNP biogenesis. Although SMN is required in all cells, proper stem cell function requires a substantially higher level of SMN. This study also shows snRNP defects in Drosophila SMN mutant tissue. Previous studies in Drosophila have shown no gross changes in snRNP levels, including U2 and U5, in lysates from whole smnA and smnB mutant larvae. smnA MARCM neuroblast clones and male germline mitotic clones have reduced snRNP levels, suggesting snRNP assembly may be particularly sensitive to SMN reduction during CNS and germline development (Grice, 2011).
SMN mutant neuroblasts have abnormal Miranda localisation. Miranda, an adaptor protein, forms a complex with the RNA binding protein Staufen which binds to prospero mRNA. In addition to snRNPs, SMN protein has been implicated in the biogenesis of numerous RNP subclasses, including proteins involved in the transport and localisation of β-actin mRNA at the synapse. Whether Miranda mislocalisation is due to direct or indirect associations with SMN should be addressed (Grice, 2011).
SMN mutant larvae have been previously shown to have synaptic defects which include enlarged and fewer boutons and a reduction in the number of GluR-IIA clusters - the neurotransmitter receptor at the Drosophila neuromuscular junction. In addition, numerous developmental defects are observed including pupation and growth defects. Complementing this work, Drosophila Gemin5 a member of the Drosophila SMN-Gemin complex has been shown to interact with members of the ecdysone signalling pathway responsible for initiating pupation and growth. Drosophila Gemin5 is also enriched in pNBs, in a pattern comparable to SMN. There is increasing evidence that suggests the Drosophila SMN complex plays an important role in pupation. Ubiquitous overexpression of SMN using da-GAL4 advances CNS development and causes premature entry into pupation. The ecdysone pathway has been identified to play an important part in the regulation of neuroblast division and neuronal differentiation during development. How the Drosophila SMN complex plays a part in stem cell biology, and how the SMN complex interacts with specific signalling pathways should be the subject of further study (Grice, 2011).
Larval and adult testes exhibit the most distinct SMN gradients in Drosophila tissues. Drosophila testes have a constant population of germline stem cells that start to divide in the late larval stages and produce sperm throughout life. The removal of SMN from male germline stem cells results in stem cell loss. In the smnB mutant testis, the reduction of SMN causes a contraction of the SMN gradient towards the apical stem cells. As SMN is lost from the primary spermatoctyes, more mature sperm are observed. Increasing SMN levels leads to an increase in primary spermatocytes and a reduction in mature sperm in the adult. This result is the first to demonstrate that high SMN levels in undifferentiated cells can repress differentiation in sperm development. Interestingly, along with the CNS, Drosophila testes have the highest number of alternative splicing events and the most differentially expressed splicing factors during development. Understanding if differential expression of SMN in specific cell types controls a shift in splicing factors as cells switch from proliferation to differentiation will be the target of future study. A recent study has identified defects in gametogenesis and testis growth in mice lacking the Cajal body marker coilin, a binding partner of SMN. The authors speculated that coilin may facilitate the fidelity and timing of RNP assembly in the cell and coilin loss may limit rapid and dynamic RNA processing. It will be important to understand how SMN and coilin genetically interact in stem cells and developing tissues (Grice, 2011).
The Drosophila CNS and male germline offer two new tractable systems that can be used to study SMN biology in development and stem cells. It also offers a system to study how SMN, a protein associated with neuronal development, could cause SMA. Although SMA is classically a disease of the motor neuron, a severe reduction of SMN protein affects a wide spectrum of cells including stem cells. Consistent with this idea, symptoms in mild forms of SMA (type III or IV) are predominately limited to motor neurons. However, patients with the most severe type (type I), suffer from defects in multiple tissues including congenital heart defects, multiple contractures, bone fractures, respiratory insufficiency, or sensory neuronopathy. Elucidating the differential requirements of SMN in individual cell types, and how their sensitivity to SMN loss can mediate the disease, can contribute to the understanding of the selectivity of SMA (Grice, 2011).
The anterodorsal projection neuron lineage of Drosophila melanogaster produces 40 neuronal types in a stereotypic order. This study takes advantage of this complete lineage sequence to examine the role of known temporal fating factors, including Chinmo and the Hb/Kr/Pdm/Cas transcriptional cascade, within this diverse central brain lineage. Kr mutation affects the temporal fate of the neuroblast (NB) itself, causing a single fate to be skipped, whereas Chinmo null only elicits fate transformation of NB progeny without altering cell counts. Notably, Chinmo operates in two separate windows to prevent fate transformation (into the subsequent Chinmo-indenpendent fate) within each window. By contrast, Hb/Pdm/Cas play no detectable role, indicating that Kr either acts outside of the cascade identified in the ventral nerve cord or that redundancy exists at the level of fating factors. Therefore, hierarchical fating mechanisms operate within the lineage to generate neuronal diversity in an unprecedented fashion (Kao, 2012).
Knocking down Kr from the NB led to skipping of a single temporal fate during adPN neurogenesis. Removing Kr from specific GMCs further revealed that GMC, which normally makes the missing adPN, had precociously adopted the next temporal fate in the absence of Kr. These observations indicate that Kr regulates temporal fate transitions in the adPN NB and is continuously required in the GMC to suppress the next temporal fate. Despite no evidence for the involvement of Hb/Pdm/Cas, Kr's role in delaying fate transition in the Kr-positive GMC suggests an analogous role in an alternate temporal cascade that confers a specific temporal fate from a set of contiguous fates. Furthermore, loss of Kr exerted no detectable effect on the remaining cascade, reminiscent of the chain control in the sequential expression of Hb/Kr/Pdm/Cas (Kao, 2012).
Kr confers the VA7l fate in adPN lineage. Notably, the temporal fate that precedes VA7l fate defines a polyglomerular PN with a rather diffuse AL elaboration. It is challenging to definitely locate the embryonic-born polyglomerular adPN due to colabeling with a large number of uniglomerular siblings. To exclude Hb as the temporal factor that precedes Kr, whether the embryonic-born polyglomerular adPN exists and has properly differentiated in hb mutant NB clones was reexamined. A combination of two sparse GAL4 drivers that collectively label three adPNs, including the embryonic polyglomerular PN plus two earlier-born uniglomerular siblings, was used to identify NB clones generated near the beginning of the lineage and simultaneously to assess the pre-VA7l polyglomerular PN. The same three adPNs were observed in the wild-type, hb, as well as Kr mutant NB clones. These results strengthen the conclusion that Kr acts alone without Hb/Pdm/Cas to specify only one middle temporal fate in the protracted adPN lineage (Kao, 2012).
In contrast to Kr defining only one temporal fate, Chinmo acts in two windows to support eight temporal fates in the adPN lineage. The two windows are separated by only one Chinmo-independent adPN that happens to split two otherwise indistinguishable VM3-targeting adPNs. Interestingly, the fate transformation of the last two embryonic adPNs (transformed from the VM3[b] and DL4 types to larval-born D type) is similar to the chinmo-elicited fate transformation of larval-born adPNs (Kao, 2012).
Chinmo has previously been implicated in governing neuronal temporal identity in the MB lineage and one partially resolved neuronal lineage. This study observed a distinct pattern of Chinmo requirement in the adPN lineage. Notably, chinmo mutant neurons aberrantly adopt later temporal cell fates within their original lineages in all cases. Moreover, Chinmo governs multiple continuous fates in MB as well as in adPN lineages. Despite these similarities, detailed mechanisms of Chinmo actions are apparently distinct. In the MB lineages, reducing Chinmo expression elicits systematical early-to-late MB temporal fate transformations, and ectopic Chinmo can specify early MB fates in late siblings. By contrast, a partial reduction in Chinmo sometimes conferred hybrid adPN fate showing features of both the prospective cell fate and the chinmo-null default fate, rather than exhibiting the morphologies reminiscent of the fates in between. And ectopic Chinmo also failed to promote early fates in late-born adPNs, providing no evidence for dosage-dependent Chinmo-mediated fate determination in the adPN lineage. Therefore, both loss- and gain-of-function genetic mosaic studies suggest that Chinmo does not directly determine any temporal cell fate in adPN lineage, but rather it suppresses a later temporal fate in early siblings to allow further neuronal diversification. Further, mechanism(s) must exist to restrict the activities of Chinmo to specific windows, because ectopic Chinmo exerted no detectable effect on adPNs within the rest of the lineage. It is also not clear whether and how Chinmo directly diversifies neuron fate (Kao, 2012).
Unlike Kr that regulates temporal fate transition in the NB, Chinmo apparently acts in the offspring and potentially downstream of some NB transcriptional cascade to increase neuron diversity. This distinction is supported by the follwing: (1) postmitotic expression of transgenic Chinmo restored proper temporal cell fates in chinmo mutant adPNs, arguing that Chinmo acts in newborn neurons to regulate adPN temporal identity; (2) deleting chinmo from NB through the entire lineage did not affect overall temporal fate transitions, as evidenced by no change in total cell count or length of the lineage; and (3) ectopic expression of chinmo exerted no detectable effect on the NB temporal fate transitions. All these observations indicate that Chinmo acts in postmitotic neurons to refine temporal identity. Temporal patterning by the Kr-containing transcriptional cascade in the NB and via Chinmo in newborn neurons exemplifies a hierarchical mode of temporal cell-fate specification. Identifying additional genes controlling adPN temporal identity and determining their mechanisms of action by iterative use of the strategy used in this paper will allow elucidation of developmental mechanisms specifying the great diversity of neuron types in the complex brain (Kao, 2012).
A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast produces a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, lineage-specific neuroblasts were marked and isolated for RNA sequencing. Particular neuroblasts were labeled throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single-cell picking. Transcriptome analysis of mushroom body, antennal lobe and type II neuroblasts compared with non-selective neuroblasts, neurons and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. Besides transcription factors that are likely to be pan-neuroblast, many transcription factors exist that are selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates (Yang, 2016).
Human outer subventricular zone (OSVZ) neural progenitors and Drosophila type II neuroblasts both generate intermediate neural progenitors (INPs) that populate the adult cerebral cortex or central complex, respectively. It is unknown whether INPs simply expand or also diversify neural cell types. This study shows that Drosophila INPs sequentially generate distinct neural subtypes, that INPs sequentially express Dichaete, Grainy head and Eyeless transcription factors, and that these transcription factors are required for the production of distinct neural subtypes. Moreover, parental type II neuroblasts also sequentially express transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. It is concluded that neuroblast and INP temporal patterning axes act together to generate increased neural diversity within the adult central complex; OSVZ progenitors may use similar mechanisms to increase neural diversity in the human brain (Bayraktar, 2013).
Tests were carried out to determine whether D, Grh and Ey exhibit cross-regulation in INPs. wor-gal4, ase-gal80 was used to drive UAS-DRNAi in a Dichaete heterozygous background (subsequently called D RNAi, in which RNAi denotes RNA interference), which removed detectable D from INP lineages. Compared to wild type, D RNAi resulted in a significant loss of early born Grh+ Ey− INPs, without altering the number of later-born Grh+ Ey+ INPs. The same result was observed in D mutant clones. By contrast, misexpression of D did not lead to ectopic Grh expression. Thus, D is necessary for the timely activation of Grh in INP lineages, although D-independent inputs also exist (Bayraktar, 2013).
To test whether Grh regulates D or Ey, R9D11-gal4 was used to drive UAS-grhRNAi in a grh heterozygous background (subsequently called grh RNAi), which significantly reduced Grh levels in middle-aged INPs. grh RNAi increased the number of D+ INPs at the expense of Ey+ INPs without altering the total number of INPs. As expected, grh RNAi did not change the numbers of D+ and Ey+ INPs in the DM1 lineage, which lacks Grh expression, nor did misexpression of Grh lead to ectopic Ey expression. It is concluded that Grh represses D and activates Ey within INP lineages (Bayraktar, 2013).
To determine whether Ey regulates D or Grh, R12E09D-gal4 UAS-FLP actin-FRT-stop-FRT-gal4 was used to drive permanent expression UAS-eyRNAi within INPs (subsequently called R12E09D) act-gal4 or INP-specific ey RNAi. It was confirmed that INP-specific ey RNAi removed Ey expression from INPs, without affecting Ey in the mushroom body or optic lobes. ey RNAi resulted in a notable increase in the number of old D-Grh+ INPs, without affecting the number of young D+ INPs. Conversely, Ey misexpression in INPs significantly reduced the number of Grh+ INPs without altering the total number of INPs. An increase was observed in D+ INPs, consistent with a regulatory hierarchy in which Ey represses Grh, which represses D. This effect was not due to ectopic Ey directly activating D because misexpression of Ey had no effect on D+ INP numbers in the DM1 lineage, which lacks Grh expression. It is concluded that Ey is necessary and sufficient to terminate the Grh expression window in INPs. A 'feedforward activation/feedback repression' model is proposed for D-to-Grh-to-Ey cross-regulation (Bayraktar, 2013).
Next, it was asked whether distinct neuronal or glial subtypes were generated during each transcription factor expression window. To determine the cell types produced by young D+ INPs or old Ey+ INPs, permanent lineage tracing was used. Cells labelled by R12E09D but not OK107ey are generated by young INPs, whereas cells labelled by OK107ey are generated by old INPs. A collection of 60 transcription factor antibodies was screened and two were found that labelled subsets of young INP progeny, and two that labelled subsets of old INP progeny. The transcription factors D and Brain specific homeobox (Bsh) labelled sparse, non-overlapping subsets of young INP progeny, but not old INP progeny. Thus, young INPs generate Bsh+ neurons, D+ neurons, and many neurons that express neither gene. By contrast, the glial transcription factor Reverse polarity (Repo) and the neuronal transcription factor Twin of eyeless (Toy) labelled sparse, non-overlapping subsets of old INP progeny, but not young INP progeny. Additional mechanisms must restrict each marker (D, Bsh, Repo and Toy) to small subsets of young or old INP progeny; for example, each population could arise from just early or late born INPs within a type II neuroblast lineage. It is concluded that INPs sequentially express the D, Grh and Ey transcription factors, and they generate distinct neuronal and glial cell types during successive transcription factor expression windows. These data provide the first evidence in any organism that INPs undergo temporal patterning (Bayraktar, 2013).
Experiments were designed to determine whether D, Grh and Ey act as temporal identity factors that specify the identity of INP progeny born during their window of expression. First, the role of Ey in the specification of late born INP progeny was investigated. INP-specific ey RNAi resulted in the complete loss of the late born Toy+ neurons and Repo+ neuropil glia, but did not alter the number of early born D+ and Bsh+ neurons. Removal of Toy+ neurons (using toy RNAi) does not alter the number of Repo+ glia, and conversely removal of Repo+ glia (using gcm RNAi) does not alter the number of Toy+ neurons; thus Ey is independently required for the formation of both classes of late INP progeny. Conversely, permanent misexpression of Ey in early INPs increased late born Toy+ neurons and decreased early born Bsh+ neurons, consistent with Ey specifying late INP temporal identity. Unexpectedly, ectopic Ey reduced the number of late born Repo+ glia. Itis concluded that Ey is an INP temporal identity factor that promotes the independent specification of late born Toy+ neurons and Repo+ glia (Bayraktar, 2013).
Next tests were performed to see whether D and Grh specify early and mid INP temporal identity. INP-specific D RNAi led to a small but significant reduction in the number of early born Bsh+ neurons, whereas INP-specific grh RNAi severely reduced the number of early born Bsh+ neurons without impairing INP proliferation or late INP progeny. This is consistent with the Bsh+ neurons deriving from the D+ Grh+ expression window. Interestingly, misexpression of D or Grh did not increase Bsh+ neuron numbers; perhaps D/Grh co-misexpression is required to generate Bsh+ neurons. It is concluded that both D and Grh are required, but not sufficient, for the production of Bsh+ early INP progeny (Bayraktar, 2013).
The function of early or late born INP progeny in adult brain development is unknown. This study determined the role of late born INP neurons and glia in the development and function of the adult central complex (CCX), an evolutionarily conserved insect brain structure containing many type II neuroblast progeny. The CCX consists of four interconnected compartments at the protocerebrum midline: the ellipsoid body, the fan-shaped body, the bilaterally paired noduli, and the protocerebral bridge; each of these compartments is formed by a highly diverse set of neurons. First, permanent lineage tracing was used to map the contribution of late born Ey+ INP progeny to the adult CCX. Cell bodies were detected in the dorsoposterior region of the CCX, and their axonal projections extensively innervated the entire ellipsoid body, fan-shaped body, and protocerebral bridge, with much weaker labelling of the paired noduli. It is concluded that old INPs contribute neurons primarily to the ellipsoid body, fan-shaped bod and protocerebral bridge regions of the CCX. Second, INP-specific ey RNAi was used to delete the late born Toy+ neurons and Repo+ glia. Loss of late born INP progeny generated major neuroanatomical defects throughout the adult CCX: the ellipsoid body and paired noduli were no longer discernible, the fan-shaped body was enlarged, and the protocerebral bridge was fragmented. Subsets of this phenotype were observed after removal of Toy+ neurons or Repo+ glia, showing that they contribute to distinct aspects of the CCX. Previous studies have described similar or weaker morphological CCX defects in ey hypomorphs, toy mutants, and after broad glia ablation during larval stages. In addition, ey RNAi adults were found to have relatively normal locomotion, but have a significant deficit in negative geotaxis. It is concluded that Ey is a temporal identity factor that specifies late born neuron and glial identity, and that these late born neural cell types are essential for assembly of the adult central complex (Bayraktar, 2013).
Bsh+ neurons and Repo+ glia were found to be sparse within the total population of young and old INP progeny, respectively, indicating that other mechanisms must help to restrict the formation of these neural subtypes. One mechanism could be temporal patterning within type II neuroblast lineages (Bayraktar, 2013).
To determine whether type II neuroblasts change their transcriptional profiles over time, known temporal transcription factors were examined for expression in type II neuroblasts at five time points in their lineage (24, 48, 72, 96 and 120 h ALH). No type II neuroblast expression for Hunchback, Kruppel, Pdm1/2 and Broad, and Grh was expressed in all type II neuroblasts at all time points. However, three transcription factors were identified with temporal expression in type II neuroblasts. D and Castor (Cas) were specifically detected in early type II neuroblasts: 3-4 neuroblasts at 24 h ALH, 0-1 neuroblast at 48 h ALH, and none later. Although D was never detected simultaneously in all type II neuroblasts at 24 h, permanent lineage tracing with R12E09D labels all type II neuroblasts, indicating that all transiently express D. The third transcription factor, Seven up (Svp), showed a pulse of expression in a subset of type II neuroblasts at 48 h ALH, but was typically absent from younger or older type II neuroblasts. D, Cas and Svp are all detected in the anterior-most type II neuroblasts (probably corresponding to DM1-DM3), and thus at least these type II neuroblasts must sequentially express D or Cas, and Svp. It is concluded that type II neuroblasts can change gene expression over time (Bayraktar, 2013).
Next, tests were performed to determine whether type II neuroblasts produce different INPs over time. Permanently labelled clones were generated within the type II neuroblast lineages at progressively later time points. If type II neuroblasts change over time to make different INPs, early and late neuroblast clones should contain different neural subtypes. Clones were assayed for Repo+ glia and Bsh+ neurons, choosing these markers because Repo+ neuropil glia have been proposed to be born early in type II neuroblast lineages and Bsh+ neurons were positioned far from the Repo+ glia consistent with a different birth-order. Bsh+ neuron numbers began to decline only in clones induced at the latest time point, showing that they are generated late in the type II neuroblast lineage. By contrast, Repo+ glia were detected in clones induced early but not lat. This allows assigning of Repo+ glia to an 'early neuroblast, old INP' portion of the lineage, and Bsh+ neurons to a 'late neuroblast, young INP' portion of the lineage. It is concluded that type II neuroblasts undergo temporal patterning, and neuroblast temporal patterning was proposed to act together with INP temporal patterning to increase neural diversity in the adult brain (Bayraktar, 2013).
This study has shown that INPs sequentially express three transcription factors (D, Grh and then Ey), and that different neural subtypes are generated from successive transcription factor windows. It is likely that multiple GMCs are born from each of the four known INP gene expression windows; GMCs born from a particular gene expression window may have the same identity, or may be further distinguished by 'subtemporal genes' as in embryonic type I neuroblast lineages. This study also showed that each temporal factor is required for the production of a distinct temporal neural subtype. Loss of D or Grh leads to the loss of Bsh+ neurons; loss of Ey leads to loss of Toy+ neurons and Repo+ glia, although the fate of the missing cells is unknown. An unexpected finding was that Ey limits the lifespan of INPs. Mechanisms that prevent INP de-differentiation have been characterized -- loss of the translational repressor Brain tumour (Brat) or the transcription factor Earmuff (Erm) causes INPs to de-differentiate into tumorigenic type II neuroblasts, but factors that terminate normal INP proliferation have never before been identified (Bayraktar, 2013).
The D-to-Grh-to-Ey INP temporal identity factors are all used in other contexts during Drosophila development. Many embryonic neuroblasts sequentially express D and Grh. Ey is expressed in mushroom body neuroblasts, and is required for development of the adult brain mushroom body. Interestingly, mammalian orthologues of D and Ey (SOX2 and PAX6, respectively) are expressed in neural progenitors, including OSVZ progenitor, but have not been tested for a role in temporal patterning (Bayraktar, 2013).
This study has shown that there are two axes of temporal patterning within type II neuroblast lineages: both neuroblasts and INPs change over time to make different neurons and glia, thereby expanding neural diversity. It will be important to investigate whether INPs generated by OSVZ neural stem cells undergo similar temporal patterning (perhaps using SOX2 and PAX6), and whether combinatorial temporal patterning contributes to the neuronal complexity of the human neocortex (Bayraktar, 2013).
The importance of producing the correct numbers of neurons during development is illustrated by both evolutionary enhancement of cognitive capacities in larger brains, and developmental disorders of brain size. In humans, increased neuronal numbers during development is speculated to partly derive from a unique subtype of neural stem cells (NSCs) that undergo a phase of expansion through symmetric self-amplifying divisions before generating neurons. Symmetric amplification also appears to underlie adult neural stem maintenance in the mouse. However, the mechanisms regulating this behavior are unclear. This study reports the discovery of self-amplifying NSCs in Drosophila and shows that they arise by a spatiotemporal conversion of classical self-renewing NSCs. This conversion is regulated by a temporal transition in the expression of proneural transcription factors prior to cell division. A causal link as found between stem cell self-amplification and increased neuronal numbers. It was further shown that the temporal transcriptional switch controls both stem cell division and subsequent neuronal differentiation (Mora, 2018).
Larval type II neuroblasts (NBs) of the developing brain, like mammalian neural stem cells, deposit neurons through intermediate neural progenitors (INPs) that can each produce a series of neurons. Both type II NBs and INPs exhibit age-dependent expression of various transcription factors, potentially specifying an array of diverse neurons by combinatorial temporal patterning. Not knowing which mature neurons are made by specific INPs, however, conceals the actual variety of neuron types and limits further molecular studies. This study mapped neurons derived from specific type II NB lineages and found that sibling INPs produced a morphologically similar but temporally regulated series of distinct neuron types. This suggests a common fate diversification program operating within each INP that is modulated by NB age to generate slightly different sets of diverse neurons based on the INP birth order. Analogous mechanisms might underlie the expansion of neuron diversity via INPs in mammalian brain (Wang, 2013).
Twin-spot MARCM allows differential labeling and thus
independent tracking of sister clones derived from a common
precursor for enhanced cell lineage analysis. It has
been applied to resolve a protracted heterogeneous type I NB
lineage through the identification of relevant clones with lineage-characteristic
morphologies from thousands of mosaic brains (Lin, 2012). In such non-selective clonal analysis, the labeled cerebral
clones could arise from any of the ~100 NB lineages per
hemisphere. Given the anticipated extraordinary complexities in
both proliferation patterns and clone morphologies of type II NB
lineages, it would be extremely challenging to isolate clean type II
lineage-derived clones and decipher their lineage relationships from
sample brains with complex, arbitrary mixes of NB clones (Wang, 2013).
A strategy has been established for targeting specific NB
lineages for clonal labeling (Awasaki, Kao, Lee, Yang and Lee., unpublished, cited in Awasaki, 2014). The strategy is based on the
GAL4-driven excision of a stop cassette within a pan-neuronal
LexA driver in a subset of NBs, leading to a lineage-specific LexA
driver in all subsequently derived progenies. Notably, a
previously uncharacterized 975 bp genomic region (stg-14) located
8.5 kb upstream of the predicted transcription start site of string
drives transient expression in embryonic type II NBs. stg-14-GAL4 filtered through a NB-specific dpn promoter
(denoted stg14^dpn) can trigger serial recombination events
specifically in type II NBs, leading to permanent activation of
LexA drivers selectively in type II NB lineages. The
strategy thus enables targeted clonal analysis in all postembryonic
type II NB lineages without internal gaps, a feature that is hard to
verify for standard GAL4 lines (Wang, 2013).
Twin-spot MARCM with the stg14^dpn lineage-restricted driver
allowed recovery of clones selectively in the eight type II NB
lineages. The DM1 to DM6 NB clones have cell body
clusters near the midline with distributions spreading dorsoventrally
on the posterior brain surface, whereas the DL1 and DL2 NB clones
reside near the posterior dorsolateral corner (Yu, 2013). They
exhibit complex morphologies but can be readily distinguished
based on cell body distributions, neurite trajectories and neuronal
elaboration patterns (Yang, 2013). The complex
yet stereotyped clone morphologies argue that each type II NB
produces a characteristic set of diverse neuronal offspring in
addition to glia (Wang, 2013).
To better determine the offspring diversity and describe how diverse
neurons arise from serial INPs, the INP clones were examined
paired with the type II NB clones induced in newly hatched larvae
and differentially labeled with twin-spot MARCM. This allowed identification of the neuronal offspring of the first larval-born INP in each
type II NB lineage. The full-size INP clones of DM1-6 and DL1
carry six to nine neuronal cell bodies, consistent
with one INP budding off a short series of GMCs.
Interestingly, the DL2 NB clone is significantly smaller than other
type II NB clones and its paired sister clone consistently carries only
one viable neuron at the adult stage, reminiscent of some
conventional type I lineages that exist as a lone hemilineage. Nonetheless, DL2
clones were recovered at comparable frequencies to the other type II NB clones with
another type II NB-specific driver, giving confidence in the
identification of DL2 as the eighth type II NB lineage (Wang, 2013).
The first larval-born INP (denoted INP1) clones exhibit lineage-characteristic
stereotyped neurite trajectories. These
multicellular INP clones are complex and appear to carry diverse
neuron types with distinct neurite trajectories. For instance, the
DM1-4 INP1 clones acquire central complex (CX) as well as diverse
non-CX elaborations. Furthermore, the INP1 clones of
the super-exuberant DM1 and DM6 lineages exhibit most features
of the parental NB clones despite drastic differences in clone size
(fewer than ten neurons in INP clones versus more than 200 neurons
in the paired NB clones). These phenomena argue that each
INP contributes cells that include a significant fraction of the diverse
cell classes observed in a given type II NB lineage (Wang, 2013).
However, the INP1 clones of most type II NB lineages exhibit
domains of neurite elaboration that are unique to some offspring of
the first larval-born INPs, as the following INP sublineages
collectively labeled in the paired NB clones fail to innervate such
INP1-specific domains. This is particularly obvious in the reduced
DL2 lineage, where the lone mature neuron (probably one of the
dopaminergic PPL1 neurons} made by INP1
is the only larval-born DL2 neuron that innervates the tips of
mushroom body (MB) α lobes bilaterally. INP1-unique elaborations could readily be detected in the complex DM1 and DM6 lineages as well. The INP1 clone of DM1 shows dense
contralateral superior posterior slope (SPS) innervation,
whereas the DM6 INP1 clone elaborates broadly alone in the
ellipsoid body (EB) and contralateral optic lobe (OL).
These observations indicate that the INP1 clones are not only
stereotyped but also unique, arguing that the ~40 serially derived
INPs in a given type II lineage could be individually distinct (Wang, 2013).
To unravel the sibling INP diversity and reveal each neuron made
by one INP, twin-spot MARCM clones were further generated in
serial 4-hour windows from 8 hours to 48 hours after larval hatching
(ALH). The study selectively focused on the DM1 lineage for detailed
sublineage analysis. The DM1 NB clones induced in first instar
larvae prior to 16 hours ALH uniformly pair with the above
identified INP1 clone. Following clone induction
between 16 and 24 hours ALH, it was observed that all the DM1 NB
clones that are unaccompanied by the unique INP1 clone instead
pair with a different stereotyped INP clone, denoted as the INP2
clone. The INP1 and INP2 clones share morphological
features characteristic of the DM1 lineage. For example,
they both innervate the CX, rubus (RUB), posterior lateral
protocerebrum (PLP), posterior ventrolateral protocerebrum
(PVLP), vest (VES) and the contralateral OL. However, compared
with the INP1 clone, the INP2 clone carries one extra neuron and
elaborates more ventrally in both the contralateral OL and central
brain. Later derived INP clones remain analogous but
distinguishable (Wang, 2013).
The similarities and differences were determined in the
offspring composition and sequence between the INP1 and INP2
sublineages. Twin-spot MARCM clones arising during
INP self-renewing divisions were collected. The paired subclones
induced at the beginning of INP1 or INP2 proliferation could be identified based on the
known sublineage-characteristic morphological features. It was observe
that a similar two-cell GMC clone may pair with a six-cell INP
subclone that retains the INP1-unique SPS innervation or a
seven-cell INP subclone that shows INP2-characteristic broad OL and
subesophageal ganglion (SEG) elaborations. The small
GMC clones and their paired large INP subclones acquire distinct
subpatterns of full INP clone morphologies, which clearly
demonstrates both the diversity and invariant sequence in INP
progeny. Close examination of the two-cell GMC clones and the
paired single-cell clones derived from the first GMCs further reveals
that the first GMC of both INP1 and INP2 consistently makes one
strongly labeled neuron with lateral elaborations and one weakly
marked neuron with medial projections (Wang, 2013).
The second GMC offspring for both the
INP1 and INP2 sublineages were subsequently mapped. The GMC2 in both INP
sublineages makes one OL neuron paired with a CX neuron. But the OL neurons of INP1 and INP2 elaborate differently and underlie most of the sublineage distinctions. The
INP1 OL neuron innervates the contralateral SPS and a dorsal
domain within the contralateral OL, whereas the INP2 OL
neuron arborizes broadly in the SEG and contralateral OL.
The third GMC offspring of INP1 and INP2 remain distinguishable
and account for the difference in sublineage mature neuron numbers. GMC3 of INP1 makes only one viable CX neuron, whereas GMC3 of INP2 produces an analogous CX
neuron as well as one extra OL neuron that targets VES and
contralateral OL. The subsequently derived GMCs in
both INP sublineages yield an undistinguishable set of three
neurons, including one VES-targeting neuron plus two CX neurons. Therefore, mapping the sequence
of individual neurons made by the DM1 INP1 and INP2 clearly
shows that each INP sublineage consists of a sequence of very
diverse neuron types that is repeated with slight modifications in the
sequential INPs. Not seeing the later-derived glia should not
affect the neuron birthdating (Wang, 2013).
Partial mapping of the DM6 INP1 and INP2 sublineages reveals
similar phenomena. First, the INP1 and INP2 clones share
morphological features characteristic of the DM6 lineage. They elaborate analogously in
superior medial protocerebrum (SMP), lateral accessory lobe (LAL),
SPS, inferior posterior slope (IPS) and VES. But the INP1 clone
solely targets the bulb/optic tubercle (BU/OTU) and contralateral OL,
whereas the INP2 clone selectively innervates the fan-shaped
body/superior clamp/superior intermediate protocerebrum
(FB/SCL/SIP). In addition, they acquire distinct patterns of neurite
elaborations in EB, gall and PLP. Second, the full-size nine-cell INP
clones can be analogously partitioned into a two-cell GMC clone
paired with a seven-cell INP subclone. The GMC1 clones of DM6 INPs consistently carry one
neuron extending dorsally into SMP or SMP/SIP and the other neuron
projecting ventrally into VES. The following GMCs of the same INP
sublineages, by contrast, make neurons innervating other neuropils. These observations indicate once again that Drosophila type II NBs
yield a series of analogous intermediate precursors that generate
related sequences of diverse neurons (Wang, 2013).
From this study it was learned that Drosophila type II NBs generate a series of INPs
that in turn each produce an invariant sequence of neuronal classes.
The same neuronal classes are produced in the same sequence
repeatedly by the serial INPs, suggesting the reiterated use of related
developmental fating programs in each INP sublineage. However,
serial INPs only yield slightly different neurons based on the INP birth
order, possibly governed by NB temporal factors and/or time-dependent environmental changes. It remains to be
tested whether the Drosophila type II lineages are pre-fated along two
dimensions - the sequence of INPs and the sequence of the progeny
of each INP - to generate stereotyped arrays of diverse neurons (Wang, 2013).
Fixed lineages derived from unique, genetically specified neuroblasts form the anatomical building blocks of the Drosophila brain. Neurons belonging to the same lineage project their axons in a common tract, which is labeled by neuronal markers. This paper presents a detailed atlas of the lineage-associated tracts forming the brain of the early Drosophila larva, based on the use of global markers (anti-Neuroglian, anti-Neurotactin, Inscuteable-Gal4>UAS-chRFP-Tub) and lineage-specific reporters. Sixty-eight discrete fiber bundles are described that contain axons of one lineage or pairs/small sets of adjacent lineages. Bundles enter the neuropil at invariant locations, the lineage tract entry portals. Within the neuropil, these fiber bundles form larger fascicles that can be classified, by their main orientation, into longitudinal, transverse, and vertical (ascending/descending) fascicles. 3D digital models of lineage tract entry portals and neuropil fascicles are presented, set into relationship to commonly used, easily recognizable reference structures such as the mushroom body, the antennal lobe, the optic lobe, and the Fasciclin II-positive fiber bundles that connect the brain and ventral nerve cord. Correspondences and differences between early larval tract anatomy and the previously described late larval and adult lineage patterns are highlighted. The L1 atlas will be important for a host of ongoing work that attempts to reconstruct neuronal connectivity at the level of resolution of single neurons and their synapses (Hartenstein, 2015).
The Drosophila brain consists of a relatively small number of invariant, genetically determined lineages which provide a model to study the relationship between gene function and neuronal architecture. This article focuses on the secondary phase of lineage morphogenesis, from the reactivation of neuroblast proliferation in the first larval instar to the time when proliferation ends and secondary axon tracts have fully extended in the late third larval instar. The location and projection of secondary lineages were reconstructed at close (4 h) intervals and a detailed map was produced in the form of confocal z-projections and digital three-dimensional models of all lineages at successive larval stages. Based on these reconstructions, it was possible to compare the spatio-temporal pattern of axon formation and morphogenetic movements of different lineages in normal brain development. In addition to wild type, lineage morphology was reconstructed in two mutant conditions. (1) Expressing the construct UAS-p35 which rescues programmed cell death it could be systematically determine which lineages normally lose hemilineages to apoptosis. (2) so-Gal4-driven expression of dominant-negative EGFR ablated the optic lobe, which led to a conclusion that the global centrifugal movement normally affecting the cell bodies of lateral lineages in the late larva is causally related to the expansion of the optic lobe, and that the central pattern of axonal projections of these lineages is independent of the presence or absence of the optic lobe (Lovick, 2015).
Steroid hormones control various aspects of brain development and behavior in metazoans, but how they enter the central nervous system (CNS) through the blood-brain barrier (BBB) remains poorly understood. It is generally believed that steroid hormones freely diffuse through the plasma membrane of the BBB cells to reach the brain, because of the predominant "simple diffusion" model of steroid hormone transport across cell membranes. Recently, however, the simple diffusion model was challenged by showing that a Drosophila organic anion-transporting polypeptide (OATP), which was named Ecdysone Importer (EcI), is required for cellular uptake of the primary insect steroid hormone ecdysone. As ecdysone is first secreted into the hemolymph before reaching the CNS, this finding raised the question of how ecdysone enters the CNS through the BBB to exert its diverse role in Drosophila brain development. This study demonstrates in the Drosophila BBB that EcI is indispensable for ecdysone entry into the CNS to facilitate brain development. EcI is highly expressed in surface glial cells that form the BBB, and EcI knockdown in the BBB suppresses ecdysone signaling within the CNS and blocks ecdysone-mediated neuronal events during development. In an ex vivo culture system, the CNS requires EcI in the BBB to incorporate ecdysone from the culture medium. The results suggest a transporter-mediated mechanism of steroid hormone entry into the CNS, which may provide important implications in controlling brain development and behavior by regulating steroid hormone permeability across the BBB (Okamoto, 2020).
Drosophila neuroblasts are a model system for studying stem cell self-renewal and the establishment of cortical polarity. Larval neuroblasts generate a large apical self-renewing neuroblast, and a small basal cell that differentiates. A genetic screen was performed to identify regulators of neuroblast self-renewal, and a mutation was identified in sgt1 (suppressor-of-G2-allele-of-skp1) that had fewer neuroblasts. sgt1 neuroblasts have two polarity phenotypes: failure to establish apical cortical polarity at prophase, and lack of cortical Scribble localization throughout the cell cycle. Apical cortical polarity was partially restored at metaphase by a microtubule-induced cortical polarity pathway. Double mutants lacking Sgt1 and Pins (a microtubule-induced polarity pathway component) resulted in neuroblasts without detectable cortical polarity and formation of 'neuroblast tumors.' Mutants in hsp83 (encoding the predicted Sgt1-binding protein Hsp90), LKB1 (PAR-4), or AMPKα all show similar prophase apical cortical polarity defects (but no Scribble phenotype), and activated AMPKα rescued the sgt1 mutant phenotype. It is proposed that an Sgt1/Hsp90-LKB1-AMPK pathway acts redundantly with a microtubule-induced polarity pathway to generate neuroblast cortical polarity, and the absence of neuroblast cortical polarity can produce neuroblast tumors (Anderson, 2012).
This study presents evidence that the evolutionary-conserved protein Sgt1 acts with Hsp90, LKB1 and AMPK to promote apical localization of the Par and Pins complexes in prophase neuroblasts. It is proposed that Sgt1/Hsp90 proteins function together based on multiple lines of evidence: (1) they show conserved binding from plants to humans; (2) the sgt1s2383 mutant results in a five amino acid deletion within the CS domain, which is the Hsp90 binding domain; (3) sgt1 and hsp83 have similar cell cycle phenotypes; and (4) sgt1 and hsp83 have similar neuroblast polarity phenotypes. The Sgt1/Hsp90 complex either stabilizes or activates client proteins (Zuehlke, 2010); it is suggested that Sgt1 activates LKB1, rather than stabilizing it, because it was not possible to rescue the sgt1 mutant phenotype by simply overexpressing wild type LKB1 protein. No tests were performed for direct interactions between Sgt1 and LKB1 proteins, and thus the mechanism by which Sgt1 activates LKB1 remains unknown (Anderson, 2012).
LKB1 is a 'master kinase' that activates at least 13 kinases in the AMPK family. It is suggested that LKB1 activates AMPK to promote neuroblast polarity because overexpression of phosphomimetic, activated AMPKα can rescue the lkb1 and sgt1 mutant phenotype. It remains unclear how AMPK activity promotes apical protein localization. An antibody to activated AMPKα (anti-phosphoT385-AMPKα shows spindle and cytoplasmic staining that is absent in ampkα mutants, and centrosomal staining that persists in AMPKα null mutants, but no sign of asymmetric localization in neuroblasts. AMPK activity is thought to directly or indirectly activate myosin regulatory light chain to promote epithelial polarity. AMPK is activated by a rise in AMP/ATP levels that occur under energy stress or high metabolism; AMP binds to the γ regulatory subunit of the heterotrimeric complex and results in allosteric activation of the α subunit. ampkα mutants grown under energy stress have defects in apical/basal epithelial cell polarity in follicle cells within the ovary. In contrast, AMPKα mutants grown on nutrient rich food still show defects in embryonic epithelial polarity, neuroblast apical polarity, and visceral muscle contractio. Larval neuroblasts, embryonic ectoderm, and visceral muscle may have a high metabolic rate, require low basal AMPK activity, or use a different mechanism to activate AMPK than epithelial cells. What are the targets of AMPK signaling for establishing apical cortical polarity in larval neuroblasts? AMPK could directly phosphorylate Baz to destabilize the entire pool of apical proteins, but currently there is no evidence supporting such a direct model. AMPK may act via regulating cortical myosin activity: clear defects have been seen in cortical motility, ectopic patchy activated myosin at the cortex, and failure of cytokinesis in sgt1, lkb1, and ampkα mutants. This strongly suggests defects in the regulation of myosin activity, but how or if gain/loss/mispositioning of myosin activity leads to failure to establish apical cortical polarity remains unknown. Lastly, the defects in apical cell polarity seen at prophase could be due to the prometaphase cell cycle delays (Anderson, 2012).
What activates the Sgt1-LKB1-AMPK pathway to promote cell polarity during prophase? In budding yeast, Sgt1 requires phosphorylation on Serine 361 (which is conserved in Drosophila Sgt1) for dimerization and function (Bansal, 2009); this residue is conserved in Drosophila Sgt1 but its functional significance is unknown (Anderson, 2012).
Sgt1/Hsp90/LKB1/AMPK are all required for apical Par/Pins complex localization, but Sgt1 must act via a different pathway to promote Dlg/Scrib cortical localization, because only the sgt1 mutant affects Dlg/Scrib localization, and overexpression of activated AMPKα is unable to restore cortical Scrib in sgt1 mutants. The mechanism by which Sgt1 promotes Dlg/Scrib cortical localization is unknown (Anderson, 2012).
This study has shown that sgt1 mutants lack Par/Pins apical polarity in prophase neuroblasts, but these proteins are fairly well polarized in metaphase neuroblasts. The rescue of cortical polarity is microtubule dependent, probably occurring via the previously described microtubule-dependent cortical polarity pathway containing Pins, Dlg and Khc-73. The weak polarity defects still observed in sgt1 metaphase neuroblasts may be due to the poor spindle morphology. The lack of microtubule-induced polarity at prophase, despite a robust microtubule array in prophase neuroblasts, suggests that the microtubule-induced cortical polarity pathway is activated at metaphase. Activation of the pathway could be via expression of the microtubule-binding protein Khc-73; via phosphorylation of Pins, Dlg or Khc-73 by a mitotic kinase like Aurora A; or via a yet unknown pathway (Anderson, 2012).
It was somewhat surprising that the sgt1 pins double mutants had increased numbers of brain neuroblasts, because each single mutant had reduced neuroblast numbers. The double mutant phenotype may be due to loss of both Pins and cortical Dlg/Scrib, as the sgt1 pins double mutant phenotype is similar to the dlg pins double mutant phenotype. It could also be due to a change in an unknown downstream effector of both Sgt1 and Pins. A not mutually exclusive possibility is that the sgt1 pins double mutant phenotype is due to loss of all Par/Pins cortical polarity. This model is consistent with the observation that sgt1 or pins single mutants retain some neuroblast cortical polarity, whereas the sgt1 pins double mutants lack all known neuroblast cortical polarity. It is proposed that the apolar double mutant neuroblasts partition cell fate determinants equally to both siblings, and that both siblings frequently assume a neuroblast identity. This is supported by the recent finding that when the neuroblast spindle is aligned orthogonal to a normal apical/basal polarity axis, such that both siblings inherit equal amounts of apical cortical proteins, the siblings always acquire a neuroblast identity. Thus, equal partitioning of apical/basal cell fate determinants (in spindle orientation mutants) or failure to establish any cortical polarity (sgt1 pins mutants) may result in neuroblast/neuroblast siblings and an expansion of the neuroblast population (Anderson, 2012).
Mitochondria are critical for neuronal function due to the high demand of ATP in these cell types. During Drosophila development, neuroblasts in the larval brain divide asymmetrically to populate the adult central nervous system. While many of the proteins responsible for maintaining neuroblast cell fate and asymmetric cell divisions are known, little is know about the role of metabolism and mitochondria in neuroblast division and maintenance. The gene clueless (clu) has been previously shown to be important for mitochondrial function. clu mutant adults have severely shortened lifespans and are highly uncoordinated. Part of their lack of coordination is due to defects in muscle, however, this study has identified high levels of Clu expression in larval neuroblasts and other regions of the dividing larval brain. While mitochondria in clu mutant neuroblasts are mislocalized during the cell cycle, surprisingly, this study shows that overall brain morphology appears to be normal. This is explained by the observation that clu mutant larvae have normal levels of ATP and do not suffer oxidative damage, in sharp contrast to clu mutant adults. Mutations in two other genes encoding mitochondrial proteins, technical knockout and stress sensitive B, do not cause neuroblast mitochondrial mislocalization, even though technical knockout mutant larvae suffer oxidative damage. These results suggest Clu functions upstream of electron transport and oxidative phosphorylation, has a role in suppressing oxidative damage in the cell, and that lack of Clu specific function causes mitochondria to mislocalize. These results also support the previous observation that larval development relies on aerobic glycolysis, rather than oxidative phosphorylation. Thus the Clu role in mitochondrial function is not critical during larval development, but is important for pupae and adults (Sen, 2013).
Cell diversity of the brain and how it is affected by starvation, remains largely unknown. This study introduces a single cell transcriptome atlas of the entire Drosophila first instar larval brain. Cell-type identity was assigned based on known marker genes, distinguishing five major groups: neural progenitors, differentiated neurons, glia, undifferentiated neurons and non-neural cells. All major classes were further subdivided into multiple subtypes, revealing biological features of various cell-types. Transcriptional changes in response to starvation were further assessed at the single-cell level. While after starvation the composition of the brain remains unaffected, transcriptional profile of several cell clusters changed. Intriguingly, different cell-types show very distinct responses to starvation, suggesting the presence of cell-specific programs for nutrition availability. Establishing a single-cell transcriptome atlas of the larval brain provides a powerful tool to explore cell diversity and assess genetic profiles from developmental, functional and behavioral perspectives (Brunet Avalos, 2019).
Unsupervised cluster analysis led to the identification of identify major cell-type categories as well as more resolved cell-types. Canonical markers (e.g. Notch, elav) grouped all cells into major categories, which based on subtype makers (e.g., ChAT, dpn, alrm) further divide them into different cell-types. This analysis further allowed the depiction of putatively novel and intriguing biological features. An example of this is the multiple-neurotransmitter-releasing neurons, which express more than one neurotransmitter, as previously found in the adult brain. Even though these cells represent a small fraction of the total of cells populating the larval brain, it is interesting to understand the need of a neuron to produce different neurotransmitter types. Even more, deciphering the biological processes, in which these neurons are involved and the regulation behind the co-releasing phenomenon, opens a new field that requires further investigation. Similarly, co-expression of neurotransmitters and neuropeptides appears to be a common feature among peptidergic cells, suggesting different modalities of intercellular communication (Brunet Avalos, 2019).
Interestingly cells of the mushroom body are closer to the neurogenic population in the UMAP plot than to the remaining differentiated neurons. Looking in detail to this group of cells, one can observe cells at different stages of differentiation: neuroprogenitors, cells undergoing differentiation and mature neurons. Knowing that the mushroom body neuroblasts continuously divide from the embryonic to the late pupal stage, and that they produce a specific neuron type, could partially explain the localization of this cluster of cells closer to undifferentiated cells. In addition to the MBNBs, which are constantly proliferating, a subset of NPCs showed to be actively replicating its genomic content at 16 hr ALH, indicating that neuroblasts in the brain may exit their quiescent state earlier than what has been previously reported (Brunet Avalos, 2019).
An intriguing, yet not fully understood, cell-type are the undifferentiated neurons (UNs), which in the UMAP plot locate in close proximity with NPCs. While these cells show expression of neuronal differentiation markers such as elav or Syt-1, they lack expression of genes required for neurotransmitter synthesis. Similarly, a recent study revealed a new quite abundant, neuronal cell-type. These cells identified as small undifferentiated neurons (SUs), are characterized by reduced dimensions, a dense heterochromatin and the absence of synapses (Andrade, 2019). Therefore, it is speculated that the UNs may correspond to the previously described SUs. It is intriguing to speculate that hdc and unk, two genes previously described to restrict cell cycle progression in response to nutrition restriction (Li, 2019), present in neurogenic cells but enriched in UNs, have a role in maintaining these cells undifferentiated (Brunet Avalos, 2019).
Moreover, this study explored the changes in the larval brain caused by starvation at single-cell resolution. Food intake has a critical effect on survival and development. Nevertheless, how the brain composition and cellular programs are affected upon food deprivation remains largely unknown. The cellular content showed to be almost invariable, only few clusters disappeared after starvation. The remaining cell-types clustered together, independently of the experimental condition, indicating that cell-type identity is maintained once it has been established. Moreover, and as it was observed with aging, the effects of starvation are cell specific; transcriptional programs in neurons remained unaffected, while glial cells, NPCs and undifferentiated neurons experienced changes at the transcriptional level or even disappear due to stressful conditions. Additionally, the most susceptible genes are those involved in fat metabolism as well as some lncRNAs, which may have a regulatory function. Neuropeptides are also sensitive to nutritional levels. As expected, when cells face nutrient deficiency, protein synthesis is affected. In this case, cells favor the production of those neuropeptides involved in food intake and water homeostasis, downregulating the remaining ones in an attempt to survive. Nevertheless, is important to also consider and not exclude the possibility that these cell types could have been loss during sample preparation and subsequent manipulation. To confidently address this point, further repetitions are required (Brunet Avalos, 2019).
Starvation is directly linked to TORC1, which integrates information about nutrient conditions of individual cells to trigger physiological responses. No substantial changes were observed at the transcriptional level for the genes in the TORC1 upon food deprivation, suggesting that cells regulate TORC1 activity at the protein level rather than at the transcriptional level. It is interesting to analyze those genes downstream of TORC1. This study focused on the TORC1 signaling pathway, rather than in the insulin pathway, in an attempt to better understand nutrient sensing responses in a cell-autonomous fashion and not at a systemic level. Recently, REPTOR and REPTOR-BP were shown to be transcription factors responsible for mediating some of the responses of reduced TORC1 activity. As expected, REPTOR and its binding protein are both upregulated upon starvation, as well as their target genes. Under limited nutrient conditions, larvae have to choose between anabolic or catabolic processes. This study observed that under extreme starvation condition, larvae have no other option than to catabolize their storages to obtain some energy and survive as long as possible. Even though intracellular nutrient-sensing activates TORC1, extracellular sensing must also be required to mount a complete physiological response. In this way, integration between different signaling pathways, Insulin-Foxo and TORC1-REPTOR, must be required (Brunet Avalos, 2019).
Furthermore, the availability of the wildtype brain cell atlas as well as the starvation induced changes provide a resource for further functional analysis of neural circuits of the larval brain as well as developmental studies at the fundamental unit of biological organization: the cell (Brunet Avalos, 2019).
Neuron specification and maturation are essential for proper central nervous system development. However, the precise mechanisms that govern neuronal maturation, essential to shape and maintain neuronal circuitry, remain poorly understood. This study analysed early-born secondary neurons in the Drosophila larval brain, revealing that the early maturation of secondary neurons goes through 3 consecutive phases: (1) Immediately after birth, neurons express pan-neuronal markers but do not transcribe terminal differentiation genes; (2) Transcription of terminal differentiation genes, such as neurotransmitter-related genes VGlut, ChAT, or Gad1, starts shortly after neuron birth, but these transcripts are, however, not translated; (3) Translation of neurotransmitter-related genes only begins several hours later in mid-pupa stages in a coordinated manner with animal developmental stage, albeit in an ecdysone-independent manner. These results support a model where temporal regulation of transcription and translation of neurotransmitter-related genes is an important mechanism to coordinate neuron maturation with brain development (Marques, 2023).
Stem cells continuously generate differentiating daughter cells and are essential for tissue homeostasis and development. Their capacity to self-renew as undifferentiated and actively dividing cells is controlled by either external signals from a cellular environment, the stem cell niche, or asymmetric distribution of cell fate determinants during cell division. This study reports that the protein kinase Ballchen (Ball) is required to prevent differentiation as well as to maintain normal proliferation of neuronal stem cells of Drosophila melanogaster, called neuroblasts. These results show that the brains of ball mutant larvae are severely reduced in size, which is caused by a reduced proliferation rate of the neuroblasts. Moreover, ball mutant neuroblasts gradually lose the expression of the neuroblast determinants Miranda and aPKC, suggesting their premature differentiation. These results indicate that Ball represents a novel cell intrinsic factor with a dual function regulating the proliferative capacity and the differentiation status of neuronal stem cells during development (Yakulov, 2014 PubMed).
These results establish that BALL is essential to maintain the proliferation rate as well as the undifferentiated state of postembryonic neuroblasts (pNBs) and therefore interlink these two aspects of stem cell self-renewal. The proliferation rate of ball2 mutant pNBs was reduced already at 72 h ALH, a time point when approximately half of the pNBs continued to express the stem cell determinants MIRA and aPKC. Therefore, it is plausible that the primary function of BALL is to control the proliferation rate of pNBs as a prerequisite for continuous self-renewal of neuroblastst (Yakulov, 2014).
The effects of a reduced proliferation rate were previously studied in epithelial tissue such as wing imaginal discs, which led to the discovery of a phenomenon termed cellular competition. It describes that cells with reduced cellular fitness proliferate at a lower rate and are eventually eliminated by apoptosis. This phenomenon was observed after generating ball2 mutant cells by MARCM in wing imaginal discs, showing that the mutant cells are capable to proliferate and to form cell clones. However, these cell clones fail to compete with wild type cells and subsequently undergo apoptosis. Maintenance of the stem cell character of pNBs is unlikely to be regulated through a competitive mechanism, since the pNB lineages contain only a single stem cell. The data suggest that the same process that determines competitiveness of wing disc epithelial cells is a prerequisite to maintain the self-renewal of pNBst (Yakulov, 2014).
Recent work has shown that BALL is required to sustain self-renewal of niche-controlled stem cells (Herzig, 2014). This work shows that this function of BALL is not restricted to niche-controlled stem cells but is also required in pNBs, which depend on asymmetric distribution of cell fate determinants for self-renewal. Thus, the function of BALL for stem cell self-renewal is not limited by the factors and mechanisms that mediate cell fate decisions in the different stem cell systems. This study therefore suggests that Drosophila stem cells employ cell intrinsic mechanisms to ensure stem cell self-renewal that are independent of the tissue specific modes of stem cell fate decisions and shared by diverse stem cell populations. The molecular basis of these mechanisms and how BALL is integrated in these processes remains to be established by future studiest (Yakulov, 2014).
The final size and function of the adult central nervous system (CNS) are determined by neuronal lineages generated by neural stem cells (NSCs) in the developing brain. In Drosophila, NSCs called neuroblasts (NBs) reside within a specialised microenvironment called the glial niche. This study explored non-autonomous glial regulation of NB proliferation. Lipid droplets (LDs) which reside within the glial niche were shown to be closely associated with the signalling molecule Hedgehog (Hh). Under physiological conditions, cortex glial Hh is autonomously required to sustain niche chamber formation. Upon FGF-mediated cortex glial overgrowth, glial Hh non-autonomously activates Hh signalling in the NBs, which in turn disrupts NB cell cycle progression and its ability to produce neurons. Glial Hh's ability to signal to NB is further modulated by lipid storage regulator lipid storage droplet-2 (Lsd-2) and de novo lipogenesis gene fatty acid synthase 1 (Fasn1). Together, these data suggest that glial-derived Hh modified by lipid metabolism mechanisms can affect the neighbouring NB's ability to proliferate and produce neurons (Dong, 2021).
Lipid shuttling between neurons and glia contributes to the development and function, and stress responses of the nervous system. To understand how a neuron acquires its lipid supply from specific lipoproteins and their receptors, A combined genetic, transcriptome, and biochemical analyses was performed in the developing Drosophila larval brain. This study reports, the astrocyte-derived secreted lipocalin Glial Lazarillo (GLaz), a homolog of human Apolipoprotein D (APOD), and its neuronal receptor, the brain-specific short isoforms of Drosophila lipophorin receptor 1 (LpR1-short), cooperatively mediate neuron-glia lipid shuttling and support dendrite morphogenesis. The isoform specificity of LpR1 defines its distribution, binding partners, and ability to support proper dendrite growth and synaptic connectivity. By demonstrating physical and functional interactions between GLaz/APOD and LpR1, this study elucidated molecular pathways mediating lipid trafficking in the fly brain, and provide in vivo evidence indicating isoform-specific expression of lipoprotein receptors as a key mechanism for regulating cell-type specific lipid recruitment (Yin, 2021).
Lipid trafficking and homeostasis are critical for the development and maintenance of the nervous system. These processes are mediated by a large set of molecular carriers shuttling a diverse group of lipid cargos in and out of designated cell types and cellular compartments. In the central nervous system (CNS), lipid homeostasis heavily relies on neuron-glia cross talk. Studies in mammalian systems have indicated that, besides direct anatomical interactions, glia also supply neurons with metabolic substrates, antioxidants, and trophic factors through secretion. Intriguingly, apolipoproteins are among the most abundant secretory factors that are produced and released by mammalian astrocytes, a group of glial cells with complex morphology and highly branched structures that are intimately associated with synapses, suggesting a critical role for glia-derived lipoprotein and their lipid cargos in synapse formation and function. This notion is supported by studies in cultured mammalian CNS neurons, where glia-derived cholesterol and phospholipids are essential for synaptogenesis. In addition, recent findings in the Drosophila system also indicate essential functions of glia in synapse formation and neurotransmission, although the link between neuron-glia lipid transport and synaptic function has yet to be established (Yin, 2021).
Characterized by their high metabolic rate and elaborate morphology, neurons require a continuous lipid supply throughout their lifetime. How lipoproteins and their receptors mediate neuron-glia lipid shuttling to meet that demand has been a long-standing question in the neurobiology field. Numerous studies over the past decades have demonstrated the critical functions of CNS lipid trafficking in synapse development and cognitive functions. In the mammalian system, deficiencies in either apolipoproteins or their receptors lead to both structural and functional deficits in the brain. For example, Apolipoprotein E (ApoE) delivers cholesterol, amyloid‑β, and other hydrophobic molecules to neurons through its interaction with Very Low-Density Lipoprotein Receptor (VLDLR) and Apolipoprotein E Receptor 2 (ApoER2). While the Apolipoprotein E (ApoE) knockout mice display significantly reduced dendrite size and synapse number as well as impaired learning and memory, both VLDLR and ApoER2 knockout animals also show deficits in cerebellar morphology and impaired contextual fear conditioning and long-term potentiation. Genetic studies of other lipid transport proteins and receptors, including APOD, Niemann-Pick Type C (NPC), Low-Density Lipoprotein Receptor (LDLR), and Low-density lipoprotein Receptor-related Protein 1 (LRP1), further support the importance of lipid trafficking in the proper development and function of the nervous system (Yin, 2021).
Due to the diversity of lipid transport proteins and lipoprotein receptors, as well as the complexity of their tissue- and cell-specific distributions, cellular and molecular mechanisms underlying neuron-glia lipid shuttling have not been well characterized in vivo under physiological conditions. Recent findings in Drosophila highlight the protective functions of neuron-glia metabolic coupling in neurons experiencing oxidative stress or enhanced activity, demonstrating how neurons transfer lipids into glia for detoxification and storage. Similarly, observations made in the mammalian system also provided evidence illustrating fatty acid (FA) transport into astrocytes mediated by ApoE and the importance of neuronal lipid clearance. In contrast, much less is known about how neurons acquire lipid cargos from glia-derived lipoproteins under normal conditions, especially during development, when neurite outgrowth and synapse formation produce a high lipid demand. Attempts were made to fill this gap by determining the functional significance and regulatory mechanisms underlying neuronal lipid uptake using the Drosophila larval brain as a model system (Yin, 2021).
In Drosophila, the Apolipoprotein B (ApoB) family lipoprotein Apolipophorin (ApoLpp) is a major hemolymph lipid carrier and has two closely related lipophorin receptors (LpRs), LpR1 and LpR2, both of which are homologs of mammalian LDLR family proteins. Notably, Drosophila LpRs have multiple isoforms produced by alternative splicing and differential promoter usage. In the fly imaginal disc and oocyte, long isoforms of LpRs (LpR-long) interact with lipid transfer particles (LTP, Apoltp) and mediate endocytosis-independent neutral lipid uptake, while short isoforms of LpRs (LpR-short) neither bind to LTP, nor mediate lipid uptake in these peripheral tissues. In contrast, previous genetic studies revealed specific expression of LpR-short in larval ventral lateral neurons (LNvs), a group of visual projection neurons, and its functions in supporting dendrite development and synaptic transmission (Yin, 2018). This observation is validated by a recent study performed in a cultured Drosophila neuronal cell line, where the LpR-dependent lipid uptake was directly visualized using fluorescently labeled ApoLpp (Matsuo, 2019). Is there a functional significance behind the isoform-specific expression of LpRs? How do short isoforms of LpRs mediate lipid uptake in neurons? These are the questions this study aims to address (Yin, 2021).
This study focused on the LpR1 gene and uncover its isoform-specific expression in neurons and its functions in regulating brain lipid content. Through systematic genetic and biochemical analyses, Glial Lazarillo (GLaz), an astrocyte-derived secreted lipocalin and a homolog of human APOD, was identified as a binding partner for the brain-specific LpR1-short; their cooperative functions are revealed in supporting dendrite morphogenesis, synaptic transmission, and lipid homeostasis in the developing larval brain. In adult Drosophila, GLaz/APOD is found in CNS glia and has been shown to regulate stress resistance and contribute to longevity. Recent studies also demonstrated that GLaz mediates neuron to glia lipid transfer and facilitates neuronal lipid clearance. By identifying GLaz's function in neural development and its direct interaction with LpR1, this study not only uncovered a pair of molecular carriers mediating neuron-glia lipid shuttling in the Drosophila CNS but also presents evidence supporting isoform-specific expression as a key mechanism for regulating the tissue distribution and ligand specificity of neuronal lipoprotein receptors. This in turn leads to the stage- and cell-type-specific regulation of lipid uptake (Yin, 2021).
Lipid-mediated communication between glia and neurons is essential for brain lipid homeostasis and serves critical functions in neural development and synaptic function. Using the developing Drosophila larval brain as a model, this study investigated how neurons acquire their lipid supply from neighboring astrocytes and the regulatory mechanisms associated with the neuron-glia lipid trafficking. Genetic and RNA-seq analyses reveal that lipid uptake in fly neurons is mediated largely by short isoforms of the LpR1 receptor, which recruits a lipid complex through direct interactions with astrocyte-derived apolipoprotein GLaz/APOD. This study identifies specific molecular carriers mediating neuron-glia lipid shuttling and reveals the isoform-specific expression of lipoprotein receptors as a mechanism that determines cell-type-specific recruitment of distinct lipid cargos (Yin, 2021).
Exon mapping of cell-specific RNA-seq libraries revealed that only short isoforms of LpR1 are expressed in LNvs and are upregulated by chronic elevation of neuronal activity. Expanding upon those studies, additional tissue-specific RNA-seq datasets were examined and qFISH analyses was proformed, which strengthened the conclusion that the short-isoforms of LpR1 are CNS-specific and are predominately expressed in neurons, while the long-isoforms of LpR1 are mainly expressed in peripheral tissues and mediate endocytosis-independent lipid uptake. In addition, genetic and biochemical analyses further reveal the functional significance of isoform specificity, including its impact on the receptor's distribution, binding partners, and ability to support specific types of lipid trafficking. These distinctions highlight transcriptional regulation as a key mechanism controlling the cell-type-specific distribution of lipoprotein receptors, their lipid cargos, and uptake efficacy. Results obtained from this study clearly indicate that the molecular and functional diversity of lipoprotein receptors far exceeds current understandings, which are mostly based on DNA sequence analyses. Single-cell transcriptome analyses with sufficient resolution to identify isoform-specific splicing events could potentially reveal the capacity and specificity of the lipid uptake machinery within each individual cell type, helping arrive at a better understanding of the regulatory mechanisms underlying the cell-type-specific lipid recruitment (Yin, 2021).
Lipid shuttling between neurons and glia contributes to energy balance, neural protection, synapse development, and function, and potentially utilizes conserved molecular machinery that is shared among different organisms. Studies in Drosophila and murine neuron-glia coculture systems have demonstrated that neuronal activity alters the metabolic programs of both neurons and glia, leading to the transfer of neuronal lipids into glia for detoxification and storage in the form of lipid droplets. Notably, in both systems, vertebrate ApoE is able to function as the lipid carrier supporting neuronal lipid transfer and lipid droplet accumulation in glia (Yin, 2021).
On the other hand, in the developing larval CNS, this study found an increased lipid demand in neurons coping with chronically elevated input activity. This increase is likely met, at least partially, by enhancing neuronal lipid uptake through the activity-induced upregulation of LpR1 expression. These observations demonstrate a strong influence imposed by activity on neurons' capacity for lipid recruitment through its effects on lipoprotein receptors. Together with earlier studies, these findings suggest that both sides of neuron-glia lipid shuttling are regulated by neuronal activity, and the regulatory mechanisms controlling the expression level of lipoprotein receptors and their interactions with specific ligand molecules likely have functional significance in activity-dependent structural and functional plasticity in the nervous system (Yin, 2021).
Recent studies in adult Drosophila compound eyes have demonstrated that GLaz is involved in lipid transfer from neuron to glia, while the current studies illustrated the interaction between GLaz and LpR1 and a possible role for GLaz in delivering lipid to neurons. Therefore, GLaz appears to be involved in both sides of the lipid trafficking and potentially serves as a key molecular target for regulatory mechanisms controlling lipid homeostasis in the fly brain (Yin, 2021).
The basic structure, molecular composition, and developmental processes of a synapse are shared among vertebrate and invertebrate systems. While synaptogenesis in mammalian neurons relies on cholesterol production by glial cells and its delivery by ApoE-containing lipoprotein complexes, whether the cholesterol and/or ApoE-like lipid carriers are required for building synapses in Drosophila neurons is not known. Importantly, the Drosophila genome does not contain a homolog of ApoE. It is also reported that flies do not have the ability to synthesize cholesterol, and only obtain it through their diet to produce essential hormones. The contrast between these two systems suggests exciting possibilities for studying the lipids involved in synapse construction by understanding the differences and similarities between lipid recruitment in Drosophila vs. mammalian neurons (Yin, 2021).
This study demonstrates that the short isoforms of LpR1 recruit lipids and support dendrite morphogenesis through their interactions with the astrocyte-secreted lipoprotein GLaz, the homolog of human APOD. Given the strong dendrite development phenotype, as well as the reduced life span and stress resistance observed in GLaz loss-of-function mutants, GLaz's hypothesized lipid cargo is likely a critical contributor towards synapse development and neuronal functions in the fly CNS. Currently, only a limited number of putative lipid ligands have been identified for GLaz's homologs; grasshopper Lazarillo binds to retinoic acid and fatty acids, and human APOD binds to arachidonic acid (AA) and progesterone (PG). Whether these lipids or other types of hydrophobic ligands bind to Drosophila GLaz has not been studied. Similar to human APOD, this study observed dimeric and tetrameric GLaz in the larval brain extract. This suggests that, instead of only being able to accommodate a small hydrophobic ligand as a single unit, the GLaz protein could participate in different types of lipoprotein complexes and exhibit distinctive behaviors in vivo through its multimeric forms (Yin, 2021).
APOD's function in the CNS has long been underestimated, despite the fact that APOD is highly elevated during aging and neural injury. When examining recent human and mouse astrocyte RNA-seq data, it was found that, although ApoE is the highest expressing apolipoprotein in mouse astrocytes, the most abundant apolipoprotein expressed in human astrocytes is APOD, strongly suggesting its functional importance in the CNS. In both Drosophila and mammalian systems, GLaz/APOD are produced by astrocytes and have both anti-oxidation and anti-inflammatory protective functions. Given the similarities between GLaz's and APOD's functional and biochemical properties, these findings on the GLaz-LpR1 interaction in Drosophila may facilitate the identification of a mammalian lipoprotein receptor that interacts with APOD and provide new insights into its protective role in aging and neurodegenerative disorders (Yin, 2021).
This study shows that the conserved Hippo pathway, a key regulator of epithelial organ size, restricts neuroblast proliferative potential and neuronal cell number to regulate brain size. The inhibition of Hippo pathway activity via depletion of the core kinases Tao-1, Hippo, or Warts regulates several key characteristics of neuroblasts during postembryonic neurogenesis. The Hippo pathway is required to maintain timely entry and exit from neurogenesis by regulating both neuroblast reactivation from quiescence and the time at which neuroblasts undergo terminal differentiation. Further, it restricts neuroblast cell-cycle speed, specifies cell size, and alters the proportion of neuron types generated during postembryonic neurogenesis. Collectively, deregulation of Hippo signaling in neuroblasts causes a substantial increase in overall brain size. It was shown that these effects are mediated via the key downstream transcription co-activator Yorkie and that, indeed, Yorkie overexpression in neuroblasts is sufficient to cause brain overgrowth. These studies reveal a novel mechanism that controls stem cell proliferative potential during postembryonic neurogenesis to regulate brain size (Poon, 2016).
DNA damaging agents, such as ionizing radiation (IR), induce cell cycle arrest, senescence, differentiation, or cell death of stem cells, which may affect tissue homeostasis. Drosophila larval brain neuroblasts (NBs) and maintaining an appropriate number of NBs is critical to maintain brain size. Irradiation of larvae at early larval stage results in microcephaly, whereas the DNA damage response of NBs that could explain this small brain size is not clearly understood. This study observed that the irradiation of larvae in the second instar retarded brain growth, accompanied by fewer NBs. The IR-induced microcephaly does not seem to result from apoptosis since the irradiated larval brain was not stained with activated Caspase nor was the microcephaly affected by the ectopic expression of the apoptosis inhibitor. When analyzed for the percentage of mitotic cells, irradiated NBs recovered their proliferative potential within 6 h post-irradiation after transient cell cycle arrest. However, IR eventually reduced the proliferation of NBs at later time points and induced the premature differentiation of NBs. In summary, IR-induced microcephaly occurs by NB loss due to premature differentiation, rather than apoptotic cell death (Wagle, 2019).
Drosophila neuroblasts are an excellent model for investigating how neuronal diversity is generated. Most brain neuroblasts generate a series of ganglion mother cells (GMCs) that each make two neurons (type I lineage), but sixteen brain neuroblasts generate a series of intermediate neural progenitors (INPs) that each produce 4-6 GMCs and 8-12 neurons (type II lineage). Thus, type II lineages are similar to primate cortical lineages, and may serve as models for understanding cortical expansion. Yet the origin of type II neuroblasts remains mysterious: do they form in the embryo or larva? If they form in the embryo, do their progeny populate the adult central complex, as do the larval type II neuroblast progeny? This study presents molecular and clonal data showing that all type II neuroblasts form in the embryo, produce INPs, and express known temporal transcription factors. Embryonic type II neuroblasts and INPs undergo quiescence, and produce embryonic-born progeny that contribute to the adult central complex. These results provide a foundation for investigating the development of the central complex, and tools for characterizing early-born neurons in central complex function (Walsh, 2017).
Neural stem cells (NSCs) have the ability to exit quiescence and reactivate in response to physiological stimuli. In the Drosophila brain, insulin receptor (InR)/phosphatidylinositol 3-kinase (PI3K)/Akt pathway triggers NSC reactivation. However, intrinsic mechanisms that control the InR/PI3K/Akt pathway during reactivation remain unknown. This study identified heat shock protein 83 (Hsp83/Hsp90), a molecular chaperone, as an intrinsic regulator of NSC reactivation. Hsp83 is both necessary and sufficient for NSC reactivation by promoting the activation of InR pathway in larval brains in the presence of dietary amino acids. Both Hsp83 and its co-chaperone Cdc37 physically associate with InR. Finally, reactivation defects observed in brains depleted of hsp83 were rescued by over-activation of the InR/PI3K/Akt pathway, suggesting that Hsp83 functions upstream of the InR/PI3K/Akt pathway during NSC reactivation. Given the conservation of Hsp83 and the InR pathway, this finding may provide insights into the molecular mechanisms underlying mammalian NSC reactivation (Huang, 2018).
How the InR/PI3K/Akt pathway is regulated during NSC reactivation is poorly understood. This study shows that molecular chaperone Hsp83/Hsp90, together with its co-chaperone Cdc37, play a role in the reactivation of Drosophila NSCs. Mechanistically, Hsp83 and Cdc37 physically associate with InR and are important for the activation of the InR/PI3K/Akt pathway in NSCs. Therefore, this study demonstrates that Hsp83 serves as an intrinsic factor within NSCs that is necessary for the activation of the InR/PI3K/Akt pathway and, in turn, reactivation of NSCs. This evidence suggests that Hsp83 and Cdc37 regulate the protein folding and activation of InR in the nervous system (Huang, 2018).
The role of Hsp83 in NSC reactivation at early larval stages is distinct from its known role in centrosomes or NSC polarity. Drosophila Hsp83 is a core centrosomal component required for proper mitotic spindle formation and chromosome segregation. In Drosophila larval CNS, Hsp83 and co-chaperone Sgt1 are required for the stabilization of Polo and centrosome organization in NSCs. Hsp83 and Sgt1 are also required for the establishment of NSC polarity via the LKB1/AMPK pathway in third-instar larvae. However, sgt1 RNAi in NSCs did not display any phenotypes during NSC reactivation, suggesting that Hsp83 interacts with different co-chaperones to control NSC reactivation and cortical polarity at different developmental stages. Consistent with this notion, Cdc37, but not other co-chaperones of Hsp83, was found to be required for NSC reactivation. The proliferation of MB NSCs were unaffected by hsp83 knockdown, Therefore, Hsp83 promotes NSC reactivation rather than general cell proliferation. Consistent with these observations, there is no significant difference in proliferation between hsp83 mutant and wild-type eye imaginal discs in the proliferating zone. Interestingly, in pupal eyes that undergo terminal differentiation, Hsp83 is required for cell-cycle exit by activating the anaphase-promoting complex/cyclosome. This study found cytokinesis defects in cdc37-depleted NSCs, but not in NSCs depleted of Hsp83. This observation is consistent with a known role of Cdc37 in cell division and cytokinesis in Drosophila (Huang, 2018).
Hsp90 plays a key role in signal transduction and appears to bind to its substrates in a near native state poised for activation by binding of ligand or other factors. Since Hsp83 overexpression is sufficient to drive the activation of InR/PI3K/Akt pathway and trigger premature NSC reactivation, Hsp83 likely plays an active role in promoting NSC reactivation by binding to InR at a late stage of folding poised for activation by dILP binding. Furthermore, in the absence of dietary amino acids, the expression of hsp83 is downregulated, likely partially contributing to the inactivation of the InR pathway. It is proposed that InR is a target of Hsp83 and Cdc37 during NSC reactivation. The physical association among Hsp83, Cdc37, and InR was strongly supported by PLA assays, and both in vitro and in vivo BiFC. Although tandem affinity purification-mass spectrometry in Drosophila S2 cells implied an interaction between Hsp83 and InR, this study failed to detect a consistent interaction between Hsp83 and InRintra in S2 cells in co-immunoprecipitation experiments, probably due to the transient nature of this interaction. In addition, genetic interaction experiments indicate that Hsp83 activates the InR/PI3K/Akt pathway to promote NSC reactivation. Taken together, InR is likely a client of Hsp83 in Drosophila NSCs. Consistent with these findings, in human fibroblasts, Hsp90 co-immunoprecipitated with intracellular InR β. Furthermore, Hsp90 facilitates the maturation of the InR precursor in the ER and, in turn, is required for cell surface expression of InR in both bovine adrenal medullary chromaffin cells and human kidney HEK293 cells. Therefore, the interaction between the Hsp90 chaperone family and InR may be conserved from Drosophila to humans. In mammals, the expression level of Hsp90 in the brain is the highest among all tissues. Although mammalian Hsp90 proteins are heavily implicated in neurodegenerative diseases, their function in brain development is not well understood. Hsp90/Cdc37 stabilize the intracellular domain of Ryk, a Wnt receptor required for neurogenesis. Furthermore, Hsp90 stabilizes hypoxia-inducible factor-1, which promotes NSC proliferation under hypoxia. It remains to be determined whether the interaction between mammalian Hsp90 and InR is conserved during mammalian NSC development (Huang, 2018).
Drosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila, was identified as a putative Notch target in genome-wide studies. This study examined expression and regulation of Path in the Drosophila larval brain. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference. Path is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE. This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE (Notch responsive element) was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction. This study has identified a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction (Feng, 2020).
Drosophila Neuroblasts (NBs) are neural stem cells that divide to give progeny, which differentiate into neurons and glia that later constitute the adult brain. NBs arise from neuroectoderm during embryonic development and enter quiescence at the end of the embryonic stage, until they are reactivated upon feeding during larval stages. After reactivation, around 350 NBs reside on the surface of the brain and constitute the stem cell pool, undergoing multiple rounds of asymmetric cell divisions. During division, each NB generates one larger daughter cell that retains stem cell identity and one smaller daughter cell that divides further to generate progeny that differentiate into certain types of neurons and glia. At the time of metamorphosis, the central nervous system (CNS) contains about 30,000 neurons and 10,000 glial cells. The glial cells fulfil supporting and nurturing function to neurons. Importantly they also ensure NBs receive the correct growth signal at the correct times. For example, signals from glia are necessary for NBs to exit quiescence upon feeding, and to remain proliferative during nutrition deprivation once the larva passes the critical weight time-point (Feng, 2020).
Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Notch depletion causes loss of NB lineages while Notch over-activation inhibits NBs from differentiating and induces brain tumours. In the canonical Notch signalling model, upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is cleaved and released into the nucleus. The nuclear NICD interacts with the DNA-binding protein known as Suppressor of Hairless (Su(H)) in flies, to activate the expression of target genes. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes including stem cell maintenance (Feng, 2020).
The brain, like other organs, needs to translate changing nutrition inputs into cell growth decisions. An emerging role of amino acid transporters, especially the SLC family, in coupling the nutrition signalling and growth pathways has been revealed in recent years. SLC38A9 acts as an amino acid sensor in the process of mTORC-activation in mammalian cell lines [Wang, 2015; Rebsamen, 2015]. Similarly, SLC36A4 helps to promote proliferation in colorectal cancer cells through its interaction with mTORC1 (Fan, 2016). A requirement for SLC36A4 in mice retinal pigmented epithelial cells also involved mTORC-activation (Shang, 2017). However, where and how might these transporters function in other cases of nutrient sensing, such as the Drosophila NBs, remains unknown. Also, it is unclear whether the growth-promoting role of these amino acid transporters would be adaptive to starvation. For example, the sparing mechanism in nutrient deprived NBs somehow bypasses the Tor pathway (Feng, 2020).
Pathetic (Path) is the Drosophila orthologue of SLC36A4, having the characteristics of a broad specificity transporter with multiple transmembrane domains. It interacts with Tor (Target of apamycin) pathway components in regulating eye growth and body growth of Drosophila and promotes dendritic growth in C4da neurons (Lin, 2015). path also exhibited the hall marks of a Notch regulated gene in a genome-wide study of genes upregulated during Notch-induced NB hyperplasia (Zacharioudaki, 2016). This study has followed up on this observation by analysing the role and regulation of Path in NBs under normal and abnormal nutrition conditions. path was shown to be a novel direct Notch target in NBs, and it is required for NB proliferation. Further its role in brain sparing was characterised, and it was found to be required to fully protect the CNS from nutrient restriction. However, the evidence indicates that glial-expressed Path is important for protecting brain growth under nutrient restriction, rather than its activity in the NBs themselves (Feng, 2020).
Previously, Path was found to be required for overall body growth and extreme dendrite growth, potentially through interacting with the PI3K/Tor pathway and protein synthesis pathways (Lin, 2015). This study identified an autonomous role of path in maintaining NB proliferation, which is in line with previous findings that path promotes growth. Path expression in NBs is partially dependant on Notch, as it contains an intronic enhancer, which is directly regulated by the pathway. Thus its regulation and involvement in NB proliferation argues that Path contributes to the normal function of Notch in NBs, although it remains to be established whether it has a similar essential role in Notch induced tumours. Furthermore, the striking reduction in pathNRE driven expression under nutrient restriction (NR) suggests that the NBs are sensitive to changes in the internal milieu of the animal. This argues that, while glial cells may shield NBs from many environmental effects, the NBs are nevertheless able to detect altered nutrient levels and may harbour addition pathways that contribute to brain-sparing (Feng, 2020).
Although this study found that Path is required for brain sparing (the maintenance of brain size) during NR, this appears to rely on its expression in Glial cells rather than NBs, despite the fact that the intronic pathNRE enhancer is sensitive to nutrient deprivation. Alk/Jeb is the major pathway that has been linked to brain growth under NR conditions, and glial knockdown of Jeb (repo-gal4 > jebRNAi) resulted in smaller NB-clone size as well as lineage number. It is possible that Path at the membrane of surface glial cells could detect the environmental amino acid levels and in turn regulate Alk/Jeb expression. Path is a potential amino acid transporter with multiple transmembrane domains, which exhibited high affinity for alanine and glycine with low transporting capacity when expressed in Xenopus oocytes. The closest mammalian relatives, proton-assisted transporter 4 (PAT4 or hPAT4), have a high affinity for proline and tryptophan. Although it remains to be fully determined which are the main substrates for Path in vivo, a recent study suggests that Path is required for scavenging proline to promote tumour growth in conditions of obesity enhanced tumorigenesis in Drosophila (Newton, 2020). In these conditions, Path regulation was also involved with nutrient sensing mechanisms albeit the levels of sugars were elevated rather than restricted. Further studies will be needed to determine whether proline is the primary substrate for Path in all conditions and the extent that path expression is differentially regulated by changes in nutrient levels according to the tissue and its proliferative state (Feng, 2020).
The Tor pathway is involved in NB reactivation, growth and proliferation. However, in L3, NB growth is regulated by the Alk/Jeb pathway which activates downstream PI3K/Akt signalling, but appears independent of Tor. One model to explain the current results is that Path functions as a mediator between Alk and the Tor pathway. Alk is required for and acts through the PI3K/Akt pathway. In L3 larvae brains, most Tor pathway components are dispensable for brain growth and brain size is not changed significantly when levels of Insulin-like peptides (Ilp) are manipulated. In contrast, the Tor pathway is activated in younger brains suggesting there may be a mechanism to bypass or switch-off the Tor pathway at later stages. One hypothesis, considering pathNRE is upregulated after NB reactivation, is that path helps to keep activity of the Tor pathway at restricted levels at later stages. In this case, the effects of path in NBs would be through nutrient sensing and protein synthesis, similar to its function in C4da neurons (Feng, 2020).
The regulation and expression of Path in the NBs suggests that, in this context, Path promotes cell growth and lineage size. Further studies will be needed to characterise what the consequences from Path up-regulation are in different circumstances. For example, whether Path has the same role in Notch induced NB tumours, and whether this would differ depending on nutrient status, remains to be established. Likewise, this study has investigated its role in so-called Type I NBs. Not mutant clones were obtained to distinguish whether it makes the same contribution in Type II NBs, which produce a highly proliferative transit amplifying intermediary, or whether its activity there differs. Finally, it is striking that pathNRE-GFP expression is decreased in neuroblasts under nutrient restriction, when neuroblast proliferation becomes spared. Whether this reflects a physiological response, whereby NB Path levels are reduced while glial cell Path persists under NR, or whether it reflects a regulatory feature, whereby a different enhancer becomes activated in these conditions, remains to be established. Nevertheless, these data highlight the importance of distinguishing the different regulatory inputs to Path, and the extent that they are dependent on different nutrient and/or amino acid (Feng, 2020).
Brain expression of Path, which encodes a broad specificity transporter, occurs in both NBs and glial cells where its functions appear to differ. NB expression of path, is partially dependent on Notch activity and is required for NB lineage proliferation but not for brain sparing under nutrient restrictions. In contrast Glial expression of Path appears to be independent of Notch and is essential for protecting the brain growth under nutrition restriction. In conclusion, this study has demonstrate different roles of Path in distinct parts of the brain that would together enable the larval brain to proliferate and grow in both normal and NR conditions (Feng, 2020).
The ability of neural stem cells (NSCs) to switch between quiescence and proliferation is crucial for brain development and homeostasis. Increasing evidence suggests that variants of histone lysine methyltransferases including KMT5A are associated with neurodevelopmental disorders. However, the function of KMT5A/Pr-set7/SETD8 in the central nervous system is not well established. This study shows that Drosophila Pr-Set7 is a novel regulator of NSC reactivation. Loss of function of pr-set7 causes a delay in NSC reactivation and loss of H4K20 monomethylation in the brain. Through NSC-specific in vivo profiling, this study demonstrated that Pr-set7 binds to the promoter region of cyclin-dependent kinase 1 (cdk1) and Wnt pathway transcriptional co-activator earthbound1/jerky (ebd1). Further validation indicates that Pr-set7 is required for the expression of cdk1 and ebd1 in the brain. Similar to Pr-set7, Cdk1 and Ebd1 promote NSC reactivation. Finally, overexpression of Cdk1 and Ebd1 significantly suppressed NSC reactivation defects observed in pr-set7-depleted brains. Therefore, Pr-set7 promotes NSC reactivation by regulating Wnt signaling and cell cycle progression. These findings may contribute to the understanding of mammalian KMT5A/PR-SET7/SETD8 during brain development (Huang, 2021).
The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Using asymmetrically dividing Drosophila larval neural stem cells (NSCs) as a model, it was shown that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, it was shown that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, these results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis (Gallaud, 2022).
Asymmetric cell division (ACD) allows stem cells to generate differentiating progeny while simultaneously maintaining their own pluripotent state. ACD involves coupling mitotic spindle orientation with cortical polarity cues to direct unequal segregation of cell fate determinants. In Drosophila neural stem cells (neuroblasts; NBs), spindles orient along an apical-basal polarity axis through a conserved complex of Partner of Inscuteable (Pins; human LGN) and Mushroom body defect (Mud; human NuMA). While many details of its function are well known, the molecular mechanics that drive assembly of the cortical Pins/Mud complex remain unclear, particularly with respect to the mutually exclusive Pins complex formed with the apical scaffold protein Inscuteable (Insc). This study identified Hu li tai shao (Hts; human Adducin) as a direct Mud-binding protein, using an aldolase fold within its head domain (Hts(HEAD)) to bind a short Mud coiled-coil domain (Mud(CC)) that is adjacent to the Pins-binding domain (Mud(PBD)). Hts is expressed throughout the larval central brain and apically polarizes in mitotic NBs where it is required for Mud-dependent spindle orientation. In vitro analyses reveal that Pins undergoes liquid-liquid phase separation with Mud, but not with Insc, suggesting a potential molecular basis for differential assembly mechanics between these two competing apical protein complexes. Furthermore, this study found that Hts binds an intact Pins/Mud complex, reduces the concentration threshold for its phase separation, and alters the liquid-like property of the resulting phase separated droplets. Domain mapping and mutational analyses implicate critical roles for both multivalent interactions (via Mud(CC) oligomerization) and protein disorder (via an intrinsically disordered region in Hts; Hts(IDR)) in phase separation of the Hts/Mud/Pins complex. This study identifies a new component of the spindle positioning machinery in NBs and suggests that phase separation of specific protein complexes might regulate ordered assembly within the apical domain to ensure proper signaling output (Parra, 2023).
While testing for genome instability in Drosophila as reported by unscheduled upregulation of UAS-GFP in cells that co-express GAL80 and GAL4, it was noticed that, as expected, background levels were low in most developing tissues. However, GFP-positive clones were frequent in the larval brain. Most of these clones originated from central brain neural stem cells. Using imaging-based approaches and genome sequencing, it was shown that these unscheduled clones do not result from chromosome loss or mutations in GAL80. This phenomenon was named 'Illuminati'. Illuminati is strongly enhanced in brat tumors and is also sensitive to environmental conditions such as food content and temperature. Illuminati is suppressed by Su(var)2-10, but it is not significantly affected by several modifiers of position effect variegation or Gal4::UAS variegation. It is conclude that Illuminati identifies a previously unknown type of functional instability that may have important implications in development and disease (Goupil, 2022).
Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. This study demonstrated that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, human Parafibromin was shown to rescue the ectopic NSC phenotype in Drosophila hyx mutant brains. This study also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. These data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers (Deng, 2022).
Environmental stress can cause mutation or genomic instability in stem cells which, in some cases, leads to tumorigenesis. Mechanisms to monitor and eliminate these mutant stem cells remain elusive. Using the Drosophila larval brain as a model, this study shows that X-ray irradiation (IR) at the early larval stage leads to accumulation of nuclear Prospero (Pros), resulting in premature differentiation of neural stem cells (neuroblasts, NBs). Through NB-specific RNAi screenings, the Mre11-Rad50-Nbs1 complex and the homologous recombination (HR) repair pathway, rather than non-homologous end-joining pathway that plays, a dominant role in the maintenance of NBs under IR stress. The DNA damage sensor ATR/mei-41 is shown to act to prevent IR-induced nuclear Pros in a WRNexo-dependent manner. The accumulation of nuclear Pros in NBs under IR stress, leads to NB cell fate termination, rather than resulting in mutant cell proliferation. This study reveals an emerging mechanism for the HR repair pathway in maintaining neural stem cell fate under irradiation stress (Xu, 2023).
Growth regulation must be robust to ensure correct final size, but also adaptative to adjust to less favorable environmental conditions. Developmental coordination between whole-organism and the brain is particularly important, as the brain is a critical organ with little adaptability. Brain growth mainly depends on neural stem cell (NSC) proliferation to generate differentiated neural cells, it is however unclear how organism developmental progression is coordinated with NSCs. This study demonstrates that the steroid hormone ecdysone plays a multi-step, stage specific role in regulating Drosophila NSCs, the neuroblasts. Animals were used that are unable to synthesize ecdysone, to show that the developmental milestone called 'critical weight peak', the peak that informs the body has reached minimum viable weight to survive metamorphosis, acts a checkpoint necessary to set neuroblast cell cycle pace during larval neurogenesis. The peaks of ecdysone that occur post-critical weight are no longer required to maintain neuroblast division rate. It was additionally shown that in a second stage, at the onset of pupariation, ecdysone is instead required to trigger neuroblast's proliferation exit and consequently the end of neurogenesis. It was demonstrated that, without this signal from ecdysone, neuroblasts lose their ability to exit proliferation. Interestingly, although these neuroblasts proliferate for a longer period, the number of differentiated neurons is smaller compared to wild-type brains, suggesting a role for ecdysone in neuron maintenance. This study provides insights into how neural stem cells coordinate their division rate with the pace of body growth, identifying a novel coordination mechanism between animal development and NSC proliferation (Oliveira, 2023).
Coordinated spatio-temporal regulation of the determination and differentiation of neural stem cells is essential for ">brain development. Failure to integrate multiple factors leads to defective brain structures or tumour formation. Previous studies suggest changes of chromatin state are needed to direct neural stem cell differentiation, but the mechanisms are unclear. Analysis of Snf5-related 1 (Snr1), the Drosophila orthologue of SMARCB1, an ATP-dependent chromatin remodelling protein, identified a key role in regulating the transition of neuroepithelial cells into neural stem cells and subsequent differentiation of neural stem cells into the cells needed to build the brain. loss of Snr1 in neuroepithelial cells leads to premature neural stem cell formation. Additionally, loss of Snr1 in neural stem cells results in inappropriate perdurance of neural stem cells into adulthood. Snr1 reduction in neuroepithelial or neural stem cells leads to the differential expression of target genes. Snr1 is associated with the actively transcribed chromatin region of these target genes. Thus, Snr1 likely regulates the chromatin state in neuroepithelial cells and maintains chromatin state in neural stem cells for proper brain development (Keegan, 2023).
Neural stem cells (NSCs) reside in a defined cellular microenvironment, the niche, which supports the generation and integration of newborn neurons. The mechanisms building a sophisticated niche structure around NSCs and their functional relevance for neurogenesis are yet to be understood. In the Drosophila larval brain, the cortex glia (CG) encase individual NSC lineages in membranous chambers, organising the stem cell population and newborn neurons into a stereotypic structure. CG were found to wrap around lineage-related cells regardless of their identity, showing that lineage information builds CG architecture. It was then discovered that a mechanism of temporally controlled differential adhesion using conserved complexes supports the individual encasing of NSC lineages. An intralineage adhesion through homophilic Neuroglian interactions provides strong binding between cells of a same lineage, while a weaker interaction through Neurexin-IV and Wrapper exists between NSC lineages and CG. Loss of Neuroglian results in NSC lineages clumped together and in an altered CG network, while loss of Neurexin-IV/Wrapper generates larger yet defined CG chamber grouping several lineages together. Axonal projections of newborn neurons are also altered in these conditions. Further, the loss of these 2 adhesion complexes specifically during development is linked to locomotor hyperactivity in the resulting adults. Altogether, these findings identify a belt of adhesions building a neurogenic niche at the scale of individual stem cell and provide the proof of concept that niche properties during development shape adult behaviour (Banach-Latapy, 2023).
Mammalian neural stem cells generate transit amplifying progenitors that expand the neuronal population, but these type of progenitors have not been studied in Drosophila. The Drosophila larval brain contains 100 neural stem cells (neuroblasts) per brain lobe, which are thought to bud off smaller ganglion mother cells (GMCs) that each produce two post-mitotic neurons. This study used molecular markers and clonal analysis to identify a novel neuroblast cell lineage containing transit amplifying GMCs (TA-GMCs). TA-GMCs differ from canonical GMCs in several ways: each TA-GMC has nuclear Deadpan, cytoplasmic Prospero, forms Prospero crescents at mitosis, and generates up to 10 neurons; canonical GMCs lack Deadpan, have nuclear Prospero, lack Prospero crescents at mitosis, and generate two neurons. It is concluded that there are at least two types of neuroblast lineages: a Type I lineage where GMCs generate two neurons, and a type II lineage where TA-GMCs have longer lineages. Type II lineages allow more neurons to be produced faster than Type I lineages, which may be advantageous in a rapidly developing organism like Drosophila (Boone, 2008).
During a clonal analysis of a larval neuroblast self-renewal
mutant it was realized that wild type brains
have two distinct types of neuroblast lineages. Mosaic analysis
with repressible cell marker (MARCM) was used to generate GFP-marked single cell clones in
the larval brain. Depending on the cell in which chromosomal
recombination occurs, it is possible to label
a single neuroblast and all its progeny, a single GMC
and all its progeny, or a single neuron derived from a terminal mitosis. A
low density of clones was induced randomly throughout the brain
at either mid-first or mid-second larval instar and
all clones were assayed 48 h after induction.
Two distinct neuroblast lineages were found: a 'Type I'
lineage that matches previously reported neuroblast
lineages, and a novel 'Type II' lineage that is larger and more complex (Boone, 2008).
Type I neuroblast clones always contained one large neuroblast near the surface of the brain that had nuclear Dpn and cytoplasmic Pros.
These clones always contained a column of smaller
cells that lacked Dpn and had nuclear Pros, with the occasional presence of a single
Dpn+ small cell contacting the neuroblast, which is
likely to be a newborn GMC. The cells furthest from the neuroblast were Dpn
Pros mature neurons that extend GFP1 axons into
the central brain. Type I neuroblast lineages are the sole occupants of the dorsoanterior
lateral (DAL) brain region, but can also be found in all other brain regions. To minimize regional variation in neuroblast lineages. Analysis of Type I
neuroblasts was restricted to the DAL region (Boone, 2008).
Type I GMC clones were assayed only in the DAL
region, where no Type II neuroblasts were observed.
All clones lacking a large Dpn+ neuroblast were considered
to be GMC clones, and these GMC clones generated at most just two cells. Thus, Type I lineages are identical to those reported for Drosophila embryonic neuroblasts,
larval mushroom body neuroblasts, and grasshopper neuroblasts (Boone, 2008).
Type II neuroblast clones always contained one large Dpn+ neuroblast near the surface of the brain, but also contained a distinctive group of small Dpn+ cells that lack nuclear Pros. There are also usually 1-2 small cells in direct contact with the
neuroblast that lack both Dpn and nuclear Pros. These two types of small cells are
never observed in Type I clones and are a defining
feature of Type II clones. Type II neuroblast clones
are found in several brain regions, including a cluster
within the DPM region. One Type II neuroblast appears to be the previously identified
DPMpm1 neuroblast based on its distinctive axon projection that
bifurcates over the medial lobe of the mushroom
body before crossing the midline (Boone, 2008).
Type II GMC clones were identified by the lack
of a large Dpn+ neuroblast. All brain regions that
contained Type II neuroblast lineages produced
GMC clones of greater than two cells; all brain regions that lacked
Type II neuroblast lineages never generated >2 cell
GMC clones. Type II GMC clones often contained Dpn+ Proscyto small cells that are unique
to Type II neuroblast lineages, confirming that these clones are sublineages
of a Type II neuroblast lineage. It is concluded that Type II neuroblasts generate GMCs that produce more than two neurons. Because Type II GMC
clones could generate several fold more neurons
than a Type I GMC, they were called 'transit amplifying GMCs' or TA-GMCs (Boone, 2008).
TA-GMC clones also contained small cells with nuclear Pros; it is suggested that these cells are equivalent to Type I GMCs based on their cell division
profile, and because two cell clones were observed in regions of the brain that
contained Type II neuroblast lineages. However, the possibility that some of these nuclear Pros cells are post-mitotic immature neurons cannot be ruled out (Boone, 2008).
If Type II lineages generate TA-GMCs that make
an average of twice as many neurons as a Type I lineage,
it would be expected that Type II lineages generate
approximately twice as many cells over the same
timespan compared with Type I lineages. Indeed, it was
found that when Type I or Type II clones are grown for
the same length of time (between clone induction and
analysis), Type II clones generate approximately
twice as many neurons. Type I clones in the DAL
generate 40.4 +/ 3.1 cells, whereas Type II lineages in the DPM generate 71.2 +/- 6.3 cells . In all cases the final Type I and Type II neuroblast clones contained
a single large Dpn+ neuroblast, ensuring that only single neuroblast clones were
counted. It is concluded that Type II TA-GMCs generate
more neurons than Type I GMCs, and that Type II
lineages generate more neurons than Type I lineages (Boone, 2008).
This study characterized the cell division patterns within
Type I and Type II lineages to help understand the
relationship between different cell types in a lineage.
It was first asked what cell type is directly produced by
Type I and Type II neuroblasts? Type I neuroblasts in the DAL region always segregate Pros
protein into the newborn GMC resulting in easily detectable levels of Pros in
neuroblast progeny. Thus, Type I neuroblasts in the DAL generate nuclear Pros+ GMCs,
as previously reported. In contrast, Type II neuroblasts of the DPM
region often fail to segregate Pros protein, despite proper localization of other apical/
basal proteins, which would account for reduced Pros levels in newborn
progeny. The variation in Pros localization among DPM neuroblasts could be due to
the presence of some Type I neuroblasts in the region,
or actual variation among Type II neuroblasts. It is
concluded that Type II neuroblasts divide asymmetrically,
but can fail to segregate Pros protein into their newborn progeny (Boone, 2008).
Next, the relationship between the Type II small cells that have high Dpn, low Pros
(Dpn+ Proscyto) and those that contain high Pros, but
no Dpn (Dpn- Prosnucl), was investiged. It was found that mitotic Dpn+
small cells always form Mira/Pros cortical crescents, with Pins protein localized
to the opposite cortical domain, and the spindle aligned along this cortical
polarity axis. This type of division is unique to Type II lineages, as all Type I GMCs
always showed diffuse cytoplasmic Pros during mitosis. It is concluded
that Type II Dpn+ small cells undergo molecularly
asymmetric cell divisions to generate a Pros+ sibling
and a Pros- sibling. It is proposed that the sibling with
little or no Pros remains a Dpn+ TA-GMC, whereas
the Pros+ sibling generates one or two post-mitotic
neurons, similar to Pros+ GMCs in Type I lineages (Boone, 2008).
To characterize the cell cycle kinetics of Type I
GMCs and Type II TA-GMCs, BrdU labeling experiments were performed. Larvae were exposed to a 4.5 h BrdU pulse and then immediately fixed and assayed
for BrdU incorporation. As expected, both Type I and
Type II neuroblasts always incorporated BrdU. Type I neuroblasts showed only a
few closely-associated GMCs labeled, whereas Type II neuroblasts had a much
larger number of labeled progeny. It is unlikely that the Type II neuroblasts
generate all of these progeny during the 4.5 h labeling
window, because the shortest neuroblast cell cycle time observed in any brain region was ~50 min, and thus it is concluded that Type II neuroblast progeny
undergo more rounds of cell division that Type I GMCs (Boone, 2008).
To determine if the proliferative Type II neuroblast
progeny are competent to differentiate into neurons,
a BrdU pulse/chase experiment was performed. Larvae
were fed BrdU for 4.5 h as described above, but then
allowed to develop for 18 h without BrdU. Type I neuroblasts
lacked BrdU incorporation, as expected due to
label dilution during the chase interval, but BrdU was
maintained in the Elav1 post-mitotic neurons born
during the pulse window. Type II neuroblasts and most of their progeny also diluted out
BrdU, confirming their status as proliferative cells, and some Elav1 post-mitotic neurons were born during the pulse interval and maintained BrdU
labeling. It is concluded that Type II neuroblast progeny are proliferative but can still
give rise to differentiated neurons (Boone, 2008).
There are currently no molecular markers that can
be used to unambiguously identify Type II neuroblasts. The inability to form Pros crescents may be shared by all Type II neuroblasts, but even so, it
would only be a useful marker for mitotic neuroblasts.
In the DPM brain region (enriched for Type II lineages)
it was found about 50% of the mitotic neuroblasts
have little or no Pros crescent, and based on the distinctive
lack of Pros in some Type II neuroblast progeny,
it is concluded that these are Type II neuroblasts.
(The 50% of the DPM neuroblasts that form Pros crescents
may be Type I neuroblasts within the region, a
special subset of Type II neuroblasts, or there may be
stochastic variability in Pros crescent-forming ability
among Type II neuroblasts.) In any case, these findings
may explain why some labs report seeing Pros crescents whereas others
report that neuroblasts do not form Pros crescents; both are correct because there are
two types of larval neuroblast lineages (Boone, 2008).
It is unknown whether neuroblasts can switch back
and forth between Type I and Type II modes of cell
lineage. If the level of Pros in the neuroblast is the key
factor distinguishing these modes of division, then
experimentally raising Pros levels in Type II lineages
may switch them to Type I lineages; conversely,
reducing Pros levels in Type I lineages may switch
them to Type II lineages. As more brain neuroblasts
become uniquely identifiable it will be interesting to
address this question. It will also be interesting to
search for Type II neuroblast lineages in other insects
or crustaceans where Type I neuroblast lineages have been documented (Boone, 2008).
What terminates the TA-GMC lineage? The TA-GMC
may fall below a size threshold for continued
proliferation. Alternatively, TA-GMCs may lose contact
with a niche-derived signal that maintains their
proliferation; Hedgehog, Fibroblast growth factor, and Activin
are all required for larval brain neuroblast proliferation,
but none have been tested for a role in TA-GMC
proliferation. Lastly, there may be lineage-specific
factors segregated into the TA-GMCs that limit their
mitotic potential. TA-GMCs may die at the end of
their lineage, as do some neuroblasts, or they may differentiate. It has been shown that loss of Pros and Brat together can generate a more severe neuroblast tumor phenotype
than either alone. This suggests
that the Type II lineages may be especially sensitive
to further loss of differentiation promoting factors
due to their low levels of endogenous Pros. Indeed, a dramatic neuroblast tumor phenotype
has been observed in type II lineages in lethal giant discs mutants. This raises the question of how Type II lineages benefit the fly. They have the
ability to generate more neurons in a faster period of
time, due to the presence of TA-GMCs, and may be an
evolutionary adaptation to the rapid life cycle of Drosophila. Slower developing insects may not require such rapid modes of neurogenesis (Boone, 2008).
Specific dorsomedial (DM) neuroblast lineages of the Drosophila brain amplify their proliferation through generation of transit amplifying intermediate progenitor cells. Together, these DM neuroblast lineages comprise over 5,000 adult-specific neural cells and thus represent a substantial part of the brain. However, no information is currently available about the structure or function of any of the neural cells in these DM lineages. This report uses MARCM-based clonal analysis together with immunocytochemical labeling techniques to investigate the type and fate of neural cells generated in the DM lineages. Genetic cell lineage-tracing and immunocytochemical marker analysis reveal that DM neuroblasts are multipotent progenitors that produce a set of postembryonic brain glia as well as a large number of adult-specific protocerebral neurons. During larval development the adult-specific neurons of each DM lineage form several spatially separated axonal fascicles, some of which project along larval brain commissural structures that are primordia of midline neuropile. By taking advantage of a specific Gal4 reporter line, the DM-derived neuronal cells can be identified and followed into early pupal stages. During pupal development the neurons of the DM lineages arborize in many parts of the brain and contribute to neuropile substructures of the developing central complex, such as the fan-shaped body, noduli and protocerebral bridge.
These findings provide cellular and molecular evidence for the fact that DM neuroblasts are multipotent progenitors; thus, they represent the first identified progenitor cells in the fly brain that have neuroglioblast functions during postembryonic development. Moreover, these results demonstrate that the adult-specific neurons of the DM lineages arborize widely in the brain and also make a major contribution to the developing central complex. These findings suggest that the amplification of proliferation that characterizes DM lineages may be an important requirement for generating the large number of neurons required in highly complex neuropile structures such as the central complex in the Drosophila brain (Izergina, 2009).
The Drosophila brain is a highly complex structure composed of tens of thousands of neurons that are interconnected in numerous exquisitely organized neuropile structures, such as the mushroom bodies, antennal lobes and central complex. The neurons of the central brain, defined as the supraesophageal ganglion without the optic lobes, derive from approximately 100 bilaterally symmetrical pairs of neural stem cell-like neuroblasts, each of which is thought to generate a characteristic lineage of neural progeny. Several studies have indicated that each developing neuroblast acquires an intrinsic capacity for neuronal proliferation in a cell-autonomous manner and generates a specific lineage of neural progeny that is nearly invariant and unique. This implies that each neuroblast acquires a specific identity that determines the number and types of neural progeny it generates. This specification of neuroblasts has been shown to occur through a combination of positional information, and temporal and combinatorial cues provided by the suite of developmental control genes expressed by each precursor (Izergina, 2009).
Neuroblasts begin to proliferate during embryonic development and during this initial phase of proliferation they generate the primary neurons of the larval brain. After a period of mitotic quiescence during the early larval period, most brain neuroblasts reactivate proliferation and produce secondary neurons that make up the bulk of the adult brain; these are referred to as adult-specific neurons. Indeed, 95% of the neurons in the adult brain are secondary neurons generated during postembryonic development. These adult-specific neurons initially form a lineage-related cluster of immature neurons that extend fasciculated primary neurites into the neuropile but wait until metamorphosis to complete their extension to synaptic targets and final morphogenesis (Izergina, 2009).
Most neuroblasts in the central brain generate lineages comprising, on average, 100 to 120 adult-specific cells. (The neuroblasts that generate the intrinsic cells of the mushroom bodies each produce an average of approximately 200 adult-specific cells; these neuroblasts do not enter a quiescent state in early larva.) In contrast, remarkably large neuroblast lineages are generated in the dorsomedial (DM) area of the larval brain. The number of adult-specific cells in these DM neuroblast lineages averages 450, more than twice the average number of cells in the mushroom body lineages. The large number of neurons in these lineages is achieved by an amplification of neuroblast proliferation through generation of intermediate progenitor cells. Most neuroblasts in the central brain divide asymmetrically in a stem cell mode whereby they self-renew and generate smaller daughter cells called ganglion mother cells, which divide once to produce two postmitotic progeny. In contrast, dividing DM neuroblasts (also referred to as posterior asense-negative (PAN) neuroblasts or type II neuroblasts) self-renew and generate intermediate progenitor cells that act as transit amplifying cells and can generate numerous ganglion mother cell-like cells by retaining their ability to divide several more times. In this respect, neurogenesis in DM lineages is similar to that seen in the mammalian central nervous system in which the primary progenitors amplify the progeny they produce through the generation of proliferating intermediate progenitors. (In addition to the six pairs of DM neuroblasts located in the dorsomedial area of the brain, there are two additional pairs of PAN (type II) neuroblasts located more laterally in the brain; because they are easier to identify, this study focused on the six DM neuroblasts (Izergina, 2009).
The six bilaterally symmetrical pairs of DM (type II) neuroblast lineages together generate over 5,000 adult-specific cells due to the amplification of neuroblast proliferation. Given current estimates of total cell number in the Drosophila brain, this cell number would roughly correspond to one-fourth of the total number of cells in the central brain. The DM lineages thus represent a substantial part of the brain. However, no information is currently available about the phenotypic fate of any of the neural cells in the DM lineages. It is not known if the cells in these lineages are exclusively neuronal or if glial cells are also generated. Nor is it known if the neurons in these lineages are involved in the formation of specific complex neuropile structures or if they project widely throughout the brain. This total lack of information on the type of cells generated and their roles in brain circuitry thus represents a major obstacle in understanding the development of the fly brain (Izergina, 2009).
Three main findings are reported. First, DM lineages comprise both adult-specific neurons and glia; DM neuroblasts thus have features of neuroglioblasts. Second, during larval development, adult-specific neurons form complex secondary axon tracts composed of separate commissural and longitudinal fascicles. Third, during pupal development the commissural fascicles arborize in and contribute to the central complex neuropile. In the following, the major implications of these three findings are discussed (Izergina, 2009).
In Drosophila, as in other insects, glial cells fall into three classes, surface glia, cortex glia and neuropile glia, each of which is represented in the larval brain. The glial cells of the early larval brain, also referred to as primary glia, arise from a small number of embryonically active neuroglioblasts. Glial numbers in the brain increase during larval development and this increase in cell number is due, at least in part, to mitotic divisions of glial cells; however, the bulk of added (secondary) glial cells has been postulated to stem from the proliferation of unidentified secondary neuroglioblasts. However, the identity of these postulated multipotent precursors in the postembryonic brain was unknown (Izergina, 2009).
A recent developmental analysis by Awasaki (2008) has shown that among the different types of glial cells in the Drosophila brain, only the perineurial surface glia and the neuropile glia are extensively generated during postembryonic development, whereas most of the subperineurial surface glia and cortex glial cells are thought to have their origin in embryogenesis. Moreover, this analysis has provided evidence that perineurial glial precursors are distributed around the brain surface, whereas neuropile (ensheathing and astrocyte-like) glial cells are derived from specific proliferation centers within the brain. However, the progenitors of these postembryonically generated glial cells as well as the mode of postembryonic glial proliferation still remained elusive. This study has identified the first postembryonic neuroglioblasts in the Drosophila brain. (Embryonic neuroglioblasts have been described previously in the ventral thoracic ganglia.) All six DM lineages generate a set of glial cells with anatomical features of neuropile glia in addition to numerous neuronal cells during postembryonic development (Izergina, 2009).
DM neuroblasts proliferate through asymmetric division that involves intermediate progenitors with transit-amplifying cell features. This implies that DM lineage-derived glia, unlike any other glial cell type in Drosophila, are generated by amplifying intermediate progenitors. However, an exact clonal analysis of the relationship between glial cells and intermediate progenitors in DM lineages will be required to validate this notion. There is some evidence that DM derived glial are generated early in the lineage. If this is indeed the case, it is conceivable that these glial cells might be important for the differentiation of the subsequently generated neuronal cells in these lineages. For example, the extended processes of DM-derived glia located near the emergent secondary axon tracts or associated with the larval brain commissure might be important in guiding axons of DM-derived neurons (which subsequently contribute to central complex neuropile) across the midline (Izergina, 2009).
Dorsomedial lineage neurons form complex secondary axon projections
During postembryonic development, secondary, adult-specific neurons generated by reactivated neuroblasts produce secondary lineages and axons of a given secondary lineage fasciculate with each other to form a discrete and generally unbranched secondary axon tract within the brain cortex and neuropile. In contrast, in the case of DM secondary lineages, discrete, albeit short, secondary axon tracts were visible in the cortex but were rarely observed in the neuropile. Rather, within the neuropile, the axons of any given DM lineage split into multiple axon fascicles that projected to very different parts of the brain as commissural and longitudinal axon bundles. Thus, at the anatomical level, the DM lineage already appears to be subdivided into different neuronal subgroups with different outgrowth behaviors during the larval stages (Izergina, 2009).
The multiplicity of axonal bundles that emerge from a given DM lineage has features that are more reminiscent of a secondary axon tract system comprising the axon tracts of several lineages than a single secondary axon tract. This may be an indirect result of the fact that DM lineages contain three to five times more secondary neurons than do conventional neuroblast lineages, which, hence, would generate three to five times more axons than conventional lineages, resulting in an excessively large secondary axon tract in the neuropile if branching did not occur. While the underlying mechanisms are currently not known, it is also possible that this anatomical complexity is related to the particular mode of neurogenesis in the DM lineages, which involves amplifying intermediate progenitors. Thus, a given intermediate progenitor might produce neural progeny that develop a common type of axonal projection pattern, whereas progeny subsets derived from different intermediate progeny might develop different axonal projection types (Izergina, 2009).
Dorsomedial lineages contribute to the developing central complex
Like other adult-specific neurons, DM secondary neurons differentiate during the pupal period, when they evolve into the long tracts that characterize the adult brain and send out proximal as well as terminal arborizations that form synapse-rich neuropile circuitry. During the early pupal differentiation period, some of the neuropile structures that specifically characterize the adult brain become visible. Among these are the principle components of the central complex. The central complex, one of the most prominent neuropile structures in the adult brain, is located centrally between the two hemispheres and consists of four substructures. These are the protocerebral bridge, the fan-shaped body, the ellipsoid body, and the noduli, all of which are interconnected by sets of columnar interneurons that form regular patterns of projection. A remarkable and common feature of DM lineages is that a subset of the neurons in each lineage contributes to the developing central complex neuropile (Izergina, 2009).
In the late larval brain, distinguishing the four substructures of the adult central complex is difficult. However, a putative midline neuropile primordium of the central complex has been described in the late larval brain in the form of a large commissural neuropile domain, consisting of the DPC1 and trCM and probably part of the DPC2 commissures. All DM lineages project a subset of their axon bundles into the DPC1 during larval development. Based on their axonal projections (and the position of their cell body clusters in the central brain), the DM1-6 lineages are tentatively assigned to the DPMm1/2, DPMpm1, DPMpm2, CM4, and CM5, CM1/2 of Pereanu and Hartenstein (2006). Interestingly, in the DPC1, these commissural bundles defasciculated into smaller lattice-like projections similar to those observed in the developing central complex of the grasshopper, an insect with direct development (Izergina, 2009).
From the first day of pupal development onward, the major components of the fan-shaped body, ellipsoid body, noduli and protocerebral bridge can be clearly recognized. In the early pupal brain, DM derived commissural neurons have already contributed to the arbors in the fan-shaped body, nodulus and protocerebral bridge. Although they could not be identified individually, it is very likely that the central complex-innervating DM neurons in the pupal brain are the same neurons that projected commissural axon bundles into the DPC1 commissure of the larval brain. This observation supports the notion that the DPC1 is indeed part of the central complex primordium (Izergina, 2009).
A noteworthy feature of the DM-derived projections in the fan-shaped body is the fact that two major fascicles are formed that project in parallel into the fan-shaped body and form two separate arborization domains. This may reflect a contribution of a given DM lineage to a pair of the eight modular subdomains ('staves') that make up the fan-shaped body. A similar contribution of the DM-derived neurons to two sections ('glomeruli') of the protocerebral bridge is also likely, although single cell resolution will be required to resolve this (Izergina, 2009).
Most neurons of the central complex belong to one of two categories: large-field elements or small-field elements. A large-field neuron typically arborizes in only a single substructure and links it to one or two central brain regions outside the central complex. Small-field neurons, as a rule, connect small columnar domains of several substructures, and the majority of small-field cells are intrinsic to the central complex. In view of the specific arborization pattern of the DM-derived neurons in the fan-shaped body, noduli and protocerebral bridge, it is hypothesized that most of these neurons represent columnar small field elements of the central complex. However, it should be noted that only a subset of the neurons in any given DM lineage are likely to innervate the central complex; all DM lineages form longitudinal projections to other parts of the brain. Thus, unlike the lineages that give rise to the mushroom body intrinsic neurons, the neuronal progeny of the DM lineages are not dedicated to a single neuropile center. Rather, the unusually large number of neurons in these lineages appears to contribute to multiple, spatially separated neuropile areas in the developing brain (Izergina, 2009).
This study has used MARCM-based clonal analysis together with immunocytochemical labeling techniques to investigate the type and fate of cells generated in the DM lineages. With a combination of neuronal and glial cell markers it is shown that most of the cells in the DM lineages are neuronal but that glial cells are also generated in these lineages. The DM neuroblasts thus represent the first identified precursor cells in the fly brain that have neuroglioblast functions during postembryonic development. It was also show that the adult-specific neurons of each DM lineage form several spatially widely separated axonal fascicles, some of which project along larval brain commissural structures that are primordia of midline neuropile. By taking advantage of a specific Gal4 reporter line, DM-derived neuronal cells identified and followed into early pupal stages, and it was demonstrated that neurons of the DM lineages make a major contribution to the developing central complex, in that the numerous columnar elements are likely to be DM lineage-derived. These findings suggest that the amplification of proliferation that characterizes DM lineages may be an important requirement for generating the large number of neurons required in highly complex neuropile structures such as the central complex in the Drosophila brain (Izergina, 2009).
To ensure normal development and maintenance of homeostasis, the extensive developmental potential of stem cells must be functionally distinguished from the limited developmental potential of transit amplifying cells. Yet the mechanisms that restrict the developmental potential of transit amplifying cells are poorly understood. This study shows that the evolutionarily conserved transcription factor dFezf/Earmuff (Erm) functions cell-autonomously to maintain the restricted developmental potential of the intermediate neural progenitors generated by type II neuroblasts in Drosophila larval brains. Although erm mutant intermediate neural progenitors are correctly specified and show normal apical-basal cortical polarity, they can dedifferentiate back into a neuroblast state, functionally indistinguishable from normal type II neuroblasts. Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation. It is concluded that Erm dependence functionally distinguishes intermediate neural progenitors from neuroblasts in the Drosophila larval brain, balancing neurogenesis with stem cell maintenance (Weng, 2010).
Tissue development and homeostasis often require stem cells to
transiently expand the progenitor pool by producing transit
amplifying cells. Yet the developmental potential of transit amplifying
cells must be tightly restricted to ensure generation of
differentiated progeny and to prevent unrestrained proliferation
that might lead to tumorigenesis. Transit amplifying
cells are defined by their limited developmental capacity,
a feature specified during fate determination. It is unknown
whether an active mechanism is required to maintain restricted
developmental potential in transit amplifying cells after specification.
This study used intermediate neural progenitors (INPs) in
developing Drosophila larval brains as a genetic model to investigate
how restricted developmental potential is regulated in
transit amplifying cells (Weng, 2010).
A fly larval brain hemisphere contains eight type II neuroblasts
that undergo repeated asymmetric divisions to self-renew and to
generate immature INPs. Immature INPs are unstable in nature and are mitotically inactive, and they lack
the expression of Deadpan (Dpn) and Asense (Ase). Immature INPs commit to the INP fate through maturation, a differentiation process necessary for specification of the INP identity. INPs express Dpn and Ase, and
undergo 8-10 rounds of asymmetric divisions to self-renew
and to produce ganglion mother cells (GMCs) that typically
generate two neurons. While 5-6 immature
INPs and 1-2 young INPs are always in direct contact with their
parental neuroblasts, the older INPs become progressively displaced
from their parental neuroblasts over time (Weng, 2010).
During asymmetric divisions of type II neuroblasts, the basal
proteins Brain tumor and Numb are exclusively segregated into
immature INPs, and function cooperatively, but nonredundantly,
to ensure that immature INPs undergo maturation and
commit to the INP fate. brain tumor or numb mutant type II neuroblasts generate immature INPs that fail to mature and do not commit
to the INP fate. Instead, brain tumor or numb mutant immature
INPs adopt their parental neuroblast fate, leading to supernumerary
type II neuroblasts. Thus, brain tumor and numb specify
the INP fate, and the ectopic expansion of type II neuroblasts in
these mutant genetic backgrounds occurs due to failure to
properly specify the INP fate. Although Brain tumor is also
asymmetrically segregated into GMCs during asymmetric divisions
of INPs, the mosaic clones in brain tumor mutant INPs
contain only differentiated neurons. This result indicates that Brain tumor is dispensable for maintaining the restricted developmental potential of INPs. How
restricted developmental potential is maintained in INPs is
currently unknown (Weng, 2010).
To identify genes that regulate self-renewal of neuroblasts, a genetic screen was conducted for mutants exhibiting ectopic larval
brain neuroblasts. One
mutation, l(2)5138, specifically resulted in massive expansion of
neuroblasts in the brain but did not affect neuroblasts on the
ventral nerve cord. The l(2)5138
mutation mapped to the 22B4-7 chromosomal interval that contains the
earmuff (erm) gene (Pfeiffer, 2008). The erm transcripts
are first detected at embryonic stage 4-6 in the specific domain
preceding formation of the embryonic brain and remain highly
expressed in the brain throughout development. Tbis study reports that Erm
functions to restrict the developmental potential of INPs by
promoting Prospero-dependent termination of proliferation and
suppressing Notch-mediated dedifferentiation. By restricting
their developmental potential, Erm ensures that INPs generate
only differentiated neurons during Drosophila neurogenesis (Weng, 2010).
All neuroblasts in l(2)5138 homozygous mutant brains were
proliferative, expressed all known neuroblast markers, and
lacked neuronal and glial markers. The l(2)5138 mutation mapped
to the erm gene, which encodes a homolog of the vertebrate
Forebrain embryonic zinc-finger family (Fezf) transcription
factors.
The l(2)5138 mutants contained a single A/T nucleotide change
in the erm coding region, leading to the substitution of a leucine
for a conserved histidine in the third C2H2 zinc-finger domain. Consistent with its predicted molecular function, ectopic expression of Erm transgenic proteins tagged with a HA epitope at the amino- or carboxyl-terminus driven by
neuroblast-specific Wor-Gal4 was detected in the nuclei of neuroblasts. However, the expression of the HA-tagged
Erm transgenic protein bearing the identical leucine-to-histidine
substitution as in the l(2)5138 mutant was undetectable,
suggesting that the mutant Erm protein is unstable. It is concluded that l(2)5138 is a mutant allele of erm (Weng, 2010).
To determine whether erm mutant brains have ectopic type I
and/or type II neuroblasts, the expression pattern
was examined of Ase and Prospero (Pros), which are only expressed in type I
neuroblasts. It was found that erm mutant brains
contained over 20-fold more type II neuroblasts (Dpn+Ase-)
than wild-type brains, with no significant change in the number
of type I neuroblasts (Dpn+Ase+). Next, the localization of Prospero was examined in mitotic neuroblasts in
larval brains expressing GFP induced by Ase-Gal4 (Ase >
GFP), which mimicked the expression pattern of the endogenous
Ase protein. In erm mutant larval brains, all
mitotic type I neuroblasts (GFP+) showed formation of basal
Prospero crescents, but none of the mitotic type II neuroblasts
(GFP-) showed the expression of Prospero. Furthermore, GFP-marked erm mutant type II neuroblast
clones consistently contained multiple type II neuroblasts,
whereas erm mutant type I neuroblast clones always contained
single type I neuroblasts and neurons. It is concluded that erm mutant brains exhibit an abnormal expansion of type II neuroblasts (Weng, 2010).
To determine the cellular origin of ectopic type II neuroblasts in
erm mutant brains, the identity of cells in the
GFP-marked clones derived from wild-type or erm mutant type
II neuroblasts was examined using specific cell fate markers. At 30 hr after clone induction, wild-type and erm mutant neuroblast clones appeared
indistinguishable, containing single parental neuroblasts
(Dpn+Ase-; R10 mm) in direct contact with 5-6 immature INPs (Dpn-Ase-), while most of the INPs (Dpn+Ase+; R6 mm) were 1 cell or more away from the parental neuroblasts. At 48 hr after clone induction, the overall size of both
wild-type and erm mutant neuroblast clones increased significantly
due to an increase in cell number, reflecting continuous
asymmetric divisions of the parental neuroblasts. In both wildtype
and erm mutant clones, the parental neuroblasts remained
surrounded by 5-6 immature INPs, while INPs and differentiated
neurons (Dpn-Ase-Pros+) were found several cells away from the parental neuroblasts. However, erm mutant clones contained fewer INPs than the wild-type clones. Importantly, erm mutant clones consistently contained 4-6 smaller ectopic type II neuroblasts (Dpn+Ase-; 6-8 mm in diameter). Thus, Erm is dispensable for both the generation and maturation of immature INPs (Weng, 2010).
Ectopic type II neuroblasts in 48 hr erm mutant clones were
always several cells away from the parental neuroblasts. This result strongly suggests that ectopic type II neuroblasts in erm mutant clones likely originate from INPs and Erm likely functions in INPs. However, it was not possible to assess the spatial expression pattern of the endogenous Erm
protein in larval brains due to lack of a specific antibody and
low signals by fluorescent RNA in situ. Alternatively,
the expression of the R9D series of Gal4
transgenes was analyzed, in which Gal4 is expressed under the control of overlapping erm promoter fragments (Pfeiffer, 2008). The
expression of R9D11-Gal4 was clearly detected in INPs, but
was undetectable in type II neuroblasts and immature INPs
even when two copies of the UAS-mCD8-GFP transgenes
were driven by two copies of R9D11-Gal4 at 32°C for 72 hr after
larval hatching. Consistently, the expression
of Erm-Gal4 was virtually undetectable in brain tumor
mutant brains that contain thousands of type II neuroblasts
and immature INPs. While the expression of UAS-erm
induced by the neuroblast-specific Wor-Gal4 driver led to
premature loss of type II neuroblasts, expression of UAS-erm
driven by Erm-Gal4 failed to exert any effect on type II neuroblasts. Importantly, targeted expression of the fly Erm or mouse Fezf1 or Fezf2 transgenic protein driven by R9D11-Gal4 restored the function of Erm and efficiently rescued the ectopic neuroblast phenotype in erm mutant brains. Therefore, R9D11-Gal4 (Erm-Gal4) contains the enhancer element sufficient to restore the Erm function in INPs leading to suppression of ectopic type II neuroblasts in erm mutant brains (Weng, 2010).
Mutant clonal analyses and overexpression studies strongly
suggest that Erm functions to suppress reversion of INPs back
into a neuroblast state. This study directly tested whether INPs in
erm mutant brains can dedifferentiate back into type II neuroblasts.
βgal-marked lineage clones originating exclusively
from INPs were induced via FRT-mediated recombination. A short pulse of flipase (FLP) expression was targeted in INPs by heat-shocking
larvae carrying a UAS-flp transgene under the control of Erm-
Gal4 and tub-Gal80ts at 30°C for 1 hr. At 72 hr after heat shock, INP clones in wildtype brains contained only differentiated neurons (Dpn-Ase-). In contrast, INP clones in erm mutant brains contained one or more type II neuroblasts as well as immature
INPs, INPs, GMCs, and neurons. This result indicates
that while INPs in wild-type larval brains can give rise to only
neurons, INPs in erm mutant brains can dedifferentiate into type II
neuroblasts that can give rise to all cell types found in a normal
type II neuroblast lineage. It is concluded that Erm functions to
maintain the restricted developmental potential of INPs and prevents
them from dedifferentiating back into a neuroblast state (Weng, 2010).
To determine how Erm maintains the restricted developmental
potential of INPs, microarray analyses was performed, and prospero mRNA was found to be drastically reduced in erm mutant
brains compared to the control brains. It was confirmed that the relative level of prospero mRNA was indeed reduced by 60%-70% in erm mutant brain extracts by using real-time PCR. These data
supported that Erm is necessary for proper transcription of prospero,
and prompted a test to see if overexpression of Erm might be
sufficient to induce ectopic Prospero expression. A short pulse of Erm expression in brain neuroblasts was induced by shifting
larvae carrying a UAS-erm transgene under the control of Wor-
Gal4 and tub-Gal80ts to from 25°C to 30°C. A 3.5 hr pulse of
Erm expression was sufficient to induce nuclear localization of
Prospero in larval brain neuroblasts. Consistent
with nuclear Prospero promoting termination of neuroblast
proliferation, ectopic expression of Erm induced by Wor-Gal4
resulted in decreased neuroblasts compared to wild-type brains (Figure 5B). Thus, it is concluded that overexpression of Erm can restrict neuroblast proliferation by triggering nuclear localization of Pros (Weng, 2010).
The data suggest that Erm might restrict the developmental
potential of INPs in part by limiting their proliferation by activating
Prospero-dependent cell cycle exit. If so, it was predicted that overexpression
of Erm should induce ectopic nuclear Prospero in INPs
and overexpression of Prospero should suppress ectopic neuroblasts
in erm mutant brains. In wild-type brains, 9.6% of INPs
(32/325) showed nuclear localization of Prospero. However,
overexpression of Erm driven by Erm-Gal4 led to nuclear localization
of Prospero in 41.5% of INPs (105/253), likely restricting their
proliferation potential and resulting in some parental type II neuroblasts
surrounded only by differentiated neurons. Importantly, ectopic expression of Prospero induced
by Erm-Gal4 efficiently suppressed ectopic neuroblasts and
restored neuronal differentiation in erm mutant brains. Thus, Erm likely restricts the proliferation of INPs
by promoting nuclear localization of Prospero. To confirm that
Prospero indeed functions downstream of Erm to restrict the
proliferation of INPs, genetic epistatic analyses were performed.
Consistent with previously published results, prospero mutant
type I neuroblast clones contained ectopic type I neuroblasts. In contrast, prospero mutant
type II neuroblast clones exhibited accumulation of ectopic
INPs while maintaining single parental neuroblasts.
Furthermore, overexpression of Erm failed to suppress ectopic
INPs in prospero mutant type II neuroblast clones, consistent
with Prospero functioning downstream of Erm. These
results indicate that blocking differentiation is not sufficient to
trigger the dedifferentiation of INPs back into type II neuroblasts.
Thus, Erms restriction on the proliferation of INPs is dependent
on Prospero function, but its suppression of the dedifferentiation
of INPs is independent of Prospero (Weng, 2010).
Previous studies showed that overexpression of constitutively
active Notch (Notchintra) in both type I and II neuroblasts is sufficient
to trigger ectopic neuroblasts. This study tested whether Erm suppresses the dedifferentiation
of INPs by inhibiting Notch signaling. Indeed, knockdown
of Notch function by RNAi in erm mutant brains led to
a dramatic reduction in ectopic type II neuroblasts compared
to erm mutant brains alone. Complementarily,
ectopic expression of constitutively active Notch (Notchintra)
induced by Erm-Gal4 transforms INPs into ectopic type II neuroblasts. Thus, reduced Notch function suppresses the
dedifferentiation of INPs in erm mutant brains whereas ectopic
activation of Notch induces the dedifferentiation of INPs. Next, whether Erm suppresses the dedifferentiation of INPs by
antagonizing a Notch-activated mechanism was tested. Coexpression of
Erm under the control of Erm-Gal4 is sufficient to suppress
ectopic neuroblasts induced by the expression of Notchintra. Thus, it is concluded that Erm can suppress the dedifferentiation of INPs by negatively regulating a Notch-activated signaling mechanism (Weng, 2010).
This study has reported a mechanism that actively maintains the
restricted developmental potential of transit amplifying cells after
specification of their identity. The evolutionarily
conserved transcription factor Erm/Fezf functions to maintain
the restricted developmental potential of INPs by limiting their
proliferation potential and suppressing their dedifferentiation
capacity. Combining proper specification of the transit
amplifying cell identity and active maintenance of their restricted
developmental potential ensures the generation of differentiated
progeny and prevents aberrant expansion of stem cells (Weng, 2010).
The lineage clones derived from single INPs in erm1/erm2
mutant brains contain dedifferentiated neuroblasts, immature
INPs, INPs, GMCs, and neurons. Several mechanisms could lead to the diversity of cells
within the clones. First, INPs in erm mutant brains
might generate GMCs and neurons initially due to the
presence of maternally deposited Erm. However,
erm transcripts are undetectable in both adult male
and female germlines by microarray analyses and in
stage 1-3 embryos by RNA in situ. Furthermore, the erm1/erm2 allelic
combination resulted in little to no zygotic Erm in the brain
because the erm1 mutation likely leads to the production of an
unstable Erm protein, whereas the erm2 mutation deletes the
entire erm open reading frame. Additionally,
the ectopic neuroblast phenotype in erm1/erm2 mutant brains
can be observed as early as 36-48 hr after larval hatching. Thus, generation of GMCs and differentiated
neurons by INPs in erm1/erm2 mutant brains is unlikely
due to the maternal effect. Alternatively, erm may promote
GMC differentiation in the type II neuroblast lineage, and in
erm mutant brains, GMCs might dedifferentiate back into
neuroblasts. If so, an ectopic accumulation
of INPs would be predicted in similarly staged mosaic clones derived from erm
mutant type II neuroblasts as compared to wild-type clones.
However, 48 hr erm mutant single neuroblast clones consistently
contained fewer INPs when compared to the wild-type clones. In addition, blocking GMC differentiation by
removing Prospero function resulted in ectopic accumulation
of INPs but did not lead to ectopic neuroblast formation. Therefore, the diversity of cells within erm mutant
clones is also unlikely due to blocking GMC differentiation. The interpretation is favored that erm mutant INPs dedifferentiate
into apparently normal neuroblasts that can give rise to all cell
types found in a type II neuroblast lineage. Consistently, the
dedifferentiated neuroblasts in erm mutant brains exhibited
normal cortical polarity and proliferation potential. Furthermore, the dedifferentiated neuroblasts in erm
mutant brains also lost the expression of Pros-Gal4 and
Erm-Gal4 and established ectopic type II neuroblast lineages
encapsulated by the cortex glial membrane.Thus, it is concluded that Erm likely restricts the developmental
potential of INPs by limiting proliferation and suppressing
dedifferentiation (Weng, 2010).
Although mutations in erm, brain tumor, and numb genes all
lead to ectopic type II neuroblasts, the proteins appear to regulate
INPs at distinct steps in the type II neuroblast lineage. Numb and Brain tumor function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate (Boone, 2008; Bowman, 2008). While ectopic expression of Numb induces premature
differentiation of type II neuroblasts and immature INPs, overexpression
of Numb is not sufficient to suppress ectopic neuroblasts in
brain tumor mutant brains. Thus, Numb likely promotes differentiation of immature INPs whereas Brain tumor likely prevents immature INPs, which
are unstable in nature, from adopting their parental neuroblast
fate. More studies will be necessary to discern whether ectopic
neuroblasts in brain tumor mutant brains arise from dedifferentiation
of partially differentiated immature INPs or failure of
immature INPs to initiate differentiation. In contrast, immature
INPs in erm mutant brains mature into functional INPs that
exhibit normal cortical polarity and proliferation potential and
can generate GMCs and neurons. Additionally, overexpression of Brain tumor or Numb in INPs was not sufficient to suppress ectopic neuroblasts in erm
mutant brains. Finally, lineage clones derived
from single INPs in erm mutant brains always contain ectopic
type II neuroblasts, multiple immature INPs, INPs, GMCs, and
neurons. These results indicate that Erm is
dispensable for maturation of immature INPs and is not within
the genetic hierarchy specifying the INP identity. Instead, Erm
maintains the restricted developmental potential of INPs after
specification of their identity (Weng, 2010).
Prospero encodes a homeodomain transcription factor, and
nuclear Prospero has been shown to trigger cell cycle exit and
GMC differentiation. In the wild-type brain, 9.6% of INPs showed
nuclear Prospero and were likely undergoing differentiation. prospero mutant type II neuroblast clones
showed ectopic accumulation of INPs but contained single neuroblasts,
indicating that blocking differentiation is not sufficient
to trigger the dedifferentiation of INPs. Thus, Prospero
restricts the proliferation potential of INPs but does not
suppress dedifferentiation of INPs (Weng, 2010).
While ectopic expression of Prospero in INPs can restore
neuronal differentiation in erm mutant brains, targeted expression
of Erm in neuroblasts or INPs was sufficient to induce rapid
nuclear localization of Prospero in these cells and terminate their
proliferation. In wild-type brains, Prospero is sequestered
in a basal crescent by the adaptor protein Miranda
in mitotic neural progenitors. Interestingly, mitotic neural progenitors
including neuroblasts and INPs transiently overexpressing Erm
also showed basal localization and segregation of Miranda
and Prospero. As such, Erm likely restricts
the proliferation potential of INPs by indirectly promoting nuclear
localization of Prospero. Therefore, Prospero does not localize
in the nuclei of mitotically active INPs, which express Miranda, but does localize in the nuclei of GMCs that do not express Miranda (Weng, 2010).
How does Erm suppress the dedifferentiation of INPs? The
results show that reduced Notch function can efficiently
suppress ectopic neuroblasts in erm mutant brains while
constitutive activation of Notch signaling induced the dedifferentiation
of INPs. Importantly, coexpression
of Erm is sufficient to suppress the dedifferentiation of INPs
triggered by expression of constitutively active Notchintra. Together, these results strongly suggest that Erm
prevents the dedifferentiation of INPs by antagonizing a
Notch-activated mechanism through interfering with the
assembly of the Notch transcriptional activator complex or inhibiting
the expression of Notch targets. Intriguingly, the amino
terminus of all Fezf proteins contains an engrailed homology 1
domain. This domain can mediate direct interaction with the
conserved transcriptional corepressor Groucho that can function
as a corepressor of Notch signaling. Additional experiments
will be needed to discern how Erm antagonizes Notch-activated
dedifferentiation of INPs (Weng, 2010).
The number of neurons generated by neural stem cells is dependent upon the regulation of cell proliferation and by programmed cell death. Recently, novel neural stem cells that amplify neural proliferation through intermediate neural progenitors, called type II neuroblasts, have been discovered, which are active during brain development in Drosophila. This study investigated programmed cell death in the dorsomedial (DM) amplifying type II lineages that contribute neurons to the development of the central complex in Drosophila, using clonal mosaic analysis with a repressible cell marker (MARCM) and lineage-tracing techniques. A significant number of the adult-specific neurons generated in these DM lineages were eliminated by programmed cell death. Programmed cell death occurred during both larval and pupal stages. During larval development, approximately one-quarter of the neuronal (but not glial) cells in the lineages were eliminated by apoptosis before the formation of synaptic connectivity during pupal stages. Lineage-tracing experiments documented the extensive contribution of intermediate neural progenitor-containing DM lineages to all of the major modular substructures of the adult central complex. Moreover, blockage of apoptotic cell death specifically in these lineages led to prominent innervation defects of DM-derived neural progeny in the major neuropile substructures of the adult central complex. These findings indicate that significant neural overproliferation occurs normally in type II DM lineage development, and that elimination of excess neurons in these lineages through programmed cell death is required for the formation of correct neuropile innervation in the developing central complex. Thus, amplification of neuronal proliferation through intermediate progenitors and reduction of neuronal number through programmed cell death operate in concert in type II neural stem-cell lineages during brain development (Jiang, 2012).
The insect central complex is an intricate, high-level, multimodal information- storage and -processing center that is involved in motor coordination of behaviors such as walking, flying and stridulation. In neuroanatomical terms, it represents a highly complex midline neuropile made up of several thousands of neurons, corresponding to approximately 50 cell types that are arranged in a highly ordered architecture. Remarkably, the large number of neurons that contribute to the central complex is generated by a small number of neuroblasts during a short developmental time period. Indeed, the amplification of DM neuroblast proliferation through INPs, which act as transient amplifying cells, may be an important mechanistic prerequisite for rapidly generating the large number of neurons required for complex central circuitry. Comparable amplification of neural stem-cell proliferation through transit amplifying cells is present in the developing brains of vertebrates including mammals. Notably, in mammalian cortical development, the wealth of neurons required for complex circuitry is largely generated by neural stem cells via INP-like progenitor cells called basal progenitors. Given that this type of amplification of neural proliferation may be a general strategy for increasing the size and complexity of the brain, a concurrent and potentially counter-balancing role of programmed cell death in neural stem-cell lineages may also be a general and evolutionarily conserved mechanism for generating brain complexity (Jiang, 2012).
Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. This study used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. The transcription factor Klumpfuss (Klu) was identified as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led to a proposal that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division (Xiao, 2012).
Asymmetric stem cell division provides an efficient mechanism to preserve a steady stem cell pool while generating differentiated progeny within the tissue where the stem cells reside. Precise spatial control of the stem cell determinants inherited by both sibling cells in every asymmetric cell division ensures that a daughter cell maintains the stem cell characteristics while the sibling progeny acquires the progenitor cell identity. In mitotic type II neuroblasts, the basal proteins Brat and Numb segregate into immature INPs and are required for the formation of INPs. This study significantly extends the findings from previous studies and showed that Brat and Numb function in immature INPs to prevent them from acquiring a neuroblast fate while promoting the INP identity. Identification and characterization of the klu gene led to a proposal that Brat and Numb converge to exert precise control of Klu to distinguish an immature INP from its sibling type II neuroblast. Numb also prevents a GMC from reverting to a type I neuroblast by inhibiting Notch signaling in the type I neuroblast lineage. Interestingly, although overexpression of klu was insufficient to induce supernumerary type I neuroblasts, increased function of klu can drastically enhance the reversion of GMCs to type I neuroblasts in the presence of activated Notch signaling. Thus, it is proposed that aberrant activation of Notch signaling induces reversion of GMCs by activating multiple downstream genes including klu. Together, these data lead to the conclusion that precise regulation of klu by multiple signaling mechanisms distinguishes a progenitor cell from its sibling stem cell during asymmetric stem cell division (Xiao, 2012).
The essential role of Brat and Numb in regulating the formation of INPs is well established, but lack of insight into maturation has hindered investigation into the mechanisms by which these two proteins distinguish an immature INP from its sibling type II neuroblast. A previous study defined immature INPs by the following criteria: (1) being immediately adjacent to the parental type II neuroblast, (2) lacking Dpn expression and (3) displaying a very low level of CycE expression. Based on these criteria, analyses of the spatial expression pattern of various cell fate markers in the type II neuroblast lineage clones in wild-type brains revealed that onset of Ase expression correlates with an intermediate stage of maturation. In 16-hour clones, one type II neuroblast (Dpn+ Ase- CycE+), two to three Ase- immature INPs (Dpn- Ase- CycE-), two to three Ase+ immature INPs (Dpn- Ase+ CycE-) and INPs (Dpn+ Ase+ CycE+) were reproducibly observed. Furthermore, it was shown that Ase- immature INPs maintain expression of the type II neuroblast-specific marker PntP1, whereas Ase+ immature INPs showed virtually undetectable PntP1 expression. Thus, onset of Ase expression should serve as a useful marker for an intermediate stage during maturation (Xiao, 2012).
The data lead to a proposal that Brat distinguishes an immature INP from its sibling type II neuroblast by indirectly antagonizing the function of Klu based on the following evidence. First, Klu was undetectable in Ase− immature INPs in the brat single-mutant or brat and numb double-mutant type II neuroblast clones. Thus, a Brat-independent mechanism must exist to downregulate Klu in immature INPs. Second, overexpression of a truncated Brat transgenic protein lacking the NHL domain, which is required for repression of mRNA translation, completely suppresses the formation of supernumerary neuroblasts. Thus, it is unlikely that downregulation of Klu in immature INPs occurs via a Brat-dependent translational repression of klu mRNA. It is proposed that Brat might suppress the expression of a co-factor necessary for the function of Klu, just as WT1 requires co-factors in order to regulate the expression of its target genes in vertebrates (Roberts, 2005). Further investigation will be necessary to discern how Brat establishes restricted developmental potential in immature INPs by antagonizing the function of Klu (Xiao, 2012).
WT1 requires its zinc-finger motifs to regulate transcription of its target genes and can function as an activator or a repressor of transcription in a context-dependent manner (Roberts, 2005). A previous study showed that overexpression of Klu can partially suppress the expression of a lacZ reporter transgene containing the cis-regulatory elements from the even-skipped gene, a putative direct target of Klu, in the fly embryonic central nervous system. Since Klu and WT1 display extensive homology in zinc-fingers 2-4, Klu is likely to recognize a similar DNA binding sequence as WT1. The even-skipped cis-regulatory element contains three putative WT1 binding sites, but nucleotide substitutions in these sites that were predicted to abolish Klu binding failed to render the lacZ reporter transgene unresponsive to overexpression of klu. These data led to a speculation that Klu might recognize a distinct consensus DNA binding sequence to WT1. To test this hypothesis, two UAS-WT1 transgenes were generated that encode the two most prevalent isoforms of the WT1 protein, WT1 −KTS and WT1 +KTS. Interestingly, neither WT1 transgene, when overexpressed by wor-GAL4, triggered the formation of supernumerary type II neuroblasts in larval brain. This is consistent with Klu recognizing a distinct consensus DNA binding sequence to WT1. However, it cannot be ruled out that the inability of the WT1 transgenic protein to induce supernumerary type II neuroblasts is simply due to the absence of necessary co-factors in the fly, as repression of target gene transcription by WT1 requires additional co-factors in vertebrates. More studies will be necessary to elucidate the molecular function of Klu in promoting type II neuroblast identity (Xiao, 2012).
Restricted developmental potential functionally defines progenitor cells and allows them to generate differentiated progeny through limited rounds of cell division without impinging on the homeostatic state of the stem cell pool. Despite their importance, the molecular mechanisms by which progenitor cells acquire restricted developmental potential remain experimentally inaccessible in most stem cell lineages. However, studies from various groups have paved the way for using fly larval brain neuroblast lineages as an in vivo model system for investigating how progenitor cells acquire restricted developmental potential (Xiao, 2012).
This study describes the expression pattern of additional molecular markers that allow unambiguous identification of two distinct populations of immature INPs. Furthermore, experimental evidence is provided strongly suggesting that these two groups of immature INPs possess distinct functional properties. More specifically, Ase- immature INPs readily revert to type II neuroblasts in response to misexpression of Klu, whereas Ase+ immature INPs appear much less responsive to Klu. These data lead to a proposal that the genome in immature INPs becomes reprogrammed during maturation such that these cells become progressively less responsive to neuroblast fate determinants such as Klu. As a consequence, an INP becomes completely unresponsive to Klu following maturation. Further experiments will be required to validate this model in the future (Xiao, 2012).
Prefoldin is a molecular chaperone complex that regulates tubulin function in mitosis. This study shows that Prefoldin depletion results in disruption of neuroblast polarity, leading to neuroblast overgrowth in Drosophila larval brains. Interestingly, co-depletion of Prefoldin and Partner of Inscuteable (Pins) leads to the formation of gigantic brains with severe neuroblast overgrowth, despite that Pins depletion alone results in smaller brains with partially disrupted neuroblast polarity. This study shows that Prefoldin acts synergistically with Pins to regulate asymmetric division of both neuroblasts and Intermediate Neural Progenitors (INPs). Surprisingly, co-depletion of Prefoldin and Pins also induces dedifferentiation of INPs back into neuroblasts, while depletion either Prefoldin or Pins alone is insufficient to do so. Furthermore, knocking down either α-tubulin or β-tubulin in pins- mutant background results in INP dedifferentiation back into neuroblasts, leading to the formation of ectopic neuroblasts. Overexpression of α-tubulin suppresses neuroblast overgrowth observed in prefoldin pins double mutant brains. These data elucidate an unexpected function of Prefoldin and Pins in synergistically suppressing dedifferentiation of INPs back into neural stem cells (Zhang, 2016).
Control of tissue homeostasis is a central issue during development. The neural stem cells, or neuroblasts, of the Drosophila larval brain is an excellent model for studying stem cell homeostasis. Asymmetric division of neuroblasts generates a self-renewing neuroblast and a different daughter cell that undergoes differentiation pathway to produce neurons or glia. Following each asymmetric division, apical proteins such as aPKC are segregated into the neuroblast daughter and function as 'proliferation factor', while basal proteins are segregated into a smaller daughter cell to act as 'differentiation factors'. At the onset of mitosis, the Partitioning defective (Par) protein complex that is composed of Bazooka (Baz)/Par3, Par6 and atypical protein kinase C (aPKC) is asymmetrically localized at the apical cortex of the neuroblast. Other apical proteins including Partner of Inscuteable (Pins), the heterotrimeric G protein Gαi, and Mushroom body defect (Mud) also accumulate at the apical cortex through an interaction of Inscuteable (Insc) with Par protein complex. Apical proteins control basal localization of cell fate determinants Numb, Prospero (Pros), Brain tumor (Brat) and their adaptor proteins Miranda (Mira) and Partner of Numb (Pon) that are segregated into the ganglion mother cell (GMC) following divisions. Apical proteins and their regulators also control mitotic spindle orientation to ensure correct asymmetric protein segregation at telophase. Several centrosomal proteins, Aurora A, Polo and Centrosomin, regulate mitotic spindle orientation (Zhang, 2016).
There are at least two different types of neuroblasts that undergo asymmetric division in the larval central brain. Perturbation of asymmetric division in either type of neuroblast can trigger neuroblast overproliferation and/or the induction of brain tumors. The majority of neuroblasts are type I neuroblasts that generate a neuroblast and a GMC in each division, while type II neuroblasts generate a neuroblast and an intermediate neural progenitor (INP), which undergoes three to five rounds of asymmetric division to produce GMCs. Ets transcription factor Pointed (PntP1 isoform), exclusively expressed in type II neuroblast lineages, promotes the formation of INPs. Failure to restrict the self-renewal potential of INPs can lead to dedifferentiation, allowing INPs to revert back into 'ectopic neuroblasts'. Notch antagonist Numb and Brat function cooperatively to promote the INP fate. Loss of brat or numb leads to 'ectopic type II neuroblasts' originating from uncommitted immature INPs that failed to undergo maturation. A zinc-finger transcription factor Earmuff functions after Brat and Numb in immature INPs to prevent their dedifferentiation. Earmuff also associates with Brahma and HDAC3, which are involved in chromatin remodeling, to prevent INP dedifferentiation. However, the underlying mechanism by which INPs possess limited developmental potential is largely unknown (Zhang, 2016).
Prefoldin (Pfdn) was first identified as a hetero-hexameric chaperone consisting of two α-like (PFDN3 and 5) and four β-like (PFDN 1, 2, 4 and 6) subunits, based on its ability to capture unfolded actin (Vainberg, 1998). Prefoldin promotes folding of proteins such as tubulin and actin by binding specifically to cytosolic chaperonin containing TCP-1 (CCT) and by directing target proteins to it. The yeast homologs of Prefoldin 2–6, named GIM1-5 (genes involved in microtubule biogenesis) are present in a complex that facilitates proper folding of α-tubulin and γ-tubulin. All Prefoldin subunits are phylogenetically conserved from Archaea to Eukarya. Structural study of the Prefoldin hexamer from the archaeum M. thermoautotrophicum showed that Prefoldin forms a jellyfish-like shape consisting of a double β barrel assembly with six long tentacle-like coiled coils that participate in substrate binding. The function of Prefoldin as a chaperone has also been illustrated in lower eukaryotes like C. elegans, in which loss of prefoldin resulted in defects in cell division due to reduced microtubule growth rate. Depletion of PFDN1 in mice displayed cytoskeleton-related defects, including neuronal loss and lymphocyte development defects. The only Prefoldin subunit in Drosophila that has been characterized to date, Merry-go-round (Mgr), the Pfdn3 subunit, cooperates with the tumor suppressor Von Hippel Lindau (VHL) to regulate tubulin stability (Delgehyr, 2012). However, the functions of Prefoldin in the nervous system remain elusive (Zhang, 2016).
This study describes the critical role of evolutionarily-conserved Prefoldin complex in regulating neuroblast and INP asymmetric division and suppressing INP dedifferentiation. Mutants for two Prefoldin subunits, Mgr and Pfdn2, displayed neuroblast overgrowth with defects in cortical polarity of Par proteins and microtubule-related abnormalities. Interestingly, co-depletion of Pins in mgr or pfdn2 mutants led to massive neuroblast overgrowth. Prefoldin and Pins synergistically regulate asymmetric division of both neuroblasts and INPs. Surprisingly, they also synergistically suppress dedifferentiation of INPs back into neuroblasts. Knocking down tubulins in pins mutant background resulted in severe neuroblasts overgrowth, mimicking that caused by co-depletion of Prefoldin and Pins. These data provide a new mechanism by which Prefoldin and Pins regulates neural stem cell homeostasis through regulating tubulin stability in both neuroblasts and INPs (Zhang, 2016).
pfdn2/CG6302, encoding a Prefoldin β-like subunit, was identified from a RNA interference (RNAi) screen in larval brains. Ectopic neuroblasts labeled by a neuroblast marker, Deadpan (Dpn), were formed upon knocking down pfdn2 under a neuroblast driver insc-Gal4. Only one neuroblast was observed in control type I neuroblast lineages using insc-Gal4 and type II neuroblast lineages using worniu-Gal4 with asense (ase)-Gal80. In contrast, upon pfdn2 RNAi excess neuroblasts were observed in both type I neuroblast lineages and type II neuroblast lineages, respectively. To verify the function of Pfdn2 in neuroblasts, a putative hypomorphic allele of pfdn2, pfdn201239, was analyzed that has a P element inserted at the 5′ untranslated region (UTR) of pfdn2. Hemizygous larval brains of pfdn201239 over Df(3L)BSC457 (referred to as pfdn2− thereafter) displayed 235.3 ± 31.7 neuroblasts per brain hemisphere, suggesting that Pfdn2 inhibits the formation of ectopic neuroblasts in larval brains. Consistently, an increase of EdU (5-ethynyl-2′-deoxyuridine)-incorporation was also observed in pfdn2− mutants compared to the control. To generate pfdn2 null alleles, a P element, EY06124, was mobilized. Its imprecise excision yielded two loss-of-function alleles, pfdn2Δ10 and pfdn2Δ17, both deleting the entire opening reading frame (ORF) of pfdn2. pfdn2Δ10 and pfdn2Δ17 mutants survive to pupal stage and display strong phenotypes with ectopic neuroblasts labeled by Dpn. These phenotypes in pfdn2Δ10 and pfdn2Δ17 mutant brains can be fully rescued by overexpression of wild-type pfdn2 or pfdn2-Venus transgene. Pfdn2 is abundantly expressed in neuroblasts, INPs and their immediate neural progeny- GMCs, detected by a specific antibody generated against Pfdn2 full length and a transgenic Pfdn2 with a Venus tag at the C-terminus. In addition, Pfdn2 expression under the tubulin-Gal4 fully rescued the lethality of both pfdn2Δ10 and pfdn2Δ17 mutants. Pfdn2 protein was undetectable in pfdn2Δ10 zygotic mutants, further supporting that it is a null allele. Both type I and type II MARCM (Mosaic Analysis with Repressible Cell Marker) clones of pfdn2Δ10 generated excess neuroblasts. These phenotypes were slightly weaker than pfdn2Δ10 zygotic mutants, likely due to residual Pfdn2 protein in the clones. These data indicate that Pfdn2 is required in both type I and type II neuroblast lineages to prevent the formation of ectopic neuroblasts (Zhang, 2016).
This study has identified an unexpected synergism between Prefoldin and Pins in suppressing neuroblasts overgrowth. Barious subunits of Prefoldin complex are implicated in asymmetric division of neuroblasts, especially during asymmetric protein segregation at telophase. It is known that depletion of Pins results in the formation of smaller larval brains, despite partial loss of neuroblasts polarity. Interestingly, co-depletion of Pfdn2 and Pins results in severe neuroblasts overgrowth, while Pfdn2 depletion alone only causes mild brain overgrowth. This phenotype is contributed by a combination of loss of neuroblast polarity, defects of asymmetric division of INPs, as well as INP dedifferentiation. Knocking down tubulins in pins mutant background mimics the co-depletion of Prefoldin and Pins, suggesting that tubulin stability appears to be critical for the suppression of neuroblast overgrowth in the absence of Pins function. The data also suggest that Prefoldin function and tubulin stability in INPs are important to suppress their dedifferentiation back into neuroblasts (Zhang, 2016).
How microtubules induce cortical polarity is poorly understood in Drosophila neuroblasts. Previously, one report showed that kinesin Khc-73, which localized at the plus end of astral microtubules, and Discs large (Dlg) induced cortical polarization of Pins/Gαi in neuroblasts. However, microtubules are considered not essential for neuroblast polarity. This study shows that Drosophila Prefoldin regulates asymmetric division of both neuroblasts and INPs through tubulins, suggesting an important role of microtubules in neuroblast polarity. The essential role of microtubules directly regulating cell polarity is found in various systems. During C. elegans meiosis, a microtubule-organizing center is necessary and sufficient for the establishment of the anterior-posterior polarity. In the fission yeast Schizosaccharomyces pombe, interphase microtubules directly regulate cell polarity through proteins such as tea1p. In mammalian airway cilia, microtubules are required for asymmetric localization of planer cell polarity proteins (Zhang, 2016).
This study shows that the role of Drosophila Prefoldin complex in regulating asymmetric division is very likely dependent on microtubules. This is consistent with the known essential role of Prefoldin for maintaining tubulin levels in various organisms such as yeast, C. elegans, plants and mammals. In yeast, Gim (Prefoldin) null mutants become super-sensitive to the microtubule-depolymerizing drug benomyl as a result of a reduced level of α-tubulin. In the absence of Prefoldin, the function of the chaperone pathway is damaged and unable to fold sufficient amount of tubulins for normal yeast growth. In C. elegans, reducing Prefoldin function causes defects in cell division presumably due to the reduction of tubulin levels and microtubule growth rate. Genetic analysis of mammalian Prefoldin also suggests that cytoskeletal proteins like actin and tubulin make up the major substrate of Prefoldin in mammals. These studies in different organisms together suggest that Prefoldin complex plays a conserved central role in tubulin folding (Zhang, 2016).
'Telophase rescue', a term refers to the phenomenon that protein mis-localization at metaphase is completely restored at telophase, is observed in many mutants that affect neuroblast asymmetric division. However, both apical and basal proteins are still mis-segregated in pfdn2 and mgr mutants, suggesting that 'telophase rescue' is defective in these mutants. Telophase rescue is regulated by TNF receptor-associated factor (DTRAF1), which binds to Baz and acts downstream of Egr/TNF. Telophase rescue also depends on Worniu/Escargot/Snail family proteins and a microtubule-dependent Khc-73/Dlg pathway. Pins did not form a protein complex with Mgr, α-tubulin or β-tubulin in co-immunoprecipitation assay. Given that Dlg is a Pins-interacting protein, Prefoldin appears to function in a different pathway with Dlg or Khc-73 during asymmetric division (Zhang, 2016).
Recently, merry-go-round (mgr), encoding Prefoldin 3 (Pfdn3)/VBP1/Gim2 subunit, was reported to regulate spindle assembly. Loss of mgr led to formation of monopolar mitotic spindles and loss of centrosomes because of improper folding and destabilization of tubulins. The current analysis on Pfdn2 indicates that pfdn2 mutants displayed similar spindle and centrosome abnormalities. In addition, the incorrectly folded tubulin due to loss of mgr may be eliminated by Drosophila von Hippel Lindau protein (Vhl), an E3 ubiquitin-protein ligase. Interestingly, the data suggest that Prefoldin has a tumor-suppressor like function in preventing neuroblast overgrowth. However, Drosophila Vhl is not important for brain tumor suppression, as its loss-of-function neither affects number of neuroblasts nor suppresses overgrowth observed in pfdn2 RNAi or mgr RNAi (Zhang, 2016).
This study shows a novel synergism between Prefoldin and Pins in suppressing dedifferentiation of INPs back into neuroblasts. Prefoldin and Pins apparently suppress dedifferentiation through regulating tubulin levels. It is likely that appropriate tubulin levels in INPs are important for their differentiation, while reducing tubulin levels can increase the risk of INP dedifferentiation. Currently, several cell fate determinants such as Brat, Numb and the SWI/SNF chromatin remodeling complex with its cofactors Erm and Hdac3 are critical to suppress INP dedifferentiation back into neuroblast. It is currently unknown whether or how Prefoldin/Pins are linked to these known suppressors of dedifferentiation. It is possible that symmetric division of INPs causes reduced levels of Brat and Numb in these abnormal INP daughters, leading to their dedifferentiation. Alternatively, Prefoldin might regulate transcription of genes within INPs to suppress dedifferentiation. It was reported that the human homolog of Pfdn5, MM-1, has a role in transcriptional regulation by binding to the E-box domain of c-Myc and represses E-box-dependent transcriptional activity. Interestingly, Prefoldin Subunit 5 gene is deleted in Canine mammary tumors, suggesting that it may be a tumor suppressor gene. This study has revealed a novel mechanism by which Prefoldin and Pins function through tubulin stability to suppress stem cell overgrowth. It is expected to contribute to the understanding of mammalian/human Prefoldin function in tumorigenesis (Zhang, 2016).
During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. This study reports that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification (Komori, 2013).
Asymmetric stem cell division provides an efficient mechanism to
simultaneously self-renew a stem cell and to generate a progenitor
cell that produces differentiated progeny. Because self-renewal
proteins segregate into both daughter progeny of the dividing
parental stem cell through the inheritance of its cytoplasmic content,
rapidly downregulating the activity of these proteins is essential for
the specification of progenitor cell identity. Brat plays a central role
in specifying INP identity in the Ase- immature INP by antagonizing
the function of the self-renewal transcription factor Klu (Xiao,
2012). These previous findings have been extended to show that Brat
specifies INP identity in the Ase- immature INP through two
separable, but convergent, mechanisms. A novel Brat-dependent
mode of Wnt pathway regulation was identified that prevents Ase-
immature INPs from reverting into supernumerary neuroblasts.
Brat specifies INP identity by attenuating the transcriptional activity of Arm through its N-terminal B-boxes. This negative regulation of Arm is achieved through the activity
of Apc2 and the destruction complex. Because increased
arm function alone is insufficient to induce supernumerary
neuroblasts, the ability of Wnt signaling to promote
neuroblast identity is dependent on other signaling mechanisms that
act downstream of Brat. Indeed, Arm function is essential for Klu
to induce supernumerary neuroblasts. These two Brat-regulated
mechanisms function to safeguard against the accidental reversion
of an uncommitted progenitor cell into a supernumerary stem cell
and to ensure that an uncommitted progenitor cell can only adopt
progenitor cell identity (Komori, 2013).
Physical interaction with the cargo-binding domain of Mira is
essential for the unequal segregation of Brat into the immature INP
following the asymmetric division of neuroblasts. Previous studies concluded that the NHL
domain of Brat directly interacts with the cargo-binding domain of
Mira, but the roles of the B-boxes and the coiled-coil domain in the
asymmetric segregation of Brat were unknown due to a lack of
specific mutant alleles. By combining a yeast two-hybrid interaction
assay and in vivo functional validation, it is concluded that both the
coiled-coil domain and the NHL domain are indeed required for the
asymmetric segregation of Brat into the Ase- immature INP
following the asymmetric division of neuroblasts. It is
speculated that the coiled-coil domain and the NHL domain of Brat
function cooperatively to provide a more stable binding platform for
Mira to ensure efficient protein segregation (Komori, 2013).
The severity of the supernumerary neuroblast phenotype in various
brat mutant allelic combinations correlates with the level of
endogenous brat inherited by the Ase- immature INP. The brat DG19310
mutation carries a transposable P-element inserted in the 5′ regulatory
region of the brat gene. The brat11 mutation, however, results in a premature stop codon at amino acid 779, leading to a truncated form of the protein that lacks
most of the NHL domain and is predicted to be unable to interact with
Mira. The
brat DG19310 or brat DG19310/11allelic combination most likely reduces Brat expression without affecting its binding to Mira. Thus, the
minimal threshold of Brat necessary for the proper specification of
INP identity in Ase- immature INPs is met most of the time, leading
to a mild supernumerary neuroblast phenotype in brat DG19310 or
brat DG19310/11 brains. By contrast,
the brat11 homozygous or brat11/Df mutant allelic combination impairs
the binding of Brat to Mira, rendering the Mira-based asymmetric
protein-sorting mechanism unable to segregate Brat into the Ase-
immature INP. As such, the threshold of Brat necessary for proper
specification of INP identity in Ase- immature INPs is rarely met,
leading to a severe supernumerary neuroblast phenotype in brat11 or
brat11/Df brains. Overexpression of the bratΔC-coil or
bratΔNHL transgene using the UAS/Gal4 system almost certainly results
in an abnormally high level of the transgenic protein in the cytoplasm
of neuroblasts. Thus, inheriting a portion of the neuroblast cytoplasm
containing an overwhelming abundance of the mutant transgenic
protein is likely to be sufficient to reach the threshold of Brat
necessary for proper specification of INP identity in Ase- immature
INPs. It is concluded that the mechanism that causes Brat to
asymmetrically segregate into the Ase- immature INP is functionally
separable from the mechanism that specifies INP identity (Komori, 2013).
Could the asymmetric protein segregation mechanism promote
the specification of INP identity by depleting Brat from the
neuroblast? Type II neuroblasts overexpressing brat, bratΔC-coil or
brat<ΔNHL maintained their identity and generated similar numbers of
progeny as wild-type control neuroblast. Thus, it is unlikely that Brat-dependent specification of
INP identity occurs through asymmetric depletion of Brat from the
neuroblast. Whether Brat acts redundantly with other
asymmetrically segregating determinants to specify INP identity in
Ase- immature INPs was also tested. Numb also exclusively segregates into the
immature INP during asymmetric divisions of type II neuroblasts. However, asymmetric segregation of Numb
is not dependent on Brat, and Numb-dependent specification of INP
identity also occurs independently of Brat. Thus, it is unlikely that Brat acts redundantly with other
asymmetric segregating determinants to specify INP identity in Ase- immature INPs (Komori, 2013).
A surprising finding revealed by the current study is that the B-boxes
are uniquely required for the specification of INP identity.
This raises a series of interesting questions. What are the roles of B-boxes
in the function of Brat in embryonic neuroblasts? Embryos
lacking both maternal and zygotic function of brat often lack RP2
neurons but never possess supernumerary neuroblasts. Since brat mutant alleles that specifically affect the function of B-boxes are unavailable, the roles of B-boxes in the
function of Brat during the asymmetric division of embryonic
neuroblasts remain unknown. Brat regulates embryonic pattern
formation by repressing mRNA translation through the ternary
complex that also contains Nanos and Pumilio. However, it is unlikely that Brat specifies INP
identity through the Nanos-Pumilio-Brat translational repression
complex for the following reasons. First, the NHL domain of Brat
is required for binding to Pumilio and Nanos and for the assembly
of the translational repressor complex.
However, the NHL domain is dispensable for Brat-dependent
specification of INP identity. Second, Nanos expression is
undetectable in larval brains, and pumilio mutant larval brains do not
possess supernumerary type II neuroblasts.
Together, these results are consistent with the conclusion that Brat
specifies INP identity via a novel Arm-mediated mechanism (Komori, 2013).
The amino acid sequence of the B-boxes is highly conserved
among all TRIM family proteins, including Brat, and is predicted to
adopt a 'RING-like' fold tertiary structure. The RING-like fold might
facilitate protein-protein interactions. This is a particularly
intriguing hypothesis in light of the fact that Apc2 and Brat both
localize to the basal cortex in type II neuroblasts, and overexpression
of brat, but not bratδB-boxes, can restore Apc2 protein localization in
neuroblasts. However, epitope-tagged Brat and endogenous could not be coprecipitated Apc2 from
the brain lysate extracted from brat null mutant larvae
overexpressing a Myc-tagged Brat transgenic protein. Thus, Brat might maintain Apc2 protein localization
indirectly through other factors. Future biochemical analyses of the
Brat protein and identification of the proteins that directly interact
with the B-boxes will provide insight into how Brat controls Apc2
localization (Komori, 2013).
The destruction complex targets β-catenin/Arm for degradation
during canonical Wnt signaling, so reduced function of the
destruction complex will lead to an increase in β-catenin/Arm,
which forms a complex with Tcf/LEF family transcription factors to
activate Wnt target gene expression. This study has concluded
that the Brat-Apc2 mechanism specifies INP identity by preventing
aberrant activation of Wnt target gene expression in Ase- immature
INPs. The role of the Wnt ligand was tested in the Brat-dependent
specification of INP identity by removing the function of
the Wnt ligand using a temperature-sensitive mutant allele or by
overexpressing a dominant-negative form of Frizzled (FzDN or GPIdFz2)
in brat DG19310/11 mutant brains. Interestingly, neither of these
manipulations modified the supernumerary neuroblast phenotype in
the sensitized brat genetic background (data not shown). These
results suggest that the Wnt ligand and its receptor Fz are irrelevant
in the Brat-dependent specification of INP identity and that the Brat-
Apc2 mechanism prevents Wnt target gene expression in Ase-
immature INPs by negatively regulating the activity of Arm.
However, these data do not exclude the possibility that a novel
activating mechanism of Wnt signaling might be present in type II
neuroblasts in Drosophila larval brains (Komori, 2013).
Attempts were made to directly demonstrate that loss of brat function
indeed leads to derepression of Wnt target gene expression in
supernumerary neuroblasts.The expression was examined of two
distinct Wnt reporter transgenes, WRE-lacZ and Notum-lacZ in brat mutant brains. However, it was not possible to detect the expression of these transgenes in supernumerary
neuroblasts in brat null mutant brains. Because
genetic manipulations altering the activity of Arm efficiently modify
the supernumerary neuroblast phenotype in brat mutant brains, these
two transgenes are unlikely to have the necessary regulatory
elements to reflect Wnt target gene activity in this tissue. Thus, it is
proposed that the Brat-Apc2 mechanism specifies INP identity by
antagonizing the transcriptional activity of Arm in Ase- immature
INPs via a receptor-independent mechanism (Komori, 2013).
Wnt signaling regulation plays key roles in both stem cell renewal and
the differentiation of progenitor cell types (Merrill, 2012; Habib et al.,
2013). In the mammalian intestinal epithelium, for example, loss of
Apc and activation of Wnt signaling results in the maintenance of
stem cell properties in the progenitor cells, a failure to differentiate,
and the production of intestinal polyps that progress to malignant
tumors. In the intestine, the inappropriate
activation of Wnt signaling is sufficient to elicit stem cell properties.
In the progenitor cells of larval type II neuroblasts, the activation of
Wnt signaling alone, through either the expression of stabilized Arm or the loss of Apc2,
does not drive stem cell
renewal in otherwise wild-type immature INPs. In this system, Brat
is the key regulator attenuating self-renewal through two independent,
but convergent, mechanisms in its regulation of both Klu and Wnt
signaling. Although Arm activity is required for Klu-dependent self-renewal
in immature INPs, its inability to promote self-renewal
alone suggests that Wnt signaling is likely to be playing a
permissive role rather than an instructive role in eliciting the
neuroblast identity. It is proposed that Brat downregulates the function
of Klu through both Arm-dependent and -independent mechanisms. Previous studies have demonstrated that TRIM32 and TRIM3, vertebrate orthologs of Brat, are essential
regulators of neural stem cells during brain development and brain
tumor formation (Boulay, 2009; Schwamborn, 2009). It
will be interesting to test whether TRIM32 and TRIM3 regulate
neural stem cells via a β-catenin-dependent mechanism (Komori, 2013).
Intermediate neural progenitor (INP) cells are transient amplifying neurogenic precursor cells generated from neural stem cells. Amplification of INPs significantly increases the number of neurons and glia produced from neural stem cells. In Drosophila larval brains, INPs are produced from type II neuroblasts (NBs, Drosophila neural stem cells), which lack the proneural protein Asense (Ase) but not from Ase-expressing type I NBs. To date, little is known about how Ase is suppressed in type II NBs and how the generation of INPs is controlled. This study shows that one isoform of the Ets transcription factor Pointed (Pnt), PntP1, is specifically expressed in type II NBs, immature INPs, and newly mature INPs in type II NB lineages. Partial loss of PntP1 in genetic mosaic clones or ectopic expression of the Pnt antagonist Yan, an Ets family transcriptional repressor, results in a reduction or elimination of INPs and ectopic expression of Ase in type II NBs. Conversely, ectopic expression of PntP1 in type I NBs suppresses Ase expression the NB and induces ectopic INP-like cells in a process that depends on the activity of the tumor suppressor Brain tumor. These findings suggest that PntP1 is both necessary and sufficient for the suppression of Ase in type II NBs and the generation of INPs in Drosophila larval brains (Zhu, 2011).
This study has demonstrated that Drosophila PntP1 is a key
molecule that suppresses Ase expression in type II NBs and
promotes the generation of INPs. PntP1 is specifically
expressed in type II NB lineages. Ectopic
PntP1 expression suppresses Ase expression in type I NBs
and induces ectopic INP-like cells. The generation of ectopic
INP-like cells requires prior suppression of Ase in the NBs and
Brat activity. Conversely, partial loss of PntP1 or inhibiting
PntP1 activity results in the reduction or elimination of INPs and
activation of Ase expression in type II NBs (Zhu, 2011).
How does PntP1 suppress Ase expression? Given that Pnt was
known to function mainly as a transcriptional activator, it is
likely PntP1 suppresses Ase expression indirectly by activating
the expression of yet to be identified target genes. However, Ets
family proteins with transcriptional activation activity, such as
Pnt homolog Ets1, could also function as transcriptional
repressors in a cell type- and promoter-dependent manner.
It cannot be entirely ruled out that PntP1 acts as
a transcriptional repressor to suppress Ase expression in type
II NBs (Zhu, 2011).
Although results from this study as well as others suggest
that suppressing Ase expression in NBs is necessary for the
generation of INPs, loss of Ase expression alone in type I NB
lineages does not lead to the generation of ectopic INPs.
Therefore, in addition to suppressing Ase expression, PntP1
must activate other target genes to promote the generation of
INPs. One of the major features that distinguish INPs from
GMCs is that INPs undergo several rounds of self-renewing
divisions, whereas GMCs divide terminally. Thus, among
PntP1 target genes could be cell-cycle regulators that promote
INPs to undergo self-renewing divisions. This finding is consistent
with the well-established function of Ets transcription factors
in cell-cycle control and tumorigenesis. Ets proteins
stimulate cell proliferation by inducing the expression of cell-cycle
regulators, such as Cyclin D1, Cdc2 kinase, and Myc.
In the developing Drosophila eye disk, Pnt up-regulates the expression
of String, the Drosophila homolog of Cdc25, to promote
the G2-M transition. Therefore, PntP1 likely activates the
expression of cell-cycle regulators to promote the self-renewing
division of INPs. A partial loss of PntP1 in pntδ33 mutant clones
or inhibition of PntP1 activity by Yan may lead to reduced expression
of cell-cycle regulators and subsequent precocious termination
of self-renewing divisions of INPs, resulting in a
reduction or elimination of INPs in type II NB lineages (Zhu, 2011).
Like in type II NB lineages, the results show that the induction
of INP-like cells by ectopic expression of PntP1 involves a Brat-mediated
maturation process. Brat functions as a translational
repressor, but exact targets of Brat in immature INPs remain
unknown. The results show that when Brat is knocked down via
RNAi, ectopic PntP1 expression leads to overproliferation of
type II NB-like cells in type I NB lineages, similar to the phenotype
observed in type II NB lineages when Brat is lost (Zhu, 2011).
However, neither ectopic PntP1 expression nor Brat knockdown
alone causes similar overproliferation phenotypes in type I NB
lineages. Therefore, it is possible that Brat promotes the maturation
of INPs in part by translationally suppressing the expression
of PntP1 target genes, particularly cell cycle-related genes,
in immature INPs, thus preventing PntP1 from activating cell
cycle progression in immature INPs before they fully mature (Zhu, 2011).
Although this study shows that PntP1 is able to induce the
generation of ectopic INP-like cells, not every type I NB lineage
ectopically expressing PntP1 produces INP-like cells. One explanation
could be that additional factors may be required for
the generation of INPs. These factors could be specifically
expressed in type II NB lineages and function either independently,
or together with PntP1 as cofactors, to promote the
generation of INPs. In the absence of these factors, PntP1 promotes
the generation of INPs at a much reduced efficiency. Ets
transcription factors often regulate target-gene expression by
recruiting other proteins. For example, Pnt interacts with
Jun to induce R7 cell-fate specification in the Drosophila retina. It is possible that PntP1 needs cofactors to efficiently activate the expression of target genes that are involved in the generation of INPs. However, it is also possible that Ase and
Pros in GMCs in type I NB lineages might counteract the role of PntP1 in cell-cycle progression by activating the expression of the cell-cycle inhibitor Dacapo, thus preventing the generation of self-renewing INP-like cells (Zhu, 2011).
In conclusion, this study demonstrates that PntP1 is responsible
for the suppression of Ase in type II NBs and the
generation of INPs. Suppression of Ase is likely a prerequisite
for PntP1 to induce the generation of INPs. Furthermore, PntP1
possibly activates yet-to-be identified target genes, including cellcycle
regulators, to induce the generation of INPs and to promote
their self-renewing divisions. In addition, the data suggest
that, at least in part, Brat promotes the maturation of INPs likely
by suppressing the expression PntP1-regulated cell cycle-related
genes in immature INPs, thus preventing immature INPs from
entering self-renewing divisions before they fully mature (Zhu, 2011).
Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. This study utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. The results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. This study further showa that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, thus this study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1 (Chen, 2022).
This study has dissected the molecular mechanism of how PntP1 suppresses Ase expression to specify the type II NB identity. PntP1 was demonstrated to acts as a transcriptional activator to indirectly suppress Ase expression by activating Tll, whereas Tll provides positive feedback to maintain the expression of PntP1 and the type II NB identity. The cis-elements that mediate the suppression of Ase by Tll and the activation of Tll by PntP1 were mapped. Thus, this work reveals mechanistic details of PntP1-mediated suppression of Ase expression and specification of type II NBs and identifies a novel direct target of PntP1 in type II NBs (Chen, 2022).
This study has demonstrated that PntP1 functions as a transcriptional activator by showing that the artificial chimeric repressor protein EnR-Ets antagonizes the function of endogenous PntP1 proteins when expressed in type II NB lineages and that the artificial chimeric activator proteins VP16AD-pntP11/2C-Ets could functionally mimic endogenous PntP1 protein when expressed in type I NBs. These results are in line with a previous in vitro study showing that PntP1 substantially activates bacterial chloramphenicol acetyltransferase (CAT) reporter expression under the control of Ets binding sites. Interestingly, the results show that the chimeric protein VP16AD-Ets is not sufficient to functionally mimic endogenous PntP1 proteins even though the Ets domain can bind to PntP1 target DNAs as demonstrated by the antagonization of PntP1's function by the EnR-Ets protein. Only when the C-terminal sub-fragment (aa. 256-511) of PntP1 is included can the chimeric activator protein functionally mimic endogenous PntP1 proteins. The Ets family proteins usually recruit additional cofactors to activate/repress target gene expression. This C-terminal fragment likely contains protein-protein interaction domains that are essential to recruit its cofactors. Since neither the pntP11/2C-Ets truncated protein nor the chimeric protein VP16AD-pntP11/2N-Ets is able to mimic wild type PntP1 protein's function, it is unlikely that this C-terminal sub-fragment contains the activation domain of PntP1 and the other N-terminal sub-fragment [pntP11/2N] contains the protein-protein interaction domains instead. Otherwise, with the VP16AD functioning as an activation domain and the N-terminal sub-fragment recruiting cofactors, the chimeric VP16AD-pntP11/2N-Ets protein would be able to functionally mimic wild type PntP1 protein. Given that a large number of Ets family transcription factors share highly conserved Ets domains but have diverse functions and activities in distinct cell types, the C-terminal sub-fragment of PntP1 may recruit cell-type-specific cofactors to regulate the expression of specific target genes in type II NB lineages, such as Erm and Tll, as it has been proposed as a general strategy for Ets family proteins to regulate the expression of tissue-specific target genes. However, the regions of PntP1 subfragments that was included in chimeric repressor/activator constructs were arbitrarily defined. The results do not tell the precise boundaries of the activation domains or potential protein-protein interaction domains. To precisely map these functional domains will require more detailed and systemic analyses in the future (Chen, 2022).
PntP1 performs diverse functions in different cell types in type II NB lineages. For example, in type II NBs, PntP1 is required to suppress Ase expression. In newly generated imINPs, PntP1 prevents premature differentiation of INPs, whereas late during imINP development, it promotes INP cell fate commitment and prevents dedifferentiation of imINPs. Therefore, as a transcriptional activator, PntP1 must activate the expression of many different target genes in type II NB lineages. However, Erm is the only direct target that has been identified previously. This study identified Tll as another direct target of PntP1 that functions primarily in type II NBs to suppress Ase expression. PntP1 is both necessary and sufficient for Tll expression. Seven putative binding sites were identified and EMSAs and ChIP-qPCR assays verified that all these sites can bind to PntP1 both in vitro and in vivo. Although ChIP-qPCR was done using type II NB-enriched DNAs isolated from Brat knockdown larval brains, it is unlikely the results are artifacts. A previous study has demonstrated that type II NB-enriched chromatin isolated from brat mutant larval brains maintains similar transcriptional status for multiple genes examined, including pntP1, as in wild type type II NBs. Furthermore, Brat mainly functions as an RNA-binding protein in the imINPs to promote degradation of mRNAs of self-renewing genes such as dpn and klu. Therefore, loss of Brat unlikely affects the binding of PntP1 to its target DNAs in type II NBs, but this possibility was not fully verified particularly because the supernumerary type II NBs in Brat knockdown brains are derived from dedifferentiation of imINPs and it has never been extensively evaluated whether the dedifferentiated type II NBs have the exact same gene expression profiles as wild type type II NBs. It might be helpful to further verify the results by single-cell ChIP using isolated wild type type II NBs. However, since the bioinformatic prediction of PntP1 binding sites was limited to the enhancer region R31F04, it is not certain whether any additional PntP1 binding sites exist outside this enhancer region and contribute the activation of tll by PntP1. In any event, the seven binding sites identified within this enhancer region are sufficient for tll to be activated by PntP1 in type II NBs as demonstrated by the specific expression of R31F04-GAL4 in type II NBs (Chen, 2022).
This work further demonstrates that Tll is the direct target of PntP1 that mediates the suppression of Ase in type II NBs by showing that simultaneous knockdown of Tll essentially blocks the suppression of Ase by misexpressed PntP1 in type I NBs. By fine mapping the cis-repressive elements in ase regulatory regions, two Tll binding sites were idnetified located at -1,292bp ~ - 1,262bp upstream of the ase TSS, consistent with a recent study showing that Tll binds to a 5-kb region upstream of the ase TSS. Although the core hexameric sequences of these two binding sites are not exactly the same as the typical Tll binding hexamer 5'-AAGTCA, EMSA results demonstrate that Tll can indeed bind to these sites either as a monomer or a homodimer, the latter of which is common for orphan nuclear receptors (Chen, 2022).
In addition to PntP1 binding sites, other studies report that Suppressor of Hairless [Su(H)], a binding partner of the intracellular domain of Notch, and Zelda (Zld) also bind to the enhancer region of tll, implicating that Notch and Zld could be upstream activator of Tll. However, Su(H) and Zld are not just expressed in type II NBs but also in type I NBs. Thus, Su(H) and Zld are unlikely to be sufficient to activate Tll, but whether PntP1 acts together with Su(H) and Zld to activate Tll in type II NBs is worth further investigation (Chen, 2022).
A previous study shows that PntP1 is expressed not only in type II NBs but also strongly in imINPs. However, Tll is primarily expressed in type II NBs but only very weakly in imINPs, and ectopic expression of Tll in imINPs reverts imINPs to type II NBs. Therefore, there must be a mechanism to inhibit the activation of Tll by PntP1 in imINPs. A recent study proposed that Erm and Hamlet (Ham) function sequentially to suppresses Tll expression in imINPs based on 1) decreasing or increasing the copy number of the tll gene suppresses or enhances the supernumerary type II NB phenotype in ham erm double heterozygous mutants, respectively; and 2) overexpressing Erm or Ham in type II NBs inhibits the expression of Tll in type II NBs. However, these data could be also explained by changes in the expression of PntP1 as it is demonstrated in this study that there is a positive feedback loop between PntP1 and Tll. For example, the reduction in the Tll expression resulting from Erm overexpression in type II NBs could be due to inhibition of PntP1 by Erm in type II NB as was reported previously rather than direct inhibition of Tll by Erm. Furthermore, Erm and Ham are not expressed in the newly generated imINPs, in which Tll expression is already largely suppressed. Therefore, the suppression of Tll in the newly generated imINPs cannot be explained by the inhibition by Erm and Ham. Other mechanisms are likely involved in suppressing Tll in imINPs (Chen, 2022).
The current results not only demonstrate that PntP1 is a direct upstream activator of Tll but also show that Tll is required to maintain PntP1 expression in type II NBs. Therefore, there is a positive feedback loop between PntP1 and Tll that is essential for maintaining the type II NB identity. Considering that PntP1 misexpression induces Tll expression in all type I NBs and generation of mINPs in a subset of type I NB lineages, whereas Tll misexpression induces PntP1 expression only in a small subset of type I NB lineages and does not induce the generation of mINPs, it is thought that PntP1 functions as a master regulator of type II NB lineage development and acts upstream of Tll, which in turn suppresses Ase expression in type II NBs. Since Tll mainly functions as a transcriptional repressor as was also demonstrated in this study, it is unlikely that Tll directly activates PntP1 expression. A previous study suggests that there might be an unknown feedback signal from INPs that could be required for maintaining PntP1 expression. Thus, one potential explanation for the loss of PntP1 expression in Tll knockdown type II NBs could be the loss of INPs and their feedback signal resulting from the ectopic Ase expression in type II NBs. However, since Tll misexpression is able to induce PntP1 expression albeit only in a small subset of type I NBs, it is more likely that Tll suppresses the expression of another unknown transcriptional repressor that is normally suppressed by Tll in type II NBs. When Tll is knocked down, this unknown transcriptional repressor could be turned on in type II NBs to suppress PntP1 expression. Whereas in type I NBs, this unknown repressor may be normally expressed to suppress PntP1 expression and misexpression of Tll may relieve the suppression of PntP1 expression (Chen, 2022).
The GAL4 reporter assays also identified in the ase enhancer region a ~50-bp fragment that is sufficient for activating Ase expression in both type I and type II NBs. Earlier studies show that the Achaete-Scute (AS-C) complex proteins together with Daughterlesss (Da) activate Ase expression in NBs during the initial specification of NBs at embryonic stages by directly binding to four E-boxes in the 5'-UTR of ase. But how Ase is maintained in NBs once they are specified is not known. The current study identified a distinct enhancer region for activating/maintaining Ase expression in NBs, suggesting that factor(s) other than the AS-C proteins may be involved in activating/maintaining Ase expression in NBs after they are specified. Therefore, the lack of Ase expression in type II NBs is not because of the absence of an activation mechanism, but rather this activation mechanism is actively suppressed by Tll. Identifying the transcriptional activator(s) involved in activating/maintaining Ase expression after NBs are specified may shed a new light on the mechanisms regulating the development and maintenance of type I and type II NBs (Chen, 2022).
Tissue homeostasis depends on the ability of stem cells to properly regulate self-renewal versus differentiation. Drosophila neural stem cells (neuroblasts) are a model system to study self-renewal and differentiation. Recent work has identified two types of larval neuroblasts that have different self-renewal/differentiation properties. Type I neuroblasts bud off a series of small basal daughter cells (ganglion mother cells) that each generate two neurons. Type II neuroblasts bud off small basal daughter cells called intermediate progenitors (INPs), with each INP generating 6 to 12 neurons. Type I neuroblasts and INPs have nuclear Asense and cytoplasmic Prospero, whereas type II neuroblasts lack both these transcription factors. This study tested whether Prospero distinguishes type I/II neuroblast identity or proliferation profile, using several newly characterized Gal4 lines. Prospero was misexpressed using the 19H09-Gal4 line (expressed in type II neuroblasts but no adjacent type I neuroblasts) or 9D11-Gal4 line (expressed in INPs but not type II neuroblasts). It was found that differential prospero expression does not distinguish type I and type II neuroblast identities, but Prospero regulates proliferation in both type I and type II neuroblast lineages. In addition, 9D11 lineage tracing was used to show that type II lineages generate both small-field and large-field neurons within the adult central complex, a brain region required for locomotion, flight, and visual pattern memory (Bayraktar, 2010).
The recent identification of the type II lineages containing transit amplifying intermediate progenitors provides an important new model for investigating progenitor self-renewal and differentiation. However, little is known about their development, cell biology, gene expression, and functional importance in the Drosophila central nervous system. This is primarily due to a lack of genetic tools and markers that are specifically expressed in type II NBs and/or INPs. This study characterized the 19H09-Gal4 line expressed in type II NBs, and the 9D11-Gal4 line expressed in INPs but not their parental type II NBs. Using 19H09 it was shown that Ase is upregulated before Dpn during INP maturation. Using both lines, Prospero misexpression was shown to regulate proliferation but not identity within type II lineages. And using 9D11 the majority of type II-derived neurons was permanently labelled to show they are major contributors to the adult central complex brain region (Bayraktar, 2010).
The 19H09-Gal4 and 9D11-Gal4 lines can also be used to monitor the development of type II NBs and INPs in different mutant backgrounds to help clarify the origin of a mutant phenotype. For example, early studies on tumor suppressor genes showed increases in global brain NB numbers; for some of these mutants (for example, brat, numb) this study now shows that the phenotype arises specifically within the type II lineage. The 19H09-Gal4 and 9D11-Gal4 lines can also be used to drive UAS-RNAi, UAS-GFP constructs to test the role of any gene within these lineages. In addition, because these lines are made from defined enhancer fragments driving Gal4 placed into a specific attP site in the genome, it is easy to generate different transgenes with precisely the same expression pattern. Some future uses would be: using 19H09-FLPase to generate mutant clones or MARCM genetic screens in type II lineages; using 9D11 to drive expression of uracil phosphoribosyltranferase to isolate RNA from INP sublineages; or using 9D11-grim to ablate specifically type II neurons to determine their role in larval or adult behavior (Bayraktar, 2010).
The 19H09 and 9D11 lines were used to show that misexpression of Prospero can suppress proliferation within type II NBs and INPs without altering NB identity. As 19H09 is expressed only during the late larval stages, Prospero misexpression with 19H09 clearly distinguishes the effects of Prospero on NB proliferation from its effects on NB fate specification, which occurs in the embryonic stages. Misexpression with both 19H09 and 9D11 lead to a reduction in the number of INPs and neurons made by each type II NB. This reduction is unlikely to be due to an effect on the parental type II NBs, such as slowed down cell cycle or compromised NB survival, for the following reasons: first, low levels of ectopic Prospero are cytoplasmic in type II NBs, where Prospero has no known function; second, ectopic Prospero does not transform type II lineages to a type I identity based on the failure to upregulate ase expression; and third, misexpression of Prospero with both 9D11 and 19H09 give similar phenotypes, yet 9D11 is not expressed in type II NBs. It is suggested that the reduction of clone size is due to an effect in the INP cell type. Possible mechanisms include INP apoptosis, INP cell cycle lengthening, premature cell cycle exit, or transforming INPs into central brain type II GMCs, which generate lineages with bifurcated axon fascicles. While it was not possible to distinguish between these possibilities, the mechanism of a transformation of INP to central brain type I GMC identity can now be tentatively excluded because the neurons still retained their ability to form bifurcated axon fascicles, which are not a feature of central brain type I GMCs (Bayraktar, 2010).
Type II NBs lack both Ase and Prospero, whereas type I NBs contain both proteins. Yet only misexpression of Ase can transform type II into type I NBs, suggesting that Ase is sufficient to upregulate prospero expression in NBs. However, loss of Ase does not transform type I NBs into type II NBs, so there must be additional factors promoting the expression of Prospero in type I NBs. The analysis of gene expression differences between type I and II NBs would be one way of uncovering genes that control the difference between them (Bayraktar, 2010).
Lineage-tracing of INP-derived neurons shows that type II lineages make major contributions to all aspects of the central complex of the adult brain, as well as the bulbs (BUs; also known as lateral triangles) and lateral accessory lobes (LAL) accessory structures, including both small-field and large-field neurons. Central complex neurons derived from type II lineages likely include several small-field types, such as ventral fiber system (VFS), pontine, fb-eb, fb-no, and pb-eb-no neurons, and, to a lesser extent, large-field types, such as F neurons, including Fm, Fl and ExFl subtypes and some extrinsic R neurons. A recent study found that type II NB clones in the pupal brain projected to the PB (the largest structure in the central complex, divided into several vertical staves and horizontal stratifications), FB (the fan-shaped body) and NO (paired noduli) regions, with some projections forming restricted arbors at the PB (protocerebral bridge) and innervating domains of the FB and NO, while others made widespread arborizations outside the central complex. The data showing labeling of the majority of type II neuronal progeny are consistent with those of a previous study: while this study did not have the resolution to link cell bodies with axon projections, it is possible to provide a more comprehensive view showing that type II lineages contribute to all central complex neuropils and accessory areas in the adult brain. Future studies that selectively ablate different spatial or temporal cohorts of type II neurons will be necessary to determine if all type II-derived neurons share a common function (Bayraktar, 2010).
Although a large subset of central complex neurons derive from type II lineages, there are clearly some central complex neurons that originate from type I NBs or embryonic type II lineages. For example, no projections were seen that match those of the well-characterized large-field R neurons (R1 to R4). It is not clear which small-field types are not derived from type II lineages as they are difficult to distinguish. However, it is clear that the type II lineages do not make up the entire central complex so there must be contributions from type I lineages as well (Bayraktar, 2010).
Outside the central complex, labeling was observed of the region-specific staining of both the mushroom body and ALs; staining in the ALs was restricted to a subset of glomeruli. These could be novel connections from the central complex to the mushroom body and ALs formed by large-field or poorly understood extrinsic small-field neurons, or the projections of non-central complex neurons labeled by 9D11. Previous studies have revealed no direct connection between central complex and mushroom bodies or between LALs and ALs, and very few connections from LALs to mushroom bodies. The type II projection patterns from larval and pupal brains suggest that the lineages are not dedicated to a single neuropile center, which is consistent with type II lineages giving rise to non-central complex neurons as well. Labeling of large regions in the protocerebrum was also observed outside the central complex. However, it was not possible to distinguish whether they were connected to the central complex or its accessory areas. Another caveat to this analysis is that 9D11 is also expressed in the larval optic lobes, and indeed labeling was observed in the adult optic lobes. It was not possible to distinguish the projections from these cells from those of the central brain cell bodies due to dense staining. Analysis of 1,200 Golgi-impregnated brains revealed direct connections between optic lobes and the BU neuropil, but not to the other central complex neuropils that were found to be labeled. This suggests that most if not all central complex labeling is due to type II-derived neurons (Bayraktar, 2010).
In addition to using 9D11 to lineage trace the contribution of larval-derived type II neurons to the adult brain, maintained expression of 9D11 was also detected in a small subset of adult neurons, which are likely to be P3 or P4 small-field pontine neurons, which are also detected by the Gal4 line NP2320. Thus, the 9D11 line, and others with similarly specific adult expression patterns, should be useful for future studies using TU-tagging to transcriptionally profile neuronal subsets, GRASP to identify pre/post-synaptic partner, or for expression of optogenetic modulators of neuronal activity to determine the role of specific neurons in behavior (Bayraktar, 2010).
This characterization of type II lineages suggests that as a group the type II NBs produce a wide variety of neuronal subtypes. This neural diversity can be achieved spatially if each type II NB generates just one or two types of neurons; this model is supported by clonal data showing that each type II NB produces neurons with distinct axon projection patterns. In addition, temporal identity could generate further neuronal diversity as seen in type I NB lineages. This model is supported by clonal analysis of a small central complex sublineage in the adult brain, which has revealed temporally distinct neuronal fates. Finally, hemilineages could provide a final doubling of neuronal diversity, in which each sibling neuron derived from a single GMC takes either an 'A' or a 'B' cell fate. The fact that bifurcating axon projections are seen even in the highly sparse type II lineages following Prospero overexpression is consistent with GMCs producing A/B neurons that have different fasciculation patterns. In the future, it will be important to determine the birth-order and identities of neurons in each type II lineage and the mechanisms that regulate spatial and temporal neural fate specification in these lineages (Bayraktar, 2010).
The Drosophila larval central brain contains about 10,000 differentiated neurons and 200 scattered neural progenitors (neuroblasts), which can be further subdivided into ~95 type I neuroblasts and eight type II neuroblasts per brain lobe. Only type II neuroblasts generate self-renewing intermediate neural progenitors (INPs), and consequently each contributes more neurons to the brain, including much of the central complex. Six different mutant genotypes were characterized that lead to expansion of neuroblast numbers; some preferentially expand type II or type I neuroblasts. Transcriptional profiling of larval brains from these mutant genotypes versus wild-type allowed identification of small clusters of transcripts enriched in type II or type I neuroblast,s, and these clusters were validated by gene expression analysis. Unexpectedly, only a few genes were found to be differentially expressed between type I/II neuroblasts, suggesting that these genes play a large role in establishing the different cell types. A large group of genes predicted to be expressed in all neuroblasts but not in neurons were identified. A neuroblast-specific, RNAi-based functional screen was performed and 84 genes were identified that are required to maintain proper neuroblast numbers; all have conserved mammalian orthologs. These genes are excellent candidates for regulating neural progenitor self-renewal in Drosophila and mammals (Carney, 2012).
To identify genes expressed differentially between type I and type II neuroblasts, genes were sought clustered with pros and ase, the only two genes known to be differentially expressed in type II neuroblasts. It was found that pros and ase reside together in a small sub-cluster of only 11 genes within group B. This sub-cluster as a whole exhibits reduced expression in brat, lgl, and lgl lgd mutants and enrichment in aur, aPKCCAAX, and lgl pins; remarkably, no other sub-cluster exhibits such a pattern. This suggests that the other nine genes in the cluster may also be specifically expressed in type I neuroblasts, like pros and ase, and that these are potentially the only genes that exhibit this unique pattern (Carney, 2012).
To test whether other genes in the small pros/ase cluster are also expressed in type I neuroblasts but not type II neuroblasts, an antibody was obtained to a candidate from this cluster, Retinal homeobox (Rx), a homeodomain-containing transcription factor. It was found that Rx is completely absent from type II neuroblasts, similar to Pros and Ase; Rx is detected in several type I neuroblasts as well as in a subset of differentiated type II progeny. Consistent with this expression pattern, it was found that brat mutants, which overproduce type II neuroblasts, show a loss of Rx staining. In contrast, lgl pins mutants, which have ectopic type I neuroblasts, show territories of strong Rx expression which is confined to Pros+ (likely type I-originating) cells. The fact that only a small patch of lgl pins mutant brain tissue is Rx+ is probably because Rx is normally expressed in a subset of type I neuroblasts. It is concluded that Rx, like Pros and Ase, is expressed in type I but not type II neuroblasts. Thus, most or all of the 11 genes in the pros/ase sub-cluster may be expressed in type I but not type II neuroblasts (Carney, 2012).
Genes expressed in type II neuroblasts but not type I neuroblasts were sought, as there are currently no known markers specifically expressed in type II neuroblasts. It was reasoned that transcripts expressed in type II neuroblasts should be enriched in genotypes that overproduce type II neuroblasts: brat, lgl and lgl lgd. One small cluster was found enriched in two of the three mutants (brat and lgl lgd). This cluster contains just 10 genes, seven encoding transcription factors. To verify the expression pattern of this gene cluster, the expression of one gene product, Optix, was examined. Optix is a conserved homeodomain-containing transcription factor required for eye development. Consistent with the microarray data, it was found that most of the Optix expression in the brain is indeed restricted to type II lineages; four of the six dorso-medial type II neuroblasts (DM1, 2, 3, and 6) express Optix, as do most of the INPs, GMCs, and neurons in these lineages. In addtion, recent work has shown that another gene in this cluster, pointedP1, is also preferentially expressed in type II neuroblasts (Sijun Zhu and Y.N. Jan, personal communication to Carney, 2012). The other two dorso-medial type II lineages (DM4 and 5) exhibit some expression of Optix in a subset of neuronal progeny, but it is absent from the neuroblasts and INPs in these lineages. In addition, a single dorsal type I neuroblast expresses Optix. Inspection of mutant brains further confirmed the type II-biased expression of Optix, in that brat mutant brains exhibit a marked increase in Optix+ neuroblasts, and in lgl pins, the increase in Optix is almost exclusively in a Pros− (type II-originating) region of the brain. These results indicate that the clustering relationships can be used to predict type I/type II expression bias with good accuracy. It is concluded that Optix is primarily expressed in type II but not type I neuroblasts, and that Optix and the other five genes in this cluster are excellent candidates for regulators of type II neuroblast identity (Carney, 2012).
It has previously been shown that co-clustering of genes in expression profiling data is likely to reflect physical or genetic interactions and participation in the same pathway. The current results are consistent with these conclusions. For example, a small group of 11 genes was identified containing the only two genes known to be expressed in type I but not type II neuroblasts, and a third gene was shown to have a similar pattern of expression — thus all genes in this cluster are likely to be expressed in type I but not type II neuroblasts. Furthermore, the strong enrichment of GO terms in small sub-clusters within both group A and group C indicates that genes within these clusters are likely to share similar functions or processes (Carney, 2012).
At the outset of this study, a large group of genes was expected to be differentially expressed in type II versus type I neuroblasts, because these neuroblasts have such strikingly different cell lineages. However, only a few gene clusters were identified that were differentially regulated in such a type I/type II consistent manner — the 11 genes in the pros/ase cluster depleted in type II neuroblasts and the 10 genes enriched in type II neuroblasts. This suggests that the small number of genes identified may play a disproportionately large role in generating differences between type I and type II neuroblasts. Might pros and ase be the only genes regulating type I/type II differences? Both Ase and Pros can promote cell cycle exit, which may result in the Ase+ Pros+ type I progeny taking a GMC identity and undergoing just one terminal division and the Ase− Pros− type II progeny taking an INP identity and continuing to proliferate. Indeed, the misexpression of either Ase or low levels of Pros in type II neuroblasts is sufficient to cause the loss of INPs and/or their premature cell cycle exit, thereby decreasing lineage size toward the size of type I neuroblasts. However, it is unclear what is required to fully transform these cells into type I neuroblasts; addressing this question will require additional molecular markers and tracing the axon projections of the progeny of these 'transformed' neuroblasts (e.g., do they now fail to make intrinsic neurons of the adult central complex?). The fact that mutants in ase and pros do not transform type I neuroblasts into type II neuroblasts indicates that other genes, perhaps some in the pros/ase cluster described here, are also important for specification of type I neuroblast identity (Carney, 2012).
It was found that the neuroblasts in each mutant have remarkably similar expression profiles, as shown by the extensive list of similarly expressed genes in group A and by the list of genes with depleted expression in mutant brains, represented by group C. It is believed that these categories provide lists of genes that are representative of those expressed in neuroblasts and neurons, respectively, based on all known neuroblast-specific genes showing up in group A and all known neuron- or glial-specific genes being excluded from group A (Carney, 2012).
Group B genes apparently are not expressed in all neuroblasts like the group A genes, nor in all neurons or glia like group C genes. However, group B genes are more likely to be expressed in subsets of neurons, not neuroblasts, because group B genes as a whole have an over-representation of GO terms more similar to group C than to group A. Why then are group B genes excluded from group C, the neuron cluster? One possible explanation is that different neuroblast lineages are affected in each mutant, and thus different subsets of neurons are missing in each mutant. If different neuroblast lineages express different genes (which seems likely), then each mutant would be missing a unique subset of neural differentiation genes, leading to the cluster being excluded from group C. This model raises the intriguing possibility that group B sub-clusters may represent lineage-specific genes (Carney, 2012).
It is also possible that the mutant genotypes themselves may cause unique transcriptional differences, leading to a cluster of genes in group B. For example, several small sub-clusters in group B are expressed differently only in aPKCCAAX brains. These transcriptional differences are not correlated with the number of type I or type II neuroblasts. Instead, these genes appear to be differentially expressed in response to elevated aPKC. Drosophila aPKC has been best studied as a component of the apical complex in mitotic neuroblasts, and its capacity for causing ectopic self-renewal has been shown to be reliant on both its catalytic activity and its membrane localizatio. However, aPKC has been ascribed a role in neuroblast proliferation as well as in polarit, and a vertebrate homolog, PKC-zeta, was shown to possess a nuclear role in both proliferation of neural progenitors and neuronal cell fate specification. These observations are consistent with a role of aPKC in causing transcriptional differences (Carney, 2012).
The findings of this study highlight the importance of expression profiling of multiple genotypes. This method gave a more reliable picture of the group A genes expressed in neuroblasts, because genes with lineage-specific or genetic background-specific changes in expression appeared to be focused into group B, where they do not interfere with the clustering of groups A and C. In addition, two small sub-clusters of genes were identified in group B that are excellent candidates for being preferentially expressed in type I or type II neuroblasts, for which there have been few examples to date. Finally, it is concluded that group A genes are likely to be expressed in neuroblasts, and functional studies have identified 84 genes that are conserved in mammals and required for regulating neuroblast numbers in Drosophila. Future phenotypic analysis in Drosophila will determine whether these genes regulate neuroblast survival, quiescence, asymmetric cell division, and/or self-renewal. Future studies on the expression and function of orthologous genes in mouse neural progenitors and human stem cells (IP or neural) will reveal whether they have conserved roles from flies to mammals (Carney, 2012).
The balance between stem cell self-renewal and differentiation is precisely controlled to ensure tissue homeostasis and prevent tumorigenesis. This study use genome-wide transgenic RNAi to identify 620 genes potentially involved in controlling this balance in Drosophila neuroblasts in the larval CNS. All phenotypes and derive measurements were quantified for proliferation, lineage, cell size, and cell shape. A set of transcriptional regulators essential for self-renewal was identified and hierarchical clustering and integration with interaction data were used to create functional networks for the control of neuroblast self-renewal and differentiation. The data identify key roles for the chromatin remodeling Brm complex, the spliceosome, and the TRiC/CCT-complex and show that the alternatively spliced transcription factor Lola and the transcriptional elongation factors Structure specific recognition protein (Ssrp) and Barricade (Barc) control self-renewal in neuroblast lineages. As these data are strongly enriched for genes highly expressed in murine neural stem cells, they are likely to provide valuable insights into mammalian stem cell biology as well (Neumuller, 2011).
As CG6049 had not been characterized before, this gene was chosen for in-depth analysis. CG6049 was named barricade (barc) to indicate the block in Nb lineage progression observed upon RNAi. Barc is conserved from yeast to humans. Like its vertebrate homolog Tat-SF1, it contains two RNA recognition modules (RRM), a nuclear localization signal, and a conserved region that contains two motifs that are known to bind to FF domains and that was named the Barc-Tat-SF1 (BTS) motif. To determine the specificity of the barc-RNAi phenotype, an RNAi-resistant barc construct was generated. When expressed together with barc-RNAi, this construct can rescue both lethality and the Nb phenotype. In addition, the barc-RNAi phenotype could be confirmed by a second, nonoverlapping RNAi line. Thus, barc is a regulator of lineage progression in Drosophila Nbs (Neumuller, 2011).
While barc-RNAi in type II lineages using wor-Gal4; ase-Gal80 causes overproliferation, barc-RNAi induced by ase-Gal4 has no overproliferation phenotype. The additional CD8::GFP-positive cells in the type II lineages express Cyclin E, indicating active proliferation, and do not express the neuronal marker Elav. More cells positive for Mira and Dpn were observed, that are expressed both in Nbs and in INPs. On average, the number of Mira-positive cells is approximately 4-fold increased. Since only one large Ase-negative type II Nb was observed, and the extra cells express the INP marker Ase, it is concluded that barc is required for INPs to generate differentiating neurons. Upon barc-RNAi, the daughter cells retain the INP fate, and this results in the overproliferation phenotype. Although barc-RNAi does not cause a similar overproliferation phenotype in type I lineages, it was observed that the diameter of type I Nbs is increased from 15 ± 0.31 μm to 17.16 ± 0.27 μm. This phenotype could either indicate an increase in growth rate or a decrease in cell cycle progression. Thus, barc is required for lineage progression in type II Nb lineages, but might also have a function in mitotic progression of type I Nbs (Neumuller, 2011).
To test Barc expression and subcellular localization, a peptide antibody was generated. The antibody detects a single band of approximately 75 kD on a western blot, which can be blocked by the antigenic peptide. The anti-Barc immunofluorescence signal is absent after barc-RNAi and increases upon Barc overexpression. Barc antibody staining revealed that Barc is a nuclear protein that is predominantly expressed in both type I and type II Nbs and to a lesser extent in INPs, GMCs, and differentiated neurons. Thus, this study has identified a nuclear regulator of type II Nb lineages that allows INPs to generate daughter cells, which undergo terminal neural differentiation (Neumuller, 2011).
This screen has identified a total of 620 genes that are potentially involved in controlling self-renewal in Drosophila neural stem cells. It was demonstrated that precise quantification of phenotypic data allows for a computer analysis that can lead to biological insights that are not easily obtained through classic single-gene approaches. Through network analysis, splicing control was identified as a key regulator of Nb self-renewal. Alternative splicing of lola might be one of the targets of this machinery as different isoforms of this transcription factor are differentially expressed and phenotypically distinct. It was also shown that duplicated forms of ribosomal subunits are functionally distinct, with one form being more specifically required in Nbs. Finally, it was demonstrated that genes involved in transcriptional elongation and chromatin remodeling are important regulators of Nb self-renewal and differentiation. It is known that more than one third of all Drosophila genes are in a poised state where active RNA polymerase is stalled in a promoter proximal position. Release of stalled polymerases might contribute to the rapid activation of differentiation genes during Nb ACD. Transcriptional elongation is important for controlling vertebrate stem cell lineages as well, but how stalled promoters are released in a cell-type-specific manner is currently unknown. Analysis in the simple Drosophila Nb lineage could shed some light on this important question in stem cell biology (Neumuller, 2011).
Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is maintained is not well understood. Using Drosophila larval neuroblasts as a model, this study demonstrates that the Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. These results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. These data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification (Zhu, 2012).
Dpn was initially identified as a pan-neural protein and has been widely used as a NB marker. However, the function of Dpn in NBs has been elusive. This study provides evidence that Dpn plays an important role in maintaining NB self-renewal. In type II NBs, in addition to maintaining the self-renewal, Dpn is also required to suppress Ase expression when these NB exit quiescence. Furthermore, Notch and Dpn may function independently in larval NBs. While both Dpn and Notch are required for maintaining the identity and self-renewal of type II NBs, knockdown of Notch does not affect the expression of Dpn in type II NBs (Zhu, 2012).
In a developing nervous system, NSCs must be maintained when they divide in order to generate the complete array of neurons and glia that form a functional neuronal circuit. Current studies are focused on determining how NSC self-renewal is maintained, as well as mechanisms governing NSC terminal differentiation. The findings that dpn mutant MB NBs as well as other type I NBs are prematurely, progressively, lost demonstrate that Dpn functions cell-autonomously to maintain the self-renewal of larval NBs. Interestingly, in dpn mutant larvae, the premature loss of type I NBs mainly occurred within 48 hours after larval hatching (with the exception of the MB NB). Zacharioudaki (2012) has reported recently that Dpn and E(spl) proteins function redundantly to maintain NB self-renewal but have different temporal expression patterns. Dpn expression in NBs is activated at the newly hatched larval stage, whereas E(spl)mγ expression becomes obvious only when NBs start to divide at the 2nd instar larval stage. The difference in temporal expression patterns between Dpn and E(spl) proteins probably explains why loss of type I NBs occurred mainly within 48 hours after larval hatching in dpn mutants. Interestingly, despite the redundant function of Dpn and E(spl) proteins in maintaining NB self-renewal, loss of Dpn alone resulted in the premature loss of MB NBs at late larval/early pupal stages, indicating that E(spl) proteins may not be involved in maintaining MB NBs at late larval/early pupal stages. The current findings as well as those of Zachariousdaki suggest that Dpn is required for maintaining NB self-renewal rather than NB formation or specification as was proposed by San-Juan and Baonza (2011). It is likely that differences in identifying and quantifying NBs at different developmental stages accounts for this discrepancy (Zhu, 2012).
The role of Dpn in NB self-renewal is also supported by the observation that ectopic/over-expression of Dpn promoted non-dividing immature intermediate INPs and terminally dividing GMCs to enter self-renewing divisions, and prolonged the self-renewal of both types of NBs. These ectopic self-renewing GMCs and immature INPs, which normally do not express Dpn, may de-differentiate to acquire a NB-like fate and contribute to the increased number of NBs, similar to what has been observed in brat, numband klumpfuss mutant type II NB lineages. However, type II NB lineages show more severe over-proliferation phenotypes than type I NB lineages in response to ectopic/over-expression of Dpn. The difference in degree of over-proliferation between the type I and type II NB lineages is likely related to intrinsic differences between the type I and type II NB daughters, rather than a difference in how Dpn itself is acting. Type I NBs produce GMCs that express genes such as pros and ase that limit proliferation, counteracting the pro-self-renewal function of Dpn. In contrast, type II NBs and immature INPs express the ETS family protein Pointed but do not express Ase or Pros, making them particularly susceptible to the ectopic expression of genes, such as dpn, that promote self-renewal. It is proposed that the significantly enhanced proliferation of type II NB progeny in response to ectopic/increased Dpn expression is most likely due to a disparity in the inherent self-renewal potential of the type I and type II NB daughters (Zhu, 2012).
The function of Dpn in maintaining NB self-renewal is consistent with mammalian Hes family proteins' function in maintaining NSCs. In the developing mammalian nervous system, the loss of Hes1, Hes3, and Hes5 leads to accelerated neurogenesis and premature depletion of neuroepithelial cells and radial glial cells, whereas forced expression of Hes proteins maintains NSCs (Zhu, 2012).
While Dpn is expressed in both type I and type II NBs, the data showed that the loss of Dpn not only resulted in the premature loss of type II NBs at early larval stages, but also led to the ectopic expression of type I NB markers Ase and Pros in type II NBs when they exited quiescence, making type II NBs appear as type I-like NBs. This indicates Dpn has two roles in type II NBs: Dpn maintains NB self-renewal just as it does in type I NBs, and Dpn is also required to maintain type II NB identity. Moreover, it appears that Dpn's role in maintaining type II NB identity is temporally restricted. Results from this study as well as others showed that dpn mutant embryonic brains contained a comparable number of Dpn+ Ase- NBs as wild type embryonic brains. In dpn mutant clones, our results showed that type II NBs did not ectopically express Ase even 4 days after clone induction when Dpn is no longer detectable. Therefore, it seems that Dpn's function to suppress Ase expression is limited to a narrow temporal window during the reactivation of type II NBs at the 1st instar larval stage. How might Dpn act to maintain type II NB identity? In mammals, Hes family proteins are well known for their roles in antagonizing the expression and/or activity of proneural genes (Ase is a member of the achaete-scute family of proneural genes). Negative interactions between dpn and the achaete-scute complex (AS-C) genes occur during Drosophila sex determination as well as neurogenesis. One potential model could be that a proneural protein(s) might be expressed in quiescent type II NBs and that Dpn is required to antagonize its expression and/or activity in order to promote type II NB fate when NBs exit quiescence. Since Dpn is expressed in both type I and type II NBs, it is postulated that its role in maintaining type II NB fate is associated with the differential expression and/or activity of another, currently unidentified gene (Zhu, 2012).
This work suggests that the premature loss of dpn mutant type I NBs could be mediated by Pros. This is supported by the findings that nuclear Pros precociously accumulates in dpn mutant type I NBs and that dpn mutant type I NBs are maintained even in adult brains in the absence of Pros. It has been shown that over-expressing Pros in embryonic and larval NBs is sufficient to induce ectopic nuclear Pros localization and terminal division. Therefore, one possibility is that Dpn negatively regulates pros expression. In the absence of Dpn, Pros expression increases, leading to the nuclear accumulation of Pros and thus premature terminal division. In type I NBs, dynamic cortical and cytoplasmic localization of Pros makes it difficult to compare the levels of Pros in wild type and dpn mutant type I NBs by immunostaining. However, ectopic Pros expression in dpn mutant type II NBs, which normally do not have Pros, provide evidence that Dpn negatively regulates Pros expression, either directly or indirectly. The existence of putative Dpn binding sites in the pros promoter suggests that Dpn could directly regulate pros expression. Alternatively, Dpn could indirectly regulate pros by inhibiting the expression and/or activity of proteins, such as Ase, that promote pros expression. In support of this notion, it has been shown that mammalian Hes proteins can inhibit the expression of proneural proteins such as Mash1 in the developing cortex, whereas forced expression of the proneural protein Mash1 in neuroepithelial cells is sufficient to promote the expression of Prox1, the mammalian homolog of Pros that plays an anti-proliferative and pro-differentiation role in the developing mammalian hippocampus and retina (Zhu, 2012).
Unlike the majority of mammalian Hes proteins or other members of the fly Hes family, which typically act downstream of Notch, results from this study as well as Zacharioudaki (2012) do not support a model in which Dpn functions as a direct target of Notch signaling in larval NBs as was proposed by San Juan and Baonza (2011). First, although these studies, as well as the work from other investigators, showed that the knockdown of Notch or disruption of Notch signaling led to premature loss of type II NBs and ectopic expression of Ase in type II NBs as was observed in dpn mutant larval brains, knockdown of Notch did not affect the expression of Dpn in type II NBs, which is consistent with previous findings. Second, the data and those of Zacharioudaki showed that removing Dpn did not abolish the over-proliferation of type II or type I NBs caused by over-expression of activated Notch. Nor did reducing Notch expression exacerbate the loss of type II NBs in dpn7 heterozygous animals. These genetic interaction data suggest that Dpn does not function downstream of Notch signaling. Thus, Dpn may be similar to the mammalian Hes2 and Hes3, which are not transcriptionally regulated by Notch.Notch and Dpn likely employ distinct mechanisms to maintain the self-renewal and suppress Ase expression in type II NBs. Zacharioudaki showed that some E(Spl) proteins, particularly E(spl)mγ and m8, depend on Notch signaling for their expression in larval NBs. However, loss of E(Spl) proteins does not result in ectopic expression of Ase in type II NBs. Therefore, Notch must function through molecules, which are yet to be identified, to regulate Ase expression in type II NBs (Zhu, 2012).
Neural stem cells in the adult brain exist primarily in a quiescent state but are reactivated in response to changing physiological conditions. How do stem cells sense and respond to metabolic changes? In the Drosophila CNS, quiescent neural stem cells are reactivated synchronously in response to a nutritional stimulus. Feeding triggers insulin production by blood-brain barrier glial cells, activating the insulin/insulin-like growth factor pathway in underlying neural stem cells and stimulating their growth and proliferation. This study shows that gap junction proteins, Inx1 and Inx2, in the blood-brain barrier glia mediate the influence of metabolic changes on stem cell behavior, enabling glia to respond to nutritional signals and reactivate quiescent stem cells. It is proposed that gap junctions in the blood-brain barrier are required to translate metabolic signals into synchronized calcium pulses and insulin secretion (Speder, 2014).
Changes in environmental conditions can have a significant impact on the development and function of the brain. Neural stem cells (NSCs) integrate both local and systemic signals to modulate their rate and extent of proliferation to meet the needs of the organism. Most NSCs in the vertebrate adult brain exist in a mitotically dormant state. These quiescent NSCs are reactivated in response to a variety of metabolic stimuli. Understanding how systemic and metabolic signals are sensed by the brain and converted into specific neural stem cell behaviors is essential to deciphering how the brain adapts to a changing environment (Speder, 2014).
In Drosophila, NSCs enter quiescence at the end of embryogenesis and are reactivated during early larval life in response to feeding. Amino acid availability is sensed by the fat body, the functional equivalent of the mammalian liver and adipose tissue. The fat body sends an as-yet-unidentified signal, or signals, to the brain to induce the production and secretion of insulin-like peptides (dIlps) by blood-brain barrier (BBB) glial cells. dIlps act locally to trigger the insulin/insulin-like growth factor receptor pathway in underlying NSCs. Consequently, the NSCs enlarge and re-enter the cell cycle (Speder, 2014).
NSC reactivation occurs synchronously in all neurogenic zones of the CNS, suggesting that BBB glial cells and/or NSCs are linked by an intercellular signaling mechanism. Gap junctions are intercellular channels formed by the juxtaposition of connexin hexamers. They enable the propagation and amplification of signals within or between cell populations. Gap junctions are found throughout the mammalian brain and are important regulators of stem cell behavior, controlling self-renewal, survival, and aging. This study shows that gap junction proteins play a key role in the nutrient-dependent reactivation of dormant neural stem cells in the Drosophila brain. Interestingly, gap junction proteins are required in the BBB glia, but not in neural stem cells, for reactivation. This study shows that gap junction proteins coordinate nutrient-dependent calcium oscillations within the BBB and are required for the production and secretion of insulin-like peptides. Gap junction proteins thus enable the synchronous reactivation of quiescent stem cells throughout the CNS (Speder, 2014).
To assess whether gap junctions play a role in NSC reactivation,
this study systematically targeted each of the eight members of the
innexin (Inx) family, the Drosophila functional
equivalents of connexins and pannexins, by RNAi in either NSCs or glia. Interestingly, no detectable phenotype was observed when
innexins were knocked down in NSCs. However, knockdown
of innexin 1 (inx1) or innexin 2 (inx2) in glia gave a striking phenotype
in which brain size is dramatically reduced
without affecting overall body size. This
suggests that the inx phenotype is not the result of a systemic
growth defect but that inx1 and inx2 have a specific role in the
brain. The specificy of inx1RNAi and inx2RNAi was checked out using in silico methods, which predict no off-targets.
The data are consistent with the recent results of Holcroft (2013), who showed that targeted RNAi against inx1 (ogre) or inx2
in glia disrupts development of the larval nervous system and
leads to adult behavioral phenotypes. This study demonstrates that
innexins are not required to link NSCs either to each other or to
glial cells. Instead, Inx1 and Inx2 are required within the glial population
alone for brain development (Speder, 2014).
To understand how glial gap junctions regulate growth in the
CNS, NSC behavior was examined after inx1 or inx2 knockdown
at different time points during the process of NSC reactivation.
Knockdown of inx1 or inx2 in glia did not affect the number of
NSCs in the ventral nerve cord (VNC), demonstrating
that the phenotype is not due to the loss of NSCs prior to NSC reactivation
(0 hr after larval hatching, ALH0). Next, cell
diameter was assessed because one of the earliest events in NSC exit from
quiescence is cell enlargement. It was found that NSC diameter is markedly
reduced (ALH24) after inx1 or inx2 knockdown in glia.
Finally, NSC proliferation was assessed
after inx knockdown. The
mitotic marker phosphohistone H3 (PH3) was assessed
before NSC reactivation (ALH0), just after reactivation (ALH48)
and at a time when wild-type NSCs are cycling actively
(ALH72). Knockdown of either inx1 or inx2 in glial cells resulted
in a severe reduction in the number of dividing NSCs at all times. It was found that NSC enlargement and entry into
mitosis were also dramatically impaired in inx1 and inx2 mutants, and that reactivation could be rescued in inx2 mutants by
glial expression of inx2. It is concluded that
inx1 and inx2 are required in the glia for NSC exit from quiescence (Speder, 2014).
Gap junction proteins (connexins, pannexins, or innexins) are
classically involved in forming intercellular channels or hemichannels,
which enable exchange between the cytoplasm and
the extracellular medium. Evidence also exists for channel-independent
roles, such as cell adhesion and direct gene regulation. To
test if channel function is important for NSC reactivation, brains were treated in culture with carbenoxolone, a classic blocker of
gap junction channels and hemi-channels. Carbenoxolone
completely blocked NSC reactivation,
implying a channel role for Inx1 and/or Inx2, the only innexins
required for NSC reactivation. It was also found that protein fusions
that interfere with the folding of the innexin N-terminal domain
(GFP-Inx1 and RFP-Inx2), which is essential for channel formation
(Nakagawa, 2010), act as dominant-negative mutants. This suggests that
the function of Inx1 and Inx2 in glia is channel-based (Speder, 2014).
Inx1 and Inx2 could be part of the same channel or form two
distinct channels, performing different functions that are both
required for NSC reactivation. Gap junction channels are formed
by the apposition of connexons (innexons in Drosophila) on adjacent
cells. Connexons can be homomeric, formed from six molecules
of a single subtype of connexin, or heteromeric, formed
from different subtypes. In the larval VNC prior to NSC reactivation
(ALH7), Inx1 and Inx2 were strongly expressed in glia and
colocalized in plaques typical of gap junctions. Super-resolution microscopy further demonstrated the tight association of Inx1
and Inx2, using tagged fusion proteins. This
close association is present from hatching and is not lost under
starvation conditions, demonstrating that
formation of the complex is not driven by nutrition.
Most Inx1 staining was lost after knockdown of
inx2 in glia, and vice versa. This suggests that Inx1 and Inx2
localization is interdependent and that Inx1
and Inx2 form heteromeric innexons
rather than independent gap junction channels. Inx1 and Inx2 have been shown to form functional heteromeric
channels in paired Xenopus oocytes (Holcroft, 2013).
It is concluded, therefore, that Inx1 and Inx2 form heteromeric
channels or hemi-channels in the glia (Speder, 2014).
Interestingly, a change was observed in Inx1/Inx2 colocalization
over time. By ALH24, when reactivation has taken place, Inx1
and Inx2 are still expressed but they no longer colocalize, suggesting that formation of Inx1/Inx2 channels is temporally regulated. Consistent with this observation, it was
discovered that the temporal requirement for inx1 and inx2 function
in NSC reactivation is between ALH0 and ALH24. Therefore, the formation and
maintenance of Inx1 and Inx2 heteromeric channels are developmentally
regulated and coincide with the time when the innexins
are required for NSC reactivation (Speder, 2014).
Inx1/Inx2 channels are required in glia to transmit nutritional
stimuli to quiescent NSCs, They are likely to be found, therefore,
in cells situated between the NSCs and the exterior of the brain.
To determine in which glial cells Inx1/Inx2 are required, Inx1/Inx2 was knocked down in different glial populations using subtype-
restricted GAL4 drivers to drive RNAi or express dominant-negative
constructs. inx function was found to be
necessary within the subperineurial glia because knockdown in
this glial subtype alone phenocopies knockdown in the entire
glial population, preventing NSC reactivation (Speder, 2014).
The subperineurial glia and the perineurial glia constitute the
Drosophila BBB. In vertebrates, the BBB
consists of a single layer of vascular endothelium closely associated
with astrocytic glia. The BBB shields
the brain from the external environment owing to tight junctions
between endothelial cells. It acts as a selective sieve to reject
potentially neurotoxic factors but allow the passage of nutrients, ions, or other signals to maintain brain homeostasis (Speder, 2014).
The Drosophila BBB exhibits similar
neuroprotective strategies to its vertebrate
counterpart, including a layer that limits the diffusion of
neurotoxic factors, and an array of conserved transporters that
regulates BBB permeability. The subperineurial glia
are large, flat polyploid cells that envelop the brain and are
closely apposed to the NSCs. They isolate
the brain from the hemolymph (the Drosophila equivalent of
blood) by virtue of lateral septate junctions.
Knockdown of inx did not disrupt the septate junctions, and no change was seen in
Dextran dye penetration. Although weak permeability defects
are difficult to detect at this stage and cannot be excluded, they do not prevent NSC reactivation (moodyD17 mutant). These data suggest that the inx mutant phenotype is not due to an impaired, leaky BBB. Using super-resolution microscopy, Inx1 and Inx2 were detected along the BBB membranes and the septate junctions lining the lateral cell membranes. It is concluded that Inx1/Inx2 channels are required autonomously in the BBB glial cells for NSC reactivation (Speder, 2014).
NSC reactivation requires the expression and secretion of insulin-
like peptides, dIlps, by BBB glial cells (Chell, 2010).
Of the eight identified insulin-like peptides in Drosophila, dIlp6
transcription was shown to increase dramatically in the CNS
upon feeding. Furthermore, when larvae were starved, forced
expression of dIlp6 in the glia was able to rescue NSC reactivation
(Chell, 2010). dIlp6 binds to the insulin receptor
(InR) on NSCs, activating the PI3K/Akt pathway and inducing
exit from quiescence (Chell, 2010; Speder, 2014 and references therein).
Whether gap junction proteins within the BBB are
required for insulin signaling was assayed. A significant decrease was found in
dIlp6 transcription after knocking down both inx1 and inx2 in
the glia. Next, dIlp6 secretion was assayed. In the
absence of an effective dIlp6 antiserum, a tagged,
functional version of dIlp6 (dIlp6-FLAG) was expressed in the BBB glia. dIlp6 secretion from the BBB glia was found to be strongly impaired in
inx1 loss-of-function mutants. Secretion
of dIlp6 was similarly impaired upon starvation. Therefore, both the expression and secretion of dIlp6
are regulated by nutrition and depend on gap junction proteins (Chell, 2010; Speder, 2014 and references therein).
NSC reactivation in inx mutants was rescued by forced
expression of dIlp6 in glia, as shown by the recovery of brain volume (80% of the brains) and of NSC diameter. Direct activation of the PI3K/Akt pathway in NSCs also resulted in rescue of brain volume and NSC enlargement and entry into mitosis. It is concluded that gap junction proteins in the BBB glia are required to activate insulin signaling and induce NSC reactivation (Speder, 2014).
Secretion of insulin by the pancreas is induced by glucose,
leading to synchronized calcium oscillations within gap junction-
coupled beta cells and insulin exocytosis. Gap junctions enable the passage of secondary
messengers that either trigger the release of calcium from intracellular
stores or the influx of calcium from the extracellular environment. Blocking
gap junctions inhibits coordinated intercellular calcium signaling. Gap junction
proteins are thus an important means of transmitting calcium
waves (Speder, 2014).
To further assess the extent of calcium oscillation coordination
within the BBB glia under these conditions, correlation
analysis was performed for 20 regions of
interest (ROI) chosen at random within the BBB glial layer. In fed larvae before reactivation, the central correlation peak demonstrates the synchronicity
of calcium oscillations within the BBB layer. Additional
peaks on each side reveal that this synchronicity repeats (Speder, 2014).
Next, calcium dynamics were monitored in inx1 mutants. None
of the mutant brains showed coordinated calcium oscillations. Instead, BBB glial cells
pulse independently, with no coordination between neighboring
cells. It is concluded that Inx1/Inx2 gap
junctions are required to coordinate synchronous calcium oscillations
within the BBB glia. In accordance with this observation,
the graph of correlation coefficient for inx1 mutants established
the total absence of synchronicity between BBB glial cells, showing that gap junctions are required for propagating calcium oscillations within the BBB (Speder, 2014).
To assess whether calcium oscillations in the BBB are induced
by a nutritional stimulus, calcium dynamics were first assayed in
the BBB glia of newly hatched larvae (ALH0), before they started
to feed. The calcium oscillations differed both in extent and frequency
from those seen in fed larvae. Correlation analysis revealed a partial coordination
within the BBB glia, strengthening the
idea that nutrition is important for extending and establishing
robust calcium synchronicity in the BBB (Speder, 2014).
Next, calcium dynamics were assessed after starvation, specifically
the absence of essential amino acids. Calcium dynamics in
the BBB of starved larvae resembled inx1 mutant brains. Synchronous calcium oscillations were completely abolished in all brains examined.
Individual BBB cells displayed some calcium pulses, but with different profiles to those seen in fed larvae. In addition, correlation analysis of starved larvae showed very
weak synchronicity. This suggests
that the synchronous, nutrition-dependent, calcium oscillations
are lost upon starvation. Importantly, neither Inx1 nor Inx2 is
lost under starvation conditions. It is concluded that
nutrition, in particular essential amino acids, shape calcium
dynamics. Upon feeding, calcium oscillations are amplified and
synchronized across the BBB (Speder, 2014).
Whether glial calcium oscillations arise from the
release of intracellular calcium, or from the influx of extracellular
calcium, was assayed, along with how these influence NSC reactivation. First, the importance of the inositol-triphosphate (IP3)
pathway was assessed. The IP3 pathway triggers calcium release from intracellular
stores. Stimulation of G-coupled receptors
by a wide range of signals activates phospholipase C, leading to
the production of IP3 from cleaved PIP2. IP3 then binds to its
receptor (Ins3PR), a ligand-gated Ca2+ channel found on the surface
of the ER, releasing intracellular calcium.
Ins3PR (itpr in Drosophila) was knocked down in the BBB glia by RNAi. Both NSC enlargement and proliferation were strongly impaired. It is concluded that NSC reactivation depends on IP3-mediated release of calcium from intracellular
stores (Speder, 2014).
Next, the importance of calcium influx was assessed. Membrane
depolarization triggers the entry of extracellular calcium
via voltage-gated calcium channels, whereas hyperpolarization prevents it.
BBB membranes were hyperpolarized by expressing the inward-rectifying
potassium channel, kir2.1. BBB hyperpolarization blocks NSC reactivation dramatically, as revealed by the complete failure of both enlargement and mitotic
re-entry. Interestingly, the mushroom body
neuroblasts, a small group of central brain NSCs that do not
undergo quiescence and reactivation, are not affected by BBB
hyperpolarization, suggesting that nutrition-dependent NSC reactivation
is specifically affected (Speder, 2014).
BBB hyperpolarization both decreases dIlp6 mRNA
levels and dIlp6 secretion,
similar to what is seen during starvation or gap junction loss-of-
function. In support of
the role of calcium oscillations in reactivating NSCs, it was found
that overexpressing the calcium-binding protein, calmodulin,
prevents NSC reactivation. These results
show that intracellular and extracellular calcium both contribute
to NSC reactivation (Speder, 2014).
Gap junction proteins within the BBB glia are required for insulin expression and secretion, a prerequisite for NSC exit from quiescence. This study demonstrates that gap junction proteins coordinate glial calcium oscillations that are required for NSC reactivation. Both intracellular calcium stores and calcium influx contribute to reactivation. Membrane depolarization is known to regulate exocytosis via calcium signaling, which controls stimulus-secretion coupling in secretory cells, such as in endocrine cells. Conditions that block calcium oscillation in the BBB glia (the loss of gap junction proteins, starvation) also impair insulin secretion (Speder, 2014).
The sequence of events leading to glial secretion of insulin bears a striking resemblance to the diet-induced release of insulin by the beta cells of the pancreas. In the pancreas, a nutritional stimulus is sensed by gap junction-coupled beta cells, inducing depolarization resulting in synchronized calcium oscillation and insulin secretion. Loss of gap junction coupling results in uncoordinated calcium pulses. In Drosophila inx loss of function mutants, individual subperineurial BBB glial cells oscillate independently of one another. Compared to starvation, in which the nutritional signal is absent and NSC reactivation cannot occur, the scattered signals from individual BBB cells are able to induce delayed, asynchronous, reactivation in a small number of NSCs. It is proposed that gap junction function within the BBB enables glial insulin release to reach a threshold high enough to trigger NSC reactivation throughout the central nervous system (Speder, 2014).
In both beta cells and BBB cells, membrane depolarization is crucial for generating calcium oscillations. Failure to depolarize or an active block to depolarization prevents insulin release. However, sustained depolarization of β cells can lead to desensitization and a decline in insulin release. Interestingly, this study found that forced depolarization of BBB glia only mildly enhances NSC reactivation. This could be due to desensitization or it may be that the system is already maximally active (Speder, 2014).
Insulin mRNA levels are decreased after gap junction knockdown, both in pancreatic islets (Bosco, 2011) and in the BBB glia. In both cases it remains to be determined if calcium oscillations can directly affect gene expression, as has been shown in other systems (Speder, 2014).
Insulin produced by the pancreas is distributed via the circulatory system, whereas glial insulin is secreted locally, directly to underlying NSCs. Glial insulin signaling is thus contained within the brain, enabling local, differential regulation of this organ. The BBB acts both as a niche and as a protective barrier, providing specific factors directly to the stem cell while shielding the brain from unwelcome systemic regulation. In the context of NSC reactivation, these two roles are conveniently complementary. In the vertebrate BBB, similar functions may be split between endothelial cells and astrocytic glia. The vascular endothelium provides the barrier function, while astrocytic glia have a regulatory role in sensing and adjusting barrier permeability to various stimuli. BBB endothelial cells can secrete cytokines, chemokines, and prostaglandins, suggesting that the BBB behaves like an endocrine tissue. Interestingly, calcium oscillations have been observed in cultured vertebrate BBB endothelial cells, but their function is largely unknown (Speder, 2014).
Gap junction communication can influence stem cell behavior by directly coupling stem cells to each other or to supporting cells, such as found in a stem cell niche. In the brain, connexon-mediated communication has been reported to occur between progenitor cells, within astrocytic networks, and between radial glia and neurons or progenitor cells and astrocytes). The proliferation of neural progenitors and the formation of cortical layers in the mouse brain depend on an intercellular gap junction network, and grafted human NSCs integrate into organotypic cultures through connexin coupling. This study shows that gap junction function within a niche, the BBB, can also influence NSC behavior.
The Drosophila BBB is a protective and selective barrier as well as a signaling center that orchestrates major developmental and physiological events. Here we show that gap junction communication enables cells within the BBB to act as a concerted unit, leading to coordinated calcium signaling and insulin release. Similarities between the BBB in vertebrates and invertebrates suggest that our findings are likely to have broader significance (Speder, 2014).
Innexins are one of two gene families that have evolved to permit neighbouring cells in multicellular systems to communicate directly. Innexins are found in prechordates and persist in small numbers in chordates as divergent sequences termed pannexins. Connexins are functionally analogous proteins exclusive to chordates. Members of these two families of proteins form intercellular channels, assemblies of which constitute gap junctions. Each intercellular channel is a composite of two hemichannels, one from each of two apposed cells. Hemichannels dock in the extracellular space to form a complete channel with a central aqueous pore that regulates the cell-cell exchange of ions and small signalling molecules. Hemichannels can also act independently by releasing paracrine signalling molecules. optic ganglion reduced (ogre) is a member of the Drosophila innexin family, originally identified as a gene essential for postembryonic neurogenesis. This study demonstrates, by heterologous expression in paired Xenopus oocytes, that Ogre alone does not form homotypic gap-junction channels; however, co-expression of Ogre with Innexin2 (Inx2) induces formation of functional channels with properties distinct from Inx2 homotypic channels. In the Drosophila larval central nervous system, Inx2 partially colocalises with Ogre in proliferative neuroepithelia and in glial cells. Downregulation of either ogre or inx2 selectively in glia, by targeted expression of RNA interference transgenes, leads to a significant reduction in the size of the larval nervous system and behavioural defects in surviving adults. It is concluded that these innexins are crucially required in glial cells for normal postembryonic development of the central nervous system (Holcroft, 2013).
The genome of Drosophila has eight innexin-encoding loci, transcripts from seven of which, shakB, ogre, inx2, inx3, inx5, inx6 and inx7, are found at some stage in the nervous system. Knowledge of precise spatial and temporal patterns of expression and functions of the individual genes is far from complete. ShakB, Inx6 and Inx7 mediate electrical signalling in defined adult neural circuits. These and the other innexins are widely and dynamically expressed during development and previous studies have highlighted roles for Ogre and Inx7 ) in neural development. The key findings of this study are that Ogre and Inx2 are required in glia for normal postembryonic development of the CNS. Their expression patterns in these cells partially overlap and, in vitro, channel activity of one protein is influenced by the presence of the other. Thus, the proteins could act independently and/or in concert (Holcroft, 2013).
Paired Xenopus oocytes have been used widely as a heterologous system to examine the ability of proteins to form gap-junction channels. In this system, Inx2 forms homotypic channels. Previous studies demonstrated that the properties of these channels are influenced by co-expression of Inx3, which itself is not competent to form intercellular channels. This study demonstrate that Ogre, like Inx3, does not form intercellular channels independently, but when co-expressed also modifies intercellular conductance in Inx2-expressing cells. Specifically, co-expression of Ogre with Inx2 in both cells of a pair reduces the voltage sensitivity, without affecting the mean level, of intercellular conductance. When expressed in single oocytes, magnitude of hemichannel currents varies with Ogre+Inx2>Inx2>Ogre. There are a number of possible mechanisms to explain these findings. Trafficking of Ogre to the membrane might be aided by Inx2; once at the membrane, Ogre, like Inx2, might form homomeric hemichannels and homotypic intercellular channels. Recordings from co-expressing cells would then reflect the presence of two distinct populations of channels. Alternatively, Ogre and Inx2 might preferentially assemble heteromeric hemichannels that dock to form intercellular channels with properties distinct from Inx2 homotypic channels. The latter is favored because even at high levels of RNA ogre did not induce homotypic intercellular channels although increasing the amount of RNA injected did induce non-junctional currents. There is also some precedent for such a mechanism as Inx2 and Inx3 have been shown to form hetero-oligomers (Holcroft, 2013).
Previous studies demonstrated that Ogre is expressed in the larval optic lobe proliferation centres. This is confirmed in this study, and it was found that Inx2 is also expressed in the optic anlage. The two innexins show significant overlap particularly in the opc and to a lesser extent in the ipc. Neither innexin is found in postmitotic neurons. By contrast, both proteins are extensively expressed, in a largely overlapping pattern, in several populations of glial cells in the larval brain lobes and ventral ganglion. The presence of two Inx proteins in highly overlapping patterns in different cell types in the developing CNS prompts several questions. Are both required for normal development? If so, in which cell types do they act and what is the mechanism of action (Holcroft, 2013)?
This study has demonstrated, by cell-specific knockdown, that Ogre and Inx2 are critically required in glial cells for normal development of the postembryonic CNS. Downregulation of the expression of either protein specifically in glia leads to a marked reduction in the size of the larval CNS. In the case of Inx2, the flies die as larvae. Flies with reduced glial Ogre expression, in contrast, develop to eclosion and survive briefly despite a small and morphologically abnormal CNS. Loss of ogre function in surviving adults leads to defective locomotor and sensorimotor activity. The behavioural phenotypes presumably are due to the failure of adult neural circuitry to develop and are reminiscent of those seen in various neural degeneration or neural wiring mutants. Circling behaviour was first described in pirouette mutants and associated with degeneration of the brain, particularly the optic lobes. Abnormal grooming is seen in flies with mutations in various molecules involved in axon growth and synaptogenesis. Flies with mutations in the cyclin-dependent kinase activator, p35, have defects in axon patterning at early developmental stages and as adults exhibit a supine phenotype (inability to right themselves) (Holcroft, 2013).
This is the first study to implicate inx2 (which is involved in epithelial morphogenesis) in neural development. ogre has a known role in postembryonic neurogenesis. The major defect was a reduction in the size of the CNS, particularly the optic lobes, the extent of which correlated with the severity of the mutation. Hypomorphic mutants survived to adulthood but with highly abnormal neuronal architecture, at least in the visual system, which was the focus of analysis. The CNS of putative null mutants was even smaller than that of hypomorphs and these flies died as late larvae or pupae. The site of ogre activity was mapped to the CNS but not to specific cell types therein. The strong expression of the protein in the wild-type optic proliferation centres and scattered neuroblasts, coupled with the profound reduction in the size of the nervous system in ogre mutants, was consistent with a requirement for the gene in the neural precursors. Interestingly, genetic mosaic analysis provided some evidence of cellular non-autonomy of the phenotype within the optic lobes (Lipshitz, 1985). The current data support the latter in demonstrating that downregulation of ogre in glial cells largely reproduces the ogre mutant phenotype. Downregulation of inx2 produces a remarkably similar phenotype. The accumulated data lead to the conclusion that these two innexins act in glial cells to regulate postembryonic neurogenesis and, quite possibly, subsequent stages of neuronal development (Holcroft, 2013).
Classically, gap-junction proteins act by forming intercellular channels that permit direct transfer of molecules between coupled cells. Increasingly, there is evidence that these proteins also have additional functions ranging from the formation of functional hemichannels that release signalling molecules such as ATP to acting as cell adhesion molecules. In considering how glial-expressed Ogre and Inx2 might regulate neuronal development it is possible to hypothesize both junctional and non-junctional mechanisms, which are not mutually exclusive. Intercellular and/or hemichannel communication among neighbouring glial cells might be crucial for development and maintenance of the extensive glial network, which, in turn, supports neurons. Alternatively, the primary role of glial innexins might be in transfer of signals from these cells to developing neurons. The former is an attractive hypothesis because glial cells are known to regulate neuronal development through provision of signalling molecules including Anachronism and Perlecan, E-cadherin and TGF-β family member Myoglianin. However, both scenarios are possible, and distinguishing these are key questions for future studies (Holcroft, 2013).
The control of self-renewal and differentiation of neural stem and progenitor cells is a crucial issue in stem cell and cancer biology. Drosophila type II neuroblast lineages are prone to developing impaired neuroblast homeostasis if the limited self-renewing potential of intermediate neural progenitors (INPs) is unrestrained. This study demonstrates that Drosophila SWI/SNF chromatin remodeling Brahma (Brm) complex functions cooperatively with another chromatin remodeling factor, Histone deacetylase 3 (HDAC3) to suppress the formation of ectopic type II neuroblasts. Multiple components of the Brm complex and HDAC3 physically associate with Earmuff (Erm), a type II-specific transcription factor that prevents dedifferentiation of INPs into neuroblasts. Consistently, the predicted Erm-binding motif is present in most of known binding loci of Brm. Furthermore, brm and hdac3 genetically interact with erm to prevent type II neuroblast overgrowth. Thus, the Brm-HDAC3-Erm repressor complex suppresses dedifferentiation of INPs back into type II neuroblasts (Koe, 2014).
This study reports a critical function of the Drosophila Brm remodeling complex in suppressing the formation of ectopic type II neuroblasts in larval brains. Mutants of major components of the Brm complex, including Brm and Bap55, and RNAi targeting of several Brm components formed ectopic type II neuroblasts. Therefore, the Drosophila Brm remodeling complex displays a tumor suppressor-like function in larval brains. Multiple subunits of the SWI/SNF complex are associated with various cancers. BAP47 (homologous to Snr1) is a bona fide tumor suppressor and the gene is deleted in pediatric rhabdoid tumors. Mutations in epigenetic regulators are found in approximately half of hepatocellular carcinoma and bladder cancers, and represent a significant portion of mutated genes in medulloblastoma. Drosophila Brm complex is essential for intestinal stem cell proliferation and commitment in the adult intestine. Two other chromatin remodeling factors, Iswi and Domino control germline stem cell and somatic stem cell self-renewal in the ovary (Koe, 2014).
Brm was shown to physically associate with Erm, a type II-specific transcription factor that prevents the dedifferentiation of INPs back into neuroblasts. Furthermore, Bap60 and Snr1, two other components of the Brm complex, also physically associate with Erm in a protein complex. Therefore, this study has provided the first molecular link during the regulation of type II neuroblast lineages. It is speculated that the association with Erm may provide functional specificity of the Brm remodeling complex in type II neuroblast lineages. It was also shown that brm genetically interacts with the type II-specific transcription factor erm. Ectopic neuroblast phenotype resulting from brm knockdown was dramatically enhanced by simultaneous knockdown of erm. Furthermore, brm knockdown, similar to erm-, can be partially suppressed by loss of Notch. These functional data suggest that Erm is a co-factor of the Brm remodeling complex in type II neuroblast lineages. However, it is uncertain how the Brm-Erm protein complex functions to prevent dedifferentiation in type II neuroblast lineages (Koe, 2014).
Bioinformatic analysis has identified a 14 bp-long motif as the de novo Erm DNA-binding motif and 202 sites out of the 270 known genomic loci harboring Brm also contain the de novo Erm DNA-binding motif. As there are many genes that are potentially co-occupied by Brm and Erm, it is possible that Brm-Erm complex results in a unique configuration of the chromatin 'landscape' in INPs to prevent INP dedifferentiation into neuroblasts. Therefore, disruption of chromatin remodelers may cause widespread changes to the transcriptome, thus amplifying the effect of the single genetic mutation (Koe, 2014).
Most class I HDACs are recruited into large multi-subunit co-repressor complexes for maximal activity. HDAC1 and 2 are found in multiple co-repressor complexes, while to date HDAC3 appears to be uniquely recruited to the Silencing mediator of retinoic and thyroid receptors (SMRT)/Nuclear receptor co-repressor (N-CoR) complex. This study reports that Drosophila HDAC3 is recruited to a novel multi-subunit complex containing Brm and Erm and that this co-repressor complex prevents dedifferentiation of INPs into type II neuroblasts. The SMRT complex appears not to be important for type II neuroblasts, as knockdown of smrter that encodes a core component of the SMRT complex neither resulted in any ectopic type II neuroblasts nor enhanced the phenotype of ectopic neuroblasts by brm knockdown. This study also showed that HDAC3 dramatically enhanced the phenotype of ectopic neuroblast upon loss of brm or snr1, two core components of the Brm complex. By identifying this novel repressor complex, this study has provided a mechanistic link between transcriptional repression and histone deacetylation during the suppression of dedifferentiation. HDACs are typically recruited by oncogenic protein complexes in lymphoma and leukemia and HDAC3 inhibitors are synergistic or additive with anticancer agents for therapeutics. The finding that HDAC3 functions cooperatively with the Brm complex in suppressing suppressing dedifferentiation of INPs into neuroblasts and induces tumors in the allograph transplantation revealed an unexpected potential involvement of HDAC3 in tumor suppression in brain tissue. It will be of interest to determine whether this effect is conserved in the mammalian central nervous system and whether it occurs in tissues other than the brain (Koe, 2014).
Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. This study shows that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3'UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b were highly expressed in neuroblasts of larval brain where Jigr1 expression was low. Genetic deletion of both miR-92a and miR-92b demonstrated an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. miR-92a and miR-92b directly targeted jigr1 in vivo and some phenotypes due to the absence of these miRNAs were partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development (Yuva-Aydemir, 2015).
MicroRNAs (miRNAs) are short (~21-23 nt) noncoding RNAs that regulate gene expression post-transcriptionally in many physiological and pathological processes. In the canonical miRNA biogenesis pathway, a long primary transcript (pri-miRNA) is generated by RNA polymerase II and cleaved by a nuclear complex formed by Drosha and DGCR8. Some pri-miRNAs produce miRNAs only (intergenic miRNAs) while others contain miRNAs in the intronic regions of protein-coding 'host' genes (intragenic miRNAs). Many intronic miRNAs and host gene mRNAs are likely co-expressed but others may not be. Few cases have been experimentally confirmed, and the functional significance of such a genomic arrangement is largely unknown (Yuva-Aydemir, 2015).
This study used the differentiation of Drosophila neuroblasts as a model system to examine the expression and function of specific miRNAs. Drosophila neuroblasts form during embryonic development and enter a proliferative quiescent state at the end of embryogenesis. In the early larval stage, neuroblasts reenter the cell cycle and undergo a series of proliferative symmetric and self-renewing asymmetric cell divisions to maintain the progenitor pool and generate diverse cell types. In each asymmetric cell division, neuroblasts divide to generate a neuroblast cell and a ganglion mother cell, which divides only once to generate two neurons or one neuron and one glial cell. The balance between self-renewal and differentiation is critical for normal development, but the mechanisms are incompletely understood (Yuva-Aydemir, 2015).
This study shows that the gene encoding jing-interacting gene regulatory 1 (jigr1), a putative DNA-binding protein containing MADF domain with unknown function (Sun, 2006), hosts miR-92a in the intron and miR-92b in the 3'UTR. The functional significance of this intragenic miRNA-host gene interaction was uncovered through genetic knockout of both miR-92a and miR-92b. During larval development, miR-92 family limits jigr1 expression in neuroblasts and is essential for maintenance of a neuroblast pool. It is proposed that this genomic arrangement and local feed-back regulatory loop are essential for animal development to ensure the generation of the proper number of neuronal and glial cells (Yuva-Aydemir, 2015).
This paper reports an unusual genomic arrangement in which miR-92a and miR-92b are embedded in the intron and 3'UTR of the host gene jigr1, respectively. In neuroblasts, miR-92a and miR-92b were highly expressed as a single transcriptional unit also containing jigr1 coding region. Genetic analysis in Drosophila showed that downregulation of jigr1 by intragenic miR-92a and miR-92b is required for neuroblast self-renewal, providing an example of the functional significance of miRNA-host gene interactions in animal development (Yuva-Aydemir, 2015).
Nearly half of the miRNAs in mammals and Drosophila are located within protein-coding genes. Most of these intragenic miRNAs are co-expressed with their host genes, but both positive and negative feedback regulation of host gene expression and function by miRNAs remains largely unknown. Most intragenic miRNAs are located in the introns of their host genes and are processed by the mirtron pathway, bypassing the microprocessor complex. On the other hand, miRNAs are rarely located in the 3'UTR of a protein-coding gene, and the effect of this organization on host gene expression or miRNA processing is not clear. The exonic miR-198, which is located in the 3'UTR of the gene encoding human folistatin like 1, is processed from a single transcript with its host gene in a mutually exclusive way. In contrast, direct regulation of some jigr1 isoforms by miR-92a and miR-92b largely accounts for the observed complementary expression domains of jigr1 and these miRNAs, although the possibility cannot be completely rule out that other mechanisms may also contribute to jigr1 repression (Yuva-Aydemir, 2015).
Through a genetic knockout of both miR-92a and miR-92b in Drosophila, which has not been done so far in any other model organism, this study discovered a novel function for miR-92a and miR-92b in neuroblast self-renewal. The findings are consistent with results obtained in mammals. However, unlike Drosophila, human and mouse miR-92a and miR-92b genes are not intragenic. Mouse miR-92a genes are located in two clusters: miR-17-92 and miR-106a-303. In developing mouse neocortex, the miR-17-92 cluster promotes neural stem cell expansion and regulates the transition to intermediate progenitors through repression of Pten by miR-19 and Tbr2 by miR-92a (Blan, 2013). Similarly, acute loss and gain of miR-92b function in mouse cortex showed that miR-92b restricts the generation of intermediate progenitor cells by suppressing Tbr2. Moreover, miR-92b maintains asymmetric division of neural stem cells by restricting Tis21 expression in mouse neocortex. However, phenotypes caused by loss of both miR-92a and miR-92b in mammals have not been reported yet. This study found that miR-92a and miR-92b work in concert to restrict Jigr1 expression in Drosophila larval neuroblasts and thereby maintain the neuroblast pool (Yuva-Aydemir, 2015 and references therein).
Jigr1, a putative MADF-domain-containing transcription factor of unknown function expressed ubiquitously in the larval central nervous system, is expressed at low levels in neural progenitor cells. The findings suggest that the progressive loss of neuroblasts in miR-92-/- brains is due to premature differentiation of these cells resulted from cell-autonomous effect of loss of miR-92, as shown by ectopic expression of nuclear Prospero, decreased BrdU uptake, and reduced cell size. Upregulation of Jigr1 seems to play a role in nuclear Prospero expression in miR-92-/- neuroblasts, since reducing Jigr1 expression eliminated this phenotype. Moreover, overexpression of Jigr1 in neuroblasts on a wild type background leads to ectopic Prospero expression and a premature differentiation phenotype. In summary, these results reveal a local regulatory loop in which miR-92a and miR-92b are expressed in the jigr1 transcription unit and also work in concert to prevent premature differentiation of neuroblasts by limiting expression of the host gene (Yuva-Aydemir, 2015).
Asymmetric stem cell division establishes an initial difference between a stem cell and its differentiating sibling, critical for maintaining homeostasis and preventing carcinogenesis. Yet the mechanisms that consolidate and lock in such initial fate bias remain obscure. This study used Drosophila neuroblasts to demonstrate that the super elongation complex (SEC) acts as an intrinsic amplifier to drive cell fate commitment. SEC is highly expressed in neuroblasts, where it promotes self-renewal by physically associating with Notch transcription activation complex and enhancing HES (hairy and E(spl)) transcription. HES in turn upregulates SEC activity, forming an unexpected self-reinforcing feedback loop with SEC. SEC inactivation leads to neuroblast loss, whereas its forced activation results in neural progenitor dedifferentiation and tumorigenesis. These studies unveil an SEC-mediated intracellular amplifier mechanism in ensuring robustness and precision in stem cell fate commitment and provide mechanistic explanation for the highly frequent association of SEC overactivation with human cancers (Liu, 2017).
Both normal development and tissue homeostasis rely on the remarkable capacity of stem cells to divide asymmetrically, simultaneously generating one identical stem cell and one differentiating progeny. Extensive studies have unveiled how extrinsic niche signals and intrinsic cell polarity cues ensure proper orientation of mitotic spindle and, hence, asymmetric division of stem cells. However, it remains unclear whether the initial fate bias, established by unequal exposure to niche signals or differential partitioning of cell fate determinants, can be immediately and automatically consolidated and stabilized into distinct and irreversible cell fate outcomes. In fact, in vivo timelapse imaging of the developing zebrafish hindbrain using the Notch activity reporter showed that, immediately after the asymmetric division of a radial glia progenitor, Notch activity is not noticeably biased in the paired daughter cells. Instead, the differential Notch activity in the pair of daughter cells only gradually increases afterward, over a time span of 3-8 hr, indicating the existence of a progressive and tightly regulated transition phase between the initial cell fate decision and the ultimate cell fate commitment. Stem cells and progenitors, especially the fast-cycling ones, face the daunting challenges of ensuring timely, precise, and robust cell fate determination in every cell cycle and are likely to achieve so through rapid amplification of the initial small fate bias upon their asymmetric division. In electronics, a device called an amplifier magnifies a small input signal to a large output signal until it reaches a desired level. Conceivably, a similar 'amplifier' mechanism could be employed in the stem cells or progenitors to accelerate the transition phase and drive cell fate commitment. Dysregulation of such an amplifier could cause an imbalance between self-renewal and differentiation, resulting in impaired tissue homeostasis. However, the regulatory modules governing the transition phase from stem cell fate decision to fate commitment, especially the identity and control of this putative 'amplifier,' remain largely unexplored (Liu, 2017).
Drosophila type II neural stem cells (NSCs), known as neuroblasts (NBs), provide an excellent model system for studying stem cell fate commitment. Firstly, distinct from type I NB lineages, type II NB lineages contain transit-amplifying cells called intermediate neural progenitors (INPs), similar to mammalian NSC lineages in both functional and molecular criteria, yet with much simpler anatomy and lineage composition. Each type II NB undergoes stereotypic, self-renewing divisions to produce immature INPs, which, upon maturation, undergo a few rounds of asymmetric, self-renewing divisions to give rise to ganglion mother cells (GMCs) that subsequently generate post-mitotic neurons or glia. Secondly, the identity of each cell type in the NB lineages can be unambiguously determined by a combination of cell fate makers as well as by their geological positions within the lineages. Thirdly, the molecular mechanisms underlying initial NB versus INP fate decision are well understood. Unidirectional Notch signaling is both necessary and sufficient to promote type II NB self-renewal. At each division, type II NBs asymmetrically segregate differentiation-promoting determinants, such as Notch antagonist Numb, into immature INPs. As a consequence, Notch pathway effector HES (hairy and E(spl)) genes, such as E(spl)mγ, are highly expressed in NBs but not in immature INPs. HES genes, encoding basic helix-loop-helix (bHLH) transcription factors, are crucial for promoting NB self-renewal. Importantly, numb mutant immature INPs fail to complete maturation but instead revert fate back into NBs and result in tumorigenesis, indicating that the asymmetric segregation of Numb protein is critical for establishing the initial fate bias between a type II NB and its sibling INP. However, whether such initial bias is sufficient to confer differential Notch activity and achieve definitive fate commitment is currently unclear. Lastly, type II NBs undergo fast cell divisions, dividing every 2 hr, placing them under huge pressure to timely yet precisely achieve differential fate outcomes. Therefore, within type II NB lineages, a regulatory module that drives robust cell fate commitment is likely to exist, plausibly with high activity (Liu, 2017).
Overactivation of Notch signaling leads to immature INP dedifferentiation and tumorigenesis, providing a sensitized background for identifying factors pivotal for NB or INP fate commitment. In such a genetic background a genome-wide RNAi-based screen was carried out for genes whose downregulation specifically suppresses the supernumerary NB phenotype induced by Notch overactivation, and subunits of the super elongation complex (SEC) were identified. The SEC is composed of the elongation factor ELL (eleven-nineteen lysine-rich leukemia) 1/2/3, the flexible scaffolding protein AFF (AF4/FMR2 family) 1/2/3/4, the ELL-associated factor EAF1/2, eleven-nineteen leukemia (ENL)/AF9, as well as the Pol II elongation factor P-TEFb consisting of cyclin T (CycT) and cyclin-dependent kinase 9 (CDK9). The screen identified all subunits of SEC except EAF and ENL/AF9, suggesting that SEC interplays with Notch signaling in promoting NB self renewal. The SEC subunits were originally identified as frequent translocation partners of MLL (mixed-lineage leukemia) in inducing leukemogenesis, and play key roles in c-Myc-dependent carcinogenesis and HIV viral DNA transcription. Previous studies demonstrated that SEC executes its functions by inducing rapid gene transcription, mainly through phosphorylating RNA polymerase II (Pol II) C-terminal domain and releasing it from promoter-proximal pausing (Liu, 2017).
This study shows that the SEC is specifically expressed in Drosophila NBs, where it acts as an amplifier to drive type II NB fate commitment. SEC exerts its function by physically associating with Notch transcription activation complex to stimulate dHES (Drosophila HES) transcription. dHES in turn promotes SEC expression/activity. Thus, driven by a self-reinforcing feedback loop between SEC and Notch signaling, an initial small bias of Notch activity between an NB and its sibling INP is rapidly amplified and consolidated into robust and irreversible fate commitment (Liu, 2017).
Is the establishment of an initial fate bias at the end of stem cell asymmetric division truly the end, or just the beginning of the end? The current findings revealed that a progressive and tightly controlled transition phase exists between the initial fate decision and the final definitive fate commitment. The results identified the evolutionarily conserved SEC as a crucial intrinsic amplifier, accelerating this previously overlooked fate transition phase and ensuring NSC fate commitment in Drosophila type II NB lineages. Inactivation of SEC prevents the self-reinforcing feedback loop between SEC and Notch signaling from running, resulting in NBs with ambiguous stem cell identity and ultimate fate loss. Conversely, ectopic overactivation of SEC initiates and sustains this positive feedback loop within progenitors, driving dedifferentiation and tumorigenesis. It is interesting to note that, as one of the most active P-TEFb-containing complexes in controlling rapid transcriptional induction in response to dynamic developmental or environmental cues, SEC is particularly suitable for being an amplifier in driving timely cell fate commitment. Since fast-cycling stem cells are under huge pressure to achieve robust fate determination in every cell cycle, it is not surprising that they employ SEC as a regulatory component to induce immediate activation of master fate-specifying genes that in turn form a self-amplifying loop with SEC to rapidly magnify the initial fate bias and ensure prompt fate commitment (Liu, 2017).
Such an intracellular amplifier mechanism revealed by these studies might complement the well-established intercellular lateral inhibition mechanism and represent a general, cell-autonomous paradigm to ensure robustness and precision in binary cell fate commitment. Lateral inhibition is a widely used mechanism underlying cell fate diversification, whereby unidirectional Notch signaling utilizes intercellular feedback loops to amplify an initial small difference between adjacent daughter cells, and eventually confers distinct cell fates. Lateral inhibition relies on intercellular interactions between adjacent cells. This study proposes a model whereby an intracellular amplifier mechanism may also diversify cell fates (Liu, 2017).
The intracellular amplifier and intercellular lateral inhibition mechanisms, both acting through feedback loops, are not mutually exclusive. Instead, they are complementary to each other and can be used concomitantly or sequentially to achieve differential fate outcomes in a timely, precise, and robust manner.
An amplifier design often employs negative feedback to prevent excessive amplification. In this study the dHES-Earmuff/Brahma-SEC double-negative regulatory mechanism that this study has revealed in NBs might also operate in neural progenitors, where the Erm/Brm complex could serve as a crucial 'brake' to prevent the Notch-SEC-Notch self-reinforcing positive feedback loop from starting (Liu, 2017).
Notch signaling plays a conserved role during vertebrate embryonic neurogenesis in maintaining the undifferentiated status of NSCs. Intriguingly, expression of HES-1, a primary target of Notch pathway in mammalian neural development, oscillates every 2 hr. It has been proposed that oscillations in HES-1 expression drive fluctuations in gene expression, resulting in differential expressions between neighboring cells, which needs be further amplified to confer distinct cell fates. How such an amplification step is triggered and modulated remains elusive. Given that SEC is highly conserved in mammals, it is interesting to speculate that a similar amplifier mechanism is employed to ensure mammalian NSC fate commitment. Whether SEC interplays with Notch signaling to drive cell fate commitment in other stem cell lineages also warrants future investigation (Liu, 2017).
Despite extensive studies elucidating how SEC regulates transcription elongation, the in vivo function of SEC in normal development and physiology remains enigmatic. The current results indicate that SEC is highly expressed in Drosophila NSCs, where it is recruited by the Notch transcription activation complex to stimulate the transcription of dHES genes and promote self-renewing fate. Interestingly, the dHES genes in fly larval brain NB lineages are non-pausing genes, raising the possibility that SEC promotes the transcriptional activation of dHES in the absence of paused Pol II. Consistent with this view, recent studies have demonstrated that the rapid transcriptional induction of some nonpausing genes, such as Cyp26a1 in human embryonic stem cells and a subset of pre-cellular genes in early Drosophila embryos, depends on SEC activity and Pol II occupancy. The current findings that SEC physically and genetically interplays with the dCSL-NICD-MAM transcription activation complex to
activate dHES transcription thus provide a unique physiological context for elucidating the detailed molecular mechanisms underlying transcriptional induction of non-pausing genes by SEC (Liu, 2017).
The upstream signals and molecular mechanisms controlling SEC activity in normal development or physiology are just unfolding. It has been previously shown that the activity of SEC could be regulated by modulating the kinase activity of CDK9, the catalytic subunit of SEC. The results unveil a new and unexpected mechanism underlying the control of SEC: the Notch-HES axis spatially restricts SEC activity within NSCs by cell-autonomously promoting the protein abundance of dAFF and dELL, two regulatory subunits of SEC. Consistently, overactivation of Notch signaling led to dedifferentiation of immature INPs, in which the expression levels of dAFF/dELL and, hence, the activity of SEC evidently increase (Liu, 2017).
Dysregulation of the SEC subunits is frequently associated with various human cancers including leukemia and glioblastoma. However, in most cases, whether SEC acts as a cancer driver or passenger is unclear. Furthermore, whether SEC subunits exert their oncogenic or tumor suppressive roles as a component of SEC or independent of SEC remains poorly understood. Intriguingly, the results show that overexpression of dELL and dAFF but not either alone induces a dramatic surge of dHES expression in immature INPs and causes progenitorderived tumor. These findings strongly suggest that, in NSC lineages, SEC drives tumorigenesis as an integral complex and exerts its oncogenic function in a dose-dependent manner. Supporting this view, the kinase activity of CDK9 is essential for dELL/dAFF-induced tumorigenesis. It will be interesting to investigate whether upregulation of dELL/dAFF abundance is sufficient to induce carcinogenesis in other biological contexts. Tne findings highlighting the self-reinforcing feedback loop between SEC and Notch signaling in driving tumorigenesis further suggest that CDK9 inhibitors could be pursued as an effective therapy for Notch overactivation-induced tumors (Liu, 2017).
Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, type II neuroblasts' transcriptome was profiled across time. The results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, this study reveals that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. It is therefore proposed that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning (Ren, 2017).
Temporal gradients of IGF-II mRNA-binding protein (Imp) and Syncrip (Syp) RNA-binding proteins have recently been described to promote early and late temporal fates, respectively, in mushroom body (MB) and antennal lobe (AL) lineages (Liu, 2015). Imp and Syp gradients oppose each other (Imp expression decreases and Syp increases over time), and they mutually inhibit each other's expression (Liu, 2015). It is unclear how the Imp-versus-Syp dominance is reversed and why the Imp-to-Syp switch occurs over different time courses in distinct NBs. Imp/Syp gradients control temporal fate in the MB by establishing a temporal gradient of Chinmo, a BTB-zinc finger nuclear transcription factor. In the AL lineages, while Imp/Syp gradients clearly promote early and late fates, it is not clear whether and how these RNA binding proteins specify all temporal fates in the rapidly changing AL lineages (Ren, 2017).
Canonical (type I) NBs produce post-mitotic neurons via budding off ganglion mother cells (GMCs), which each divides once into two neurons. By contrast, each of the 16 type II NBs (eight per brain hemisphere) first generates a series of intermediate neural progenitors (INPs). Each INP can, in turn, produce around five GMCs, thus giving rise to an INP sublineage that consists of a short sequence of neuronal and glial progeny. The complex type II pattern of neurogenesis mimics the production of neurons by mammalian neural stem cells through intermediate precursors (Ren, 2017).
Like type I NBs, type II NBs exhibit lineage identity and temporal fate. Labeling the progeny made by a type II NB (NB clone) reveals distinct lineage-characteristic morphology. Six type II NB lineages (DM1-DM6) originate from the dorsomedial posterior brain surface; the remaining two lineages (DL1 and DL2) arise from the dorsolateral posterior brain surface. An INP produces an invariant sequence of distinct sister neuron pairs, and successive INPs generate a similar, but not identical, neuronal series (Wang, 2014). These observations illustrate temporal fate diversification along both axes of NB and INP self-renewal. A cascade of three tTFs (Dichaete, Grainy head, Eyeless) governs temporal fates within the INP sublineages (Bayraktar, 2013). As to the extended axis of type II NB self-renewal, it is not clear whether a protracted tTF cascade exists or just gradients of proteins could guide the orderly derivation of variant INP sublineages (Ren, 2017).
In order to resolve the temporal fating mechanisms in type II NBs, this study profiled the transcriptome of type II NBs and uncovered 81 dynamic genes, including Imp and Syp. Imp and Syp were shown to promote early and late INP temporal fate, respectively. Two tTFs, Castor (Cas) and Seven-up (Svp), are critical for the initiation of Imp/Syp temporal gradients. Despite no progression of the Imp/Syp gradients, svp mutant clones carried INPs with multiple early fates. It is proposed that Cas/Svp-triggered Imp/Syp gradients confer coarse temporal fates to diversify INPs (Ren, 2017).
RNA-seq of type II NBs across larval development revealed 81 temporally dynamic genes, including Imp and Syp. Utilizing various neuronal classes characteristic of early or late INP sublineages, this study demonstrated that the opposing Imp/Syp gradients govern type II lineage temporal patterning. However, it is not clear whether absolute or relative levels of Imp and/or Syp confer specific temporal fates, or how a given Imp/Syp level is perceived by individual GMCs of the same INP origin. More sophisticated controls over Imp and Syp levels and finer temporal fate readouts are required to resolve these details. Nonetheless, svp mutant NBs, which maintain initial Imp/Syp levels, still undergo some limited temporal fate progression, as evidenced by the presence of various OL-elaboration neurons. It is therefore proposed that the rapidly progressing Imp/Syp gradients confer type II NBs with coarse temporal fates, which guide or permit subtemporal patterning among INPs born within a given Imp/Syp temporal window (Ren, 2017).
A brief series of Cas and Svp bursts initiates the prompt switch of Imp-versus-Syp dominance in type II NBs. Cas likely precedes Svp in its post-embryonic re-expression; however, the dynamic expression of Cas and Svp appear independently yet simultaneously controlled by an unknown temporal cue. Curiously, while Cas is dispensable, ectopic Cas delays the onset of Imp/Syp gradients. As the final NB tTF in the widely expressed embryonic cascade, Cas may be required for some embryonic INP fates that were not examined in this study. With regards to the role of Cas in Imp/Syp expression, it is hypothesized that the termination of Cas expression is important for proper Imp/Syp gradient progression. The Cas-GOF would thus expose the NBs to a prolonged Cas window. Precocious and continuous Svp expression did not accelerate or alter the Imp/Syp gradients, further implicating involvement of unknown temporal cues, not only in the regulation of dynamic Cas/Svp expressions, but also in confining the acute Svp action. It is speculated that the acute Svp signal can repress Imp as well as promote Syp and ultimately place Svp over Imp in their winner-take-all competition. Both the timing and intensity of the Cas/Svp bursts may vary among different NBs, which can potentially shape distinct Imp/Syp gradients in different neuronal lineages (Ren, 2017).
The Imp/Syp gradients in type II NBs are inherited by INPs born at distinct times and confer the INPs with their temporal identity. Each INP subsequently expresses a cascade of tTFs to assign its serially derived GMCs with distinct cell fates. Imp/Syp and their downstream effectors could interact with the INP tTFs to specify terminal temporal fates in post-mitotic cells. Multiple feedforward gene regulatory loops might also be involved to control the expression of terminal selector genes. Distinct Imp/Syp levels may specify different neuronal classes among the GMCs of the same birth order that inherit the same tTF from INPs. It is not possible to resolve such complex temporal fating mechanisms without further sophisticated single-cell lineage mapping tools (Ren, 2017).
Converging evidence indicates that conserved tTFs control neuronal diversity from Drosophila to mammalian species. Mammalian neural stem cells also show temporally patterned neurogenesis. The Ikaros family zinc finger 1, orthologs of Drosophila Hb, specify early temporal fate in both the retina and the cortex. A recent study showed that an ortholog of Drosophila castor, Casz1, promotes late neuronal fates in the mouse retina. The Chick Ovalbumin Upstream Promoter-Transcription Factors (COUP-TFI and II), mammalian orthologs of Drosophila svp, promote the temporal transition from neurogenesis to gliogenesis in neural stem cells. COUP-TFI also orchestrates the serial generation of distinct types of cortical interneurons. Loss of COUP-TFs resulted in overproduction of early-born neuronal fates, at the expense of late-born glial and interneuron fates. Thus, COUP-TFs seem to be functionally conserved to Drosophila svp (Ren, 2017).
Notably, a descending Imp gradient exists in mouse neural stem cells and governs temporal changes in stem cell properties. Given the use of INPs in both type II NB lineages and mammalian neurogenesis, it would be interesting to determine whether homologs of Imp/Syp/Chinmo play analogous roles in regulating the temporal fates of mammalian neural stem cells and whether these genes act with the conserved tTFs (Ren, 2017).
Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. This study shows that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, tumour formation could be blocked completely by re-expressing Asense (homologue of human ASCL1), which is a direct target of Tailless. These results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells (Hakes, 2020).
The results revealed the mechanism through which high levels of the orphan nuclear receptor Tll initiate tumours in the Drosophila CNS. Tll is expressed in Type II NSCs during larval development, where it is required for Type II NSC identity and subsequent lineage progression. In the absence of Tll, the proneural transcription factor Ase is derepressed in Type II NSCs. As a consequence, transit amplifying INPs are no longer generated and the resulting NSC lineages have a lower neurogenic potential (Hakes, 2020).
In mice, TLX is expressed in NSCs during embryonic development and in adulthood. Embryonic NSCs display defects in proliferation in the absence of TLX and the loss of TLX in adults results in the loss of transit-amplifying intermediates and reduction in neurogenesis. While these effects were previously attributed to changes in the NSC cell cycle, the current results suggest a cell fate change may occur due to the loss of TLX (Hakes, 2020).
High levels of TLX in human glioblastoma are correlated with tumour aggressiveness. High level expression of TLX results in glioblastoma-like lesions derived from SVZ NSC lineages in mouse models of glioblastoma indicating that TLX can also promote glioblastoma development. However, it was not known how high TLX leads to glioblastoma, nor had the cellular origin of TLX-induced tumours been identified. TLX and its Drosophila homologue, Tll, are highly conserved proteins and this study found that both genes are able to revert INPs to NSC fate as a first step in tumour initiation. Ectopic expression of Tll was also sufficient to induce the expansion of NSCs throughout the Drosophila CNS, demonstrating the widespread vulnerability of NSC and progenitor populations to ectopic Tll expression (Hakes, 2020).
This study found that the ectopic NSCs resulting from high Tll expression are negative for Ase. Tll binds to the ase locus, suggesting that Tll directly represses ase. The absence of Ase is a hallmark of Type II NSCs. Therefore, ectopic Tll promotes a cell fate change from INP/Type I NSC to Type II NSC and thereby initiates tumourigenesis (Hakes, 2020).
The capacity of Tll to induce NSC expansion had been reported previously as part of a study showing that Tll regulates the proliferation of larval mushroom body NSCs and GMCs (Kurusu, 2009). The authors showed that overexpressing Tll resulted in ectopic NSCs, but they did not identify the origin of these tumours and argued against a role for Tll in Type II NSC fate. Tll-induced tumourigenesis could be blocked by ectopic expression of Pros (Kurusu, 2009). However, ectopic Pros results in the loss of NSCs even in wild type brains. In contrast, Type I NSC lineages appear normal after Ase misexpression in wild type brains. Furthermore, it has been reported that high levels of the human homologue of Pros, PROX1, exacerbate glioblastoma, arguing against PROX1 expression as a therapeutic strategy (Hakes, 2020).
This study found that the tumourigenic capacity of Drosophila Tll and human TLX was highly conserved. Human TLX could also induce ectopic Type II NSCs from INPs through the repression of Ase. Analysis of scRNA seq from glioblastoma revealed that TLX and ASCL1 expression is mutually exclusive. It is notable that the origin of human glioblastoma has been mapped to the SVZ. While TLX positive NSCs have been identified in both the SVZ and dentate gyrus, high levels of TLX giving rise to glioblastoma has only been shown robustly in the SVZ. Furthermore, a recent study demonstrated that low expression levels of ASCL1 correlate with glioblastoma malignancy. Ectopic expression of ASCL1 in glioblastoma stem cells was sufficient to promote neuronal differentiation. Based on these results in Drosophila, it is predicted that introducing ASCL1 would override the repressive effect of TLX, induce neuronal differentiation and reduce tumour growth, thereby providing an effective treatment (Hakes, 2020).
The results indicate that INPs are the tumour initiating cells in Type II NSC lineages expressing high levels of the orphan nuclear receptor Tll and potentially implicate intermediate progenitors as one of the cells of origin in TLX+ glioblastomas, an aggressive and lethal brain tumour. This study found that Ase is a direct target of Tll and that Ase expression not only blocks Tll-induced tumourigenesis, but also reinstates a normal neural differentiation programme (Hakes, 2020).
Stem cells that indirectly generate differentiated cells through intermediate progenitors drives vertebrate brain evolution. Due to a lack of lineage information, how stem cell functionality, including the competency to generate intermediate progenitors, becomes extinguished during progenitor commitment remains unclear. Type II neuroblasts in fly larval brains divide asymmetrically to generate a neuroblast and a progeny that commits to an intermediate progenitor (INP) identity. This study identified Tailless (Tll) as a master regulator of type II neuroblast functional identity, including the competency to generate INPs. Successive expression of transcriptional repressors functions through Hdac3 to silence tll during INP commitment. Reducing repressor activity allows re-activation of Notch in INPs to ectopically induce tll expression driving supernumerary neuroblast formation. Knocking-down hdac3 function prevents downregulation of tll during INP commitment. It is proposed that continual inactivation of stem cell identity genes allows intermediate progenitors to stably commit to generating diverse differentiated cells during indirect neurogenesis (Rives-Quinto, 2020).
The expansion of outer subventricular zone (OSVZ) neural stem cells, which indirectly produce neurons by initially generating intermediate progenitors, drives the evolution of lissencephalic brains to gyrencephalic brains. Recent studies have revealed important insights into genes and cell biological changes that lead to the formation of OSVZ neural stem cells. However, the mechanisms controlling the functional identity of OSVZ neural stem cells, including the competency to generate intermediate progenitors, remain unknown. This study provided compelling evidence demonstrating that Tll is necessary and sufficient for the maintenance of an undifferentiated state and the competency to generate intermediate progenitors in type II neuroblasts. It was also shown that two sequentially activated transcriptional repressors, Erm and Ham, likely function through Hdac3 to silence tll during INP commitment ensuring normal indirect neurogenesis in larval brains. It is proposed that continual inactivation of stem cell functional identity genes by histone deacetylation allows intermediate progenitors to stably commit to generating sufficient and diverse differentiated cells during neurogenesis (Rives-Quinto, 2020).
Stem cell functional identities encompass the maintenance of an undifferentiated state and other unique functional features, such as the competency to generate intermediate progenitors. Because genetic manipulation of Notch signaling perturbs the regulation of differentiation during asymmetric stem cell division, the role of Notch in regulating other stem cell functions remains poorly understood. In the fly type II neuroblast lineage, overexpressing Notchintra or the downstream transcriptional repressors Dpn, E(spl)mγ, and Klumpfuss (Klu) induces the formation of supernumerary type II neuroblasts at the expense of generating immature INPs via the inhibition of erm activation. These results indicate that the Notch-Dpn/E(spl)mγ/Klu axis provides an evolutionarily conserved mechanism to maintain neural stem cells in an undifferentiated state. Although overexpressing Notchintra but not Dpn, E(spl)mγ, and Klu in combination in INPs is sufficient to drive supernumerary type II neuroblast formation, Notchintra overexpression is not sufficient to transform a type I neuroblast into a type II neuroblast. Thus, it is proposed that Notch functions as a general activator of genes expression in type II neuroblasts, and specific regulators of type II neuroblast functional identities must exist (Rives-Quinto, 2020).
This study strongly suggests that tll functions as a master regulator of type II neuroblast functional identities. Identical to Notch, tll is necessary for maintaining type II neuroblasts in an undifferentiated state and is sufficient to induce INP reversion into type II neuroblasts. Uniquely, high levels of Tll is sufficient to molecularly transform greater than 60% of type I neuroblasts in the ventral brain region into type II neuroblasts. Brain regionalization leads to distinct degrees of sensitivity to Tll overexpression. For example, type I neuroblasts in the dorsal-anterior region of the brain are resistant to Tll-induced lineage transformation. By contrast, Tll overexpression can transform most, if not all, type I neuroblasts in the ventral-lateral and ventral-medial regions of the brain into type II neuroblasts. High levels of Tll expression in ventral-lateral type I neuroblasts leads to accumulation of mostly supernumerary type II neuroblasts and very few Erm::V5+ immature INPs. This result phenocopies Tll overexpression driven by strong drivers in the type II neuroblast lineages, and suggests that neuroblast progeny rapidly re-acquire a neuroblast identity instead of an immature INP identity. Tll overexpression in ventral-medial type I neuroblasts leads to the formation of supernumerary type II neuroblasts interspersed with Erm::V5+ immature INPs, mimicking Tll overexpression driven by moderate drivers in the type II neuroblast lineages. It is speculated that progeny of transformed type II neuroblasts in the ventral-medial region of the brain can assume an immature INP identity and then revert into supernumerary type II neuroblasts. These data strongly support a model that Tll is a potent activator of type II neuroblast functional identities (Rives-Quinto, 2020).
A key question regarding the proposed function of Tll in regulating type II neuroblast functional identities is how it mechanistically links to genes previously shown to control these characteristics. ChIP-seq on fly embryonic nuclear extract using the Tll::GFP(Bac) transgenic protein identified hundreds of putative Tll target genes that include all previously characterized regulators of type II neuroblast functional identities. Tll binds dpn, E(spl)mγ and klu loci in embryos, suggesting that Tll likely regulates their expression. The phenotypic effects of loss- and gain-of-function of tll on type II neuroblasts mimic those of dpn, E(spl)mγ and klu. The vertebrate homolog of Tll, Tlx, has been shown to function as a transcriptional activator during neurogenesis. Thus, Tll might maintain type II neuroblasts in an undifferentiated state by promoting dpn, E(spl)mγ and klu expression. Tll also binds pntP1 and btd loci in embryos. Similar to Tll overexpression, mis-expression of PntP1 or Btd can transform type I neuroblasts in the ventral brain region into type II neuroblasts. Thus, it is plausible that tll functions through pntP1 or btd to regulate the competency to generate INPs in type II neuroblasts. These results strongly support the model that Tll is key component of the regulatory mechanism that endows type II neuroblasts with lineage-specific functional identities. Future experiments to validate the mechanistic links between tll and genes that regulate various lineage-specific functional characteristics will allow for the establishment of gene regulatory circuits that regulate type II neuroblast functional identities (Rives-Quinto, 2020).
The identification of ham as a putative regulator of INP commitment was unexpected given that a previously published study concluded that Ham functions to limit INP proliferation. Ham is the fly homolog of Prdm16 in vertebrates and has been shown to play a key role in regulating cell fate decisions in multiple stem cell lineages. Prdm16 contains two separately defined zinc-finger motifs, with each likely recognizing unique target genes. Prdm16 can also function through a variety of cofactors to activate or repress target gene expression, independent of its DNA-binding capacity. Thus, Ham can potentially inactivate stem cell functionality genes via one of several mechanisms. By using a combination of previously isolated alleles and new protein-null alleles, this study demonstrated that the N-terminal zinc-finger motif is required for Ham function in immature INPs. Based on the overexpression of a series of chimeric proteins containing the N-terminal zinc-finger motif, the data indicate that Ham prevents INP reversion to supernumerary type II neuroblasts by recognizing target genes via the N-terminal zinc-finger motif and possibly repressing their transcription. These results suggest that Ham prevents INPs from reverting to supernumerary type II neuroblasts by possibly repressing target gene transcription (Rives-Quinto, 2020).
A key question raised by this study is why two transcriptional repressors that seemingly function in a redundant manner are required to prevent INPs from reverting to supernumerary type II neuroblasts. INP commitment lasts approximately 6-8 hr following the generation of an immature INP; after this time, the immature INP transitions into an INP. erm is poised for activation in type II neuroblasts and becomes rapidly activated in the newly generated immature INP less than 90 min after its generation. As such, Erm-mediated transcriptional repression allows for the rapid inactivation of type II neuroblast functional identity genes. Because Erm expression rapidly declines in INPs when Notch signaling becomes reactivated, a second transcriptional repressor that becomes activated after Erm and whose expression is maintained throughout the life of an INP is required to continually inactivate type II neuroblast functional identity genes. Ham is an excellent candidate because it becomes expressed in immature INPs 3-4 hr after the onset of Erm expression and is detected in all INPs. Similar to Erm, Ham recognizes target genes and represses their transcription. Furthermore, ham functions synergistically with erm to prevent INP reversion to supernumerary type II neuroblasts, and overexpressed Ham can partially substitute for endogenous Erm. Thus, Erm- and Ham-mediated transcriptional repression renders type II neuroblast functional identity genes refractory to activation by Notch signaling throughout the lifespan of the INP, ensuring the generation of differentiated cell types rather than supernumerary type II neuroblasts instead (Rives-Quinto, 2020).
Genes that specify stem cell functional identity become refractory to activation during differentiation, but the mechanisms that restrict their expression are poorly understood due to a lack of lineage information. Researchers have proposed several epigenetic regulator complexes that may restrict neural stem-cell-specific gene expression in neurons. This study knocked-down the function of genes that were implicated in restricting neural stem cell gene expression during differentiation in order to identify chromatin regulators that are required to inactivate type II neuroblast functional identity genes during INP commitment. Surprisingly, this study found that only Hdac3 is required for both Erm- and Ham-mediated suppression of INP reversion to type II neuroblasts. This finding is consistent with a recent study showing that blocking apoptosis in lineage clones derived from PRC2-mutant type II neuroblasts did not lead to supernumerary neuroblast formation. The current data strongly suggest that genes specifying type II neuroblast functional identity, such as tll, are likely silenced rather than decommissioned in INPs. This result is supported by the finding that overexpressing Notchintra, but not Notch downstream transcriptional repressors, in INPs can re-establish a type II neuroblast-like undifferentiated state. It is speculated that continual histone deacetylation is required to counter the transcriptional activator activity of endogenous Notch and silence tll in INPs. By contrast, the chromatin in the tll locus might be close and inaccessible to the Notch transcriptional activator complex in type I neuroblasts; thus, overexpressing Notchintra cannot transform type I neuroblasts into type II neuroblasts. A key remaining question is what transcription factor is required to maintain the chromatin in the tll loci in an open state. Insights into regulation of the competency of the tll locus to respond to activated Notch signaling might improve understanding of the molecular determinants of OSVZ neural stem cells (Rives-Quinto, 2020).
During central nervous system (CNS) development, spatiotemporal gene expression programs mediate specific lineage decisions to generate neuronal and glial cell types from neural stem cells (NSCs). However, little is known about the epigenetic landscape underlying these highly complex developmental events. This study performed ChIP-seq on distinct subtypes of Drosophila FACS- purified neural stem cells (NSCs) and their differentiated progeny to dissect the epigenetic changes accompanying the major lineage decisions in vivo By analyzing active and repressive histone modifications, this study shows that stem cell identity genes are silenced during differentiation by loss of their activating marks and not via repressive histone modifications. This analysis also uncovers a new set of genes specifically required for altering lineage patterns in type II neuroblasts, one of the two main Drosophila NSC identities. Finally, it was demonstrated that this subtype specification in NBs, unlike NSC differentiation, requires Polycomb-group (PcG)-mediated repression (Abdusselamoglu, 2019).
During development of the central nervous system (CNS), neural stem cells (NSCs) divide asymmetrically to generate daughter cells with self-renewing capacity but also complex neurogenic and gliogenic lineages. Regulation of this process requires tight and highly dynamic control of multiple cell fate decisions. For cells to commit to their ultimate cell identity, spatiotemporal gene expression programs are required. It is assumed that activation of lineage-specific genes and silencing of stem cell genes is accompanied by changes in chromatin states. How histone modifications change over time during neurogenesis in vivo, however, is not very well described (Abdusselamoglu, 2019).
The Drosophila larval CNS has become a key model for the fundamental mechanisms underlying brain development and chromatin states. The larval CNS is populated by distinct types of NSCs, or neuroblasts (NBs), which vary in abundance, neuronal output and division mode. Together, these NBs give rise to the majority of the neurons of the adult brain. The majority of the central brain NBs are of type I (NBIs). Each NBI gives rise to another NBI and a ganglion mother cell (GMC), which divides once more to generate two differentiated neurons or glia. Type II NBs (NBIIs) are a rare subpopulation with only eight NBII per brain lobe. Unlike NBIs, NBIIs divide into one NBII and one transit-amplifying cell called an intermediate neural progenitor (INP). NBIIs generate many more neurons, because INPs continue to divide asymmetrically five or six times, each time giving rise to a GMC that divides into two neurons or glia cells. Other than lineage structure and size, cell markers can also be used to differentiate NB subtypes. Whereas NBIs express both Asense (Ase) and Deadpan (Dpn), NBIIs only express Dpn. During neurogenesis, both NB subtypes divide asymmetrically to give rise to their respective progeny. Brain tumors form if the asymmetric segregation of cell fate determinants during NB cell division is disrupted. Among these determinants are the TRIM-NHL protein Brain tumor (Brat) and the Notch inhibitor Numb. Although Brat depletion results in the generation of ectopic NBII-like tumor NBs (tNBs) at the expense of differentiated brain cells, simultaneous loss of Brat and Numb causes the NBI-like tNBs to overproliferate (Abdusselamoglu, 2019).
In many cell types, transitions in chromatin states are regulated by the evolutionarily conserved Polycomb group (PcG) and Trithorax group (TrxG) proteins. PcG and TrxG have emerged as antagonistic regulators that silence or activate gene expression, respectively. These multimeric protein complexes regulate the transcriptional state of genes by post-translationally modifying amino acid residues of histone tails. PcG proteins exert a repressive activity via two main complexes, the Polycomb repressive complexes 1 and 2 (PRC1 and PRC2). Although PRC1 and PRC2 can exist in various compositions and associate with context-specific accessory proteins, both have been shown to contain a specific core set of proteins including subunits with catalytic activity. Within PRC2, Enhancer of zeste [E(z) in Drosophila, EZH1/2 in mammals] catalyzes the trimethylation of lysine 27 on histone 3 (H3K27me3). H3K27me3 is recognized by PRC1, which in turn includes the histone H2A ubiquityltransferase Sce [RING1A (RING1) and RING1B (RNF2) in mammals]. Histone modifications associated with active transcription are deposited by TrxG proteins, which counteract repressive marks by histone acetylation or methylation, in particular by trimethylation of lysine 4 on histone H3 at active promoters (Abdusselamoglu, 2019).
Although well-known for their role in long-term transcriptional memory, PcG and TrxG complexes are highly dynamic during development and thus facilitate cellular plasticity. In the last decade, it has been shown that PcG and TrxG complexes are crucial for ensuring correct neurogenesis in mammals as well as in Drosophila. Despite the strength of genetic in vivo experiments, however, global analysis of the histone modifications underlying their function, and therefore target genes, has mainly been performed in vitro. This constitutes a real knowledge gap, as recent studies have demonstrated that the chromatin states may vary significantly between in vivo tissues and their related in vitro cell lines, mainly owing to culture conditions. Given also that epigenetic changes are highly context- and developmental time-dependent, providing in vivo datasets to investigate chromatin states of different cell types in complex tissues will increase understanding of how the epigenetic landscape dynamically defines cellular states (Abdusselamoglu, 2019).
In recent years, in vivo studies made use of Drosophila to shed light on the dynamics of chromatin state changes during embryonic neural differentiation and during larval stages. Profiling the binding of chromatin remodelers has highlighted the plasticity of chromatin states during differentiation. Although binding of chromatin factors is associated with active or repressive chromatin, binding does not necessarily reflect downstream histone modifications. For example, the histone marks can change drastically between parasegments of the Drosophila embryo, whereas the occupancy of PcG proteins remains unchanged. Thus, investigating the dynamics of chromatin states based on chromatin marks is crucial for understanding the functional specialization of cells during development. Moreover, how PcG/TrxG complexes target genes on the chromatin level between different subtypes of progenitor cells during neuronal differentiation or tumorigenic transformation has remained elusive (Abdusselamoglu, 2019).
This study has used the Drosophila larval CNS to track in vivo changes of histone modifications not only upon differentiation, but also between different populations of NSCs and their tumorigenic counterparts. A fluorescence-activated cell sorting (FACS)-based method was developed to sort different cell types and perform ChIP-seq for the active histone mark, H3K4me3, and the repressive mark, H3K27me3. The FACS-based approach provides an in vivo dataset that reveals dynamic histone modifications during neuronal differentiation. In particular, it was observed that self-renewal and cell-division genes are repressed independently of H3K27me3 levels. In contrast, it was further shown that H3K27me3-mediated repression is crucial for silencing lineage-specific stem cell factors, including known factors as well as a new set of genes that are specific to NBIIs. Finally, genetic evidence is presented for the requirement of these new NBII-specific factors for self-renewal and demonstrate the role of PcG complexes in defining different subtypes of neural stem cells (Abdusselamoglu, 2019).
This study provides a resource of histone modification datasets for different types of NSCs and their differentiated progeny. In combination with chromatin accessibility and binding maps of chromatin remodelers of Drosophila brain cells, it is hoped that this dataset will serve as a useful community resource. During differentiation, stem cell identity genes are silenced in a PcG-independent manner, which supports previous findings showing that these genes are silenced through HP1-enriched chromatin. In addition, PcG-mediated silencing is unlikely to instruct the stepwise inactivation of stem cell genes during differentiation as loss of H3K27me3 did not induce ectopic NBs (Abdusselamoglu, 2019).
This study has taken advantage of in vivo genetic labeling to investigate chromatin dynamics of different NB subtypes. As the type II NBs are very lowly abundant, tumor NBs of type I and type II origins were used as a proxy in order to obtain enough material to be able to compare these two cell types. Each change was further validated by comparing tumor with healthy type I NBs and excluded artifacts present because of the tumorigenic state of the cells. The data show that both TrxG and PcG are required to establish NBII identity. A set of NBII-specific genes was identified, including previously identified btd and Sp1. Dll and eya, which are specifically required for NBII maintenance, were identified. It has been previously described that btd acts as an activator of Dll in the development of the ventral imaginal discs. This suggests that in NBII-identity specification the Trithorax-target btd could act together with Dll and eya. Such a mechanism would explain why the loss of btd causes a distinct phenotype compared with the loss of Dll and eya. Interestingly, an NBI to NBII conversion is observed only in 18% of NBIs ectopically overexpressing btd, indicating that either co-factors are missing or that the chromatin of btd targets is inaccessible. The data of NB subtype-specific genes being characterized by H3K27me3 repressive chromatin favor the latter. Therefore, as opposed to TrxG-activated stem cell and mitosis genes, the repression of NBII-specific genes is ensured by PcG-mediated H3K27me3 histone modifications, suggesting that Polycomb plays a role in defining the diversity of NSC lineages. Moreover, the data indicate that PcG repression is required not only for the silencing of HOX genes but also for the self-renewal capacity of NBs. Unlike TrxG, the loss of catalytic subunits of PcG complexes did not convert NBIIs to NBIs or vice versa. This suggests that NB subtype specification cannot be explained solely by an absence of repression but requires a further activation mechanism. Strikingly, loss of PcG complexes caused a significant decrease in the number of NBs. Interestingly, across all the cell types, developmental genes such as caudal, eve, peb, scr and slp1, as well as genes involved in embryonic NB temporal patterning [hb, kr, pdm (nub), cas and grh], are heavily marked with H3K27me3. It is therefore possible that PcG-mediated repression is required to silence these developmentally crucial genes in addition to the Hox genes. Thus, the observed reduction in NB stemness might be caused by the de-repression of these genes (Abdusselamoglu, 2019).
Besides an overall decrease in NB maintenance, an increased sensitivity of NBII lineages to a reduction in PRC2 activity was observed. Interestingly, opa and ham, two previously described temporal switch genes in NBII lineages, are also enriched in H3K27me3 marks in NBs. Opa and ham are expressed in the immediate NBII progeny, the INPs, and ectopic expression of these genes limits self-renewal of NBIIs and causes NBIIs to disappear. Even though these two genes are heavily marked with H3K27me3 across all NB samples, NBIIs might be specifically sensitive to PRC2 depletion because they could be more receptive to premature de-repression of genes, the expression of which is normally restricted NBII progeny only (Abdusselamoglu, 2019).
In the future, investigating the downstream targets of PcG in different NB subtypes could reveal the underlying mechanisms of subtype specification. In conclusion, these data provide a useful resource to investigate how chromatin state dynamics orchestrate the diversity and correct progression of NSC lineages (Abdusselamoglu, 2019).
Translating advances in cancer research to clinical applications requires better insight into the metabolism of normal cells and tumour cells in vivo. Much effort has focused on understanding how glycolysis and oxidative phosphorylation (OxPhos) support proliferation, while their impact on other aspects of development and tumourigenesis remain largely unexplored. This study found that inhibition of OxPhos in neural stem cells (NSCs) or tumours in the Drosophila brain not only decreases proliferation, but also affects many different aspects of stem cell behaviour. In NSCs, OxPhos dysfunction leads to a protracted G1/S-phase and results in delayed temporal patterning and reduced neuronal diversity. As a consequence, NSCs fail to undergo terminal differentiation, leading to prolonged neurogenesis into adulthood. Similarly, in brain tumours inhibition of OxPhos slows proliferation and prevents differentiation, resulting in reduced tumour heterogeneity. Thus, in vivo, highly proliferative stem cells and tumour cells require OxPhos for efficient growth and generation of diversity (van den Ameele, 2019).
The observation that some cancer cells rely primarily on aerobic glycolysis for energy and biomass production (the Warburg effect) has often led to the assumption that the other main source of ATP, mitochondrial oxidative phosphorylation (OxPhos), is dispensable. However, it is becoming increasingly clear that many tumours do require mitochondrial activity for energy and biosynthesis and OxPhos is now frequently exploited as a therapeutic target in cancer. OxPhos takes place at the inner mitochondrial membrane in five large protein complexes (Complex I-V), which together form the respiratory chain. Complexes I-IV transfer electrons from NADH to O2 and use the released energy to translocate protons from the mitochondrial matrix into the intermembrane space. The resulting electrochemical gradient is then used by Complex V (ATP synthase) to generate ATP from ADP. Apart from the production of ATP, OxPhos is also directly involved in the generation of NAD+, orotate, fumarate and reactive oxygen species (ROS) and thus affects many cellular processes, such as nucleotide synthesis, signalling pathway activity and epigenetic modifications. The Warburg effect has since been interpreted as a normal adaptation to the metabolic requirements of proliferation, both in cancer cells and proliferating stem cells. High glycolytic flux is thought to be required for a constant supply of biomass while OxPhos, apart from its role in production of ATP, primarily maintains the cellular redox balance (van den Ameele, 2019 and references therein).
However, metabolic flux in cancer cells can be influenced by extrinsic and intrinsic factors such as substrate availability, oncogenic mutations and the tumour's tissue and cell type of origin. Brain tumours in particular recapitulate many features of their tissue of origin and grow along a hierarchy reminiscent of normal brain development. An integrated understanding of the interactions between metabolism and cell identity in vivo, during both tumourigenesis and normal development, is therefore crucial to translate advances in cancer research to clinical applications (van den Ameele, 2019).
Development of the Drosophila central nervous system (CNS) has been used extensively as a powerful reductionist model of human brain development and tumourigenesis in vivo. The CNS of Drosophila develops from rapidly cycling embryonic and larval neural stem cells (NSCs) that generate a wide variety of neurons and glia. Neuronal diversity is achieved primarily by spatial and temporal patterning, which confers specific identities on NSCs and their progeny according to their location and developmental time. Neural stem cells (NSCs) in Drosophila and mammals are thought to generate ATP through aerobic glycolysis rather than OxPhos, whereas their neuronal progeny switch to mitochondrial respiration upon differentiation. Upregulation of aerobic glycolysis, reminiscent of the Warburg effect, has also been described in a number of Drosophila tumour paradigms. However, the interpretation that mitochondrial respiration is dispensable for normal Drosophila NSCs contrasts with the clear requirement for OxPhos to support cell cycle progression in the Drosophila eye disc. This study investigated whether, and to what extent, Drosophila NSCs and brain tumours rely on oxidative phosphorylation (van den Ameele, 2019).
This study shows that the metabolic requirements of highly proliferative NSCs in the Drosophila brain, as well as the tumour cells NSCs generate upon transformation, cannot be met by aerobic glycolysis alone. Instead, Drosophila NSCs require OxPhos for key aspects of their behaviour: proliferation, generation of diversity through temporal patterning, and termination of proliferation. Respiratory activity may provide an explanation for the strong increase in ROS production that has been observed in NSCs upon hypoxia and for the developmental lethality caused by CNS-specific mutation of the mitochondrial genome. While OxPhos dysfunction affects both normal NSCs and tumour cells in the brain, inhibition of glycolysis only affects tumour growth but not normal brain development. This is reminiscent of the upregulation of aerobic glycolysis in Hipk, EGFR or PDGF/VEGF-induced tumours in the Drosophila wing disc. Future experiments will determine the origin and consequences of this tumour-specific reliance on glycolysis in the brain (van den Ameele, 2019).
The results contrast with previous findings suggesting that OxPhos is dispensable during normal NSC development and in brain tumours, and is only activated at the end of neurogenesis as part of a metabolic switch to induce termination of NSC proliferation. While the current experiments do not directly address whether this metabolic switch takes place, the results provide an alternate interpretation. Sustained OxPhos activity throughout NSC development was shown to be required for normal temporal patterning. Prolonged expression of early temporal markers makes NSCs unresponsive to the developmental cues that govern cell cycle exit and this study shows that restoring temporal progression by timely depletion of the early temporal factor Imp enhances termination of proliferation in spite of continued OxPhos inhibition. These findings thus integrate key aspects of NSC and tumour cell biology: OxPhos-dependent proliferation is required for temporal patterning and differentiation at the G1/S transition of the cell cycle. This enables NSCs to undergo normal aging and to respond to the developmental cues that instruct termination of proliferation. Interestingly, adult neurogenesis in the subventricular zone of the mammalian brain depends on p57-induced slowing of the cell cycle during embryonic development. It is not known whether p57 expression or mitochondrial dysfunction also affects the temporal identity of mammalian NSCs. Importantly, the effects observed in this study are specific to the G1/S transition: activation of the G2/M checkpoint did not affect temporal patterning or termination of proliferation. The results therefore demonstrate that the size and composition of Drosophila NSC lineages are not strictly predetermined but rather controlled by both intrinsic and extrinsic factors. Single-cell sequencing data indicate that metabolic differences exist between NSCs in different regions of the brain or at different developmental stages, and it will be interesting to assess whether all NSCs are similarly affected by OxPhos dysfunction and G1/S delay or whether specific lineages show stereotypical responses, as has been shown for entry into quiescence, where arrest in G2 or G0 is predetermined (van den Ameele, 2019).
This study indicates that OxPhos might constitute a targetable metabolic vulnerability of cancer. Small molecule inhibitors of OxPhos are currently being developed and tested in clinical trials to treat various forms of cancer. However, this study found that the in vivo impact of OxPhos dysfunction is much more complex than mere inhibition of proliferation. A better understanding of the interactions between metabolism, differentiation and tumour heterogeneity in vivo has the potential to uncover novel therapeutic approaches (van den Ameele, 2019).
Proliferating cells, typically considered 'nonexcitable,' nevertheless, exhibit regulation by bioelectric signals. Notably, voltage-gated sodium channels (VGSC) that are crucial for neuronal excitability are also found in progenitors and up-regulated in cancer. This study identified a role for VGSC in proliferation of Drosophila neuroblast (NB) lineages within the central nervous system. Loss of paralytic (para), the sole gene that encodes Drosophila VGSC, reduces neuroblast progeny cell number. The type II neuroblast lineages, featuring a population of transit-amplifying intermediate neural progenitors (INP) similar to that found in the developing human cortex, are particularly sensitive to para manipulation. Following a series of asymmetric divisions, INPs normally exit the cell cycle through a final symmetric division. The data suggest that loss of Para induces apoptosis in this population, whereas overexpression leads to an increase in INPs and overall neuroblast progeny cell numbers. These effects are cell autonomous and depend on Para channel activity. Reduction of Para expression not only affects normal NB development, but also strongly suppresses brain tumor mass, implicating a role for Para in cancer progression. These studies are the first to identify a role for VGSC in neural progenitor proliferation. Elucidating the contribution of VGSC in proliferation will advance understanding of bioelectric signaling within development and disease states (Piggott, 2019).
VGSCs play important roles during neural development where neural activity is important for axon guidance and synaptic refinement. VGSC activity within the nervous system is essential, as its absence leads to lethality in a number of mouse models. In addition, mutations in VGSC are associated with childhood diseases including autism spectrum disorders, epilepsy, mental retardation, among others, highlighting the necessity for understanding the role of VGSC during development (Piggott, 2019).
Recent work has begun to uncover functions of VGSC in development of nonexcitable cells. One such study found that NaV1. channels (encoded by SCN3A) are expressed in radial glial cells and are important for migration during cortex development. A large scale clinical study has found that 25% of individuals with SCN2A mutations presented with microcephaly, suggesting that VGSC may influence neuronal progenitor proliferation. Additionally, VGSCs are important for cardiac progenitor development in zebrafish. These studies indicate that VGSCs play a role in progenitors during development, although the nature of their contributions is unclear (Piggott, 2019).
This study has identified a role for the VGSC Para in neural development. Loss of para reduced total cell number in the type I and type II central neuroblast lineages. These effects are cell autonomous and could be rescued by cDNA expression within neuroblast lineages. The stronger phenotype observed in the type II lineage may reflect a role for Para in INPs, which are only present in the type II lineage (Piggott, 2019).
Loss of Para led to increased apoptosis that may arise from a failure of cell cycle progression. Research in other systems has found that depolarization is essential for G to M progression and it is possible that Para promotes cell cycle progression in Drosophila progenitors through its depolarizing activity. Proliferative cells tend to display more depolarized resting membrane potentials, as compared with differentiated cells, like neurons. In this context, Para may primarily exist in the inactivated state and a persistent sodium current of an amplitude significantly smaller than that of the transient current, may predominate. In addition, VGSCs are highly subjected to RNA editing and alternative splicing. Some of these variants result in window currents and persistent currents, like that found in embryonic variants of Para. Para could, therefore, contribute to a depolarized state that may be important for proliferation. Alternatively, Para could be temporally up-regulated during specific states of the cell cycle to activate downstream molecular cascades. Depolarization has been shown to influence cell cycle regulation, including gene expression, calcium signaling, protein localization, and phosphorylation states. The contribution of VGSC to axon growth cones, metastasis, and migration indicates a role for depolarization in cytoskeletal rearrangement. The cytoskeleton contributes to many aspects of cell division including subcellular localization of cell-fate determinants during asymmetric cell division, volume changes, nuclear envelope breakdown, and chromosome segregation during M-phase. If Para activity were to influence any of these processes, dysregulation of cell cycle progression would be expected (Piggott, 2019).
VGSCs drive membrane depolarization as they catalyze selective permeation of sodium ions, thereby driving the membrane potential towards the sodium equilibrium potential. A number of studies highlight the potential role of Na+signaling as a driver of proliferation. Regeneration experiments in Xenopus laevis find NaV1-mediated Na+ influx, rather than depolarization, acts through salt inducible kinase (SIK) to drive proliferation, outgrowth, and morphogenesis by activating Notch, Wnt, BMP, and FGF signaling. Moreover, a number of recent studies in Drosophila find that sodium permeable ion channels promote proliferation. These include the epithelial sodium channel ENaC whose overexpression drives gut stem cell overproliferation, ROS stimulated upregulation of cation permeable TRPA channels that induce proliferation, and cation permeable Piezo that is important for proliferation in the adult fly midgut as well as glioma proliferation in multiple fly glioma model (Piggott, 2019).
These studies have identified a role for voltage-gated sodium channel, Para, in neural development and cancer. Going forward it will be interesting to discover how the activity of other ion channels, exchangers and pumps converge to influence cellular behavior. While Para's actions were cell autonomous, it's intriguing to wonder whether signaling within progeny and the cellular niche occurs. Many progenitors are connected through gap junctions and electrical signaling likely facilitates communication and coordination, essential for tissue patterning and cellular morphogenesis during development and regeneration. Genetically tractable model systems like Drosophila provide a platform to build this knowledge and further understanding of the contribution of ion channels to proliferation and differentiation. VGSCs are promising sites for therapeutic intervention due to their druggable structure and the availability of FDA approved medications targeting them for pain and antipsychotics. These studies will offer significant insight towards future biomedical therapies for birth defects, cancer and regenerative medicine (Piggott, 2019).
Temporal patterning of neural progenitors is an evolutionarily conserved strategy for generating neuronal diversity. Type II neural stem cells in the Drosophila central brain produce transit-amplifying intermediate neural progenitors (INPs) that exhibit temporal patterning. However, the known temporal factors cannot account for the neuronal diversity in the adult brain. To search for new temporal factors, NanoDam, which enables rapid genome-wide profiling of endogenously-tagged proteins in vivo with a single genetic cross, was developed. Mapping the targets of known temporal transcription factors with NanoDam identified Homeobrain and Scarecrow (ARX and NKX2.1 orthologues) as novel temporal factors. Homeobrain and Scarecrow define middle-aged and late INP temporal windows and play a role in cellular longevity. Strikingly, Homeobrain and Scarecrow have conserved functions as temporal factors in the developing visual system. NanoDam enables rapid cell type-specific genome-wide profiling with temporal resolution and can be easily adapted for use in higher organisms (Tang, 2021).
The nervous system is generated by a relatively small number of neural stem cells (NSCs) and progenitors that are patterned both spatially and temporally. Spatial patterning confers differences between populations of NSCs, while changes in gene expression over time direct the birth order and subtype identity of neuronal progeny. Temporal transcription factor cascades determine neuronal birth order in the Drosophila embryonic central nervous system (CNS) and the larval central brain and optic lobe. In the central brain, Type II NSCs generate transit-amplifying intermediate neural progenitors (INPs), which divide asymmetrically to self-renew and generate daughter cells (ganglion mother cells or GMCs) in a manner analogous to human outer radial glial cells. GMCs in turn undergo a terminal cell division, generating neurons or glial cells that contribute to the adult central complex. The sequential divisions of INPs increase the quantity of neurons, which in turn creates a platform for generating wider neuronal diversity: eight Type II NSCs in each brain lobe give rise to at least 60 different neuronal subtypes. The tight control of progenitor temporal identity is crucial for the production of neuronal subtypes at the appropriate time and in the correct numbers. The INPs produced by the six dorsal-medial Type II lineages (DM1-6) sequentially express the temporal transcription factors Dichaete (D, a member of the Sox family), Grainy head (Grh, a Grh/CP2 family transcription factor) and Eyeless (Ey, a homologue of Pax6). These temporal factors were discovered initially by screening Type II lineages for restricted expression of neural transcription factors, using 60 different antisera. This non-exhaustive approach was able to find a fraction of the theoretically necessary temporal factors, leaving the true extent of temporal regulation and the identity of missing temporal factors open. Furthermore, the cross-regulatory interactions predicted in a temporal cascade, in which each temporal transcription factor activates expression of the next temporal factor and represses expression of the temporal factor preceding it, are not fulfilled solely by D, Grh and Ey (Tang, 2021).
Three further factors contribute to INP temporal progression, but they are expressed broadly rather than in discrete temporal windows: Osa, a SWI/SNF chromatin remodelling complex subunit, and two further transcription factors, Odd-paired (Opa) and Hamlet (Ham). Therefore, there must exist other transcription factors that are expressed in defined temporal windows and exhibit the regulatory interactions expected in a temporal cascade. It was postulated that other temporal factors, that contribute to generating the diversity of neuronal subtypes arising within each INP lineage, remain to be identified (Tang, 2021).
Given the feed-forward and feed-back transcriptional regulation previously observed in temporal transcription cascades, it was surmised that novel temporal factors would be amongst the transcriptional targets of D, Grh or Ey. Therefore, a novel approach, NanoDam, was devised to identify the genome-wide targets of transcription factors within their normal expression windows in vivo without cell isolation, cross linking or immunoprecipitation. Temporal factors are expressed transiently in a small pool of rapidly dividing progenitor cells. NanoDam provides a simple, streamlined approach to obtain genome-wide binding profiles in a cell-type-specific and temporally restricted manner (Tang, 2021).
Using NanoDam, the transcriptional targets of D, Grh and Ey were determined in INPs and, by performing single cell RNA sequencing, determined which of the directly bound loci were activated or repressed. Next, which of the target loci encoded transcription factors was assessed and whether these were expressed in restricted temporal windows within INPs. Where in the INP transcriptional cascade these factors acted was surveyed and whether they cross regulate the expression of other temporal transcription factor genes was ascertained, as expected for temporal factors. Finally, it was shown that the newly discovered temporal factors play the same roles, and exhibit the same cross regulatory interactions, in the temporal cascade in the developing visual system. This is particularly striking as theINPs and the NSCs of the developing optic lobe have different cells of origin and yet the mechanism they use to generate neural diversity is conserved (Tang, 2021).
Temporal patterning leads to the generation of neuronal diversity from a relatively small pool of neural stem or progenitor cells. Temporal regulation is achieved by the restricted expression of temporal transcription factors within precise developmental windows. The onset and duration of each temporal window in neural stem or progenitor cells must be regulated tightly in order for the appropriate subtypes of neurons to be generated at the correct time to establish functional neuronal circuits (Tang, 2021).
This study focused on the INPs of the Type II NSC lineages that generate the central complex of the Drosophila brain. Previously, INPs were shown to express sequentially the temporal factors D, Grh and Ey. Given the expectation that other temporal factors remained to be discovered, and that these were likely to be the transcriptional targets of the known temporal factors, a new technique called was used NanoDam to profile the binding targets of D, Grh and Ey with cell-type specificity and within their individual temporal windows (Tang, 2021).
NanoDam enables genome-wide profiling of any endogenously tagged chromatin-binding protein with a simple genetic cross, bypassing the need to generate Dam-fusion proteins, or the need for specific antisera or cell isolation. Furthermore, NanoDam profiles binding only in cells where the tagged protein is normally expressed. Binding within a subset of the protein's expression pattern can be achieved by controlling NanoDam with specific GAL4 drivers. To date, collaborative efforts have produced more than 3900 Drosophila lines expressing GFP-tagged proteins in their endogenous patterns. Approximately 93% of all transcription factors have been GFP-tagged in lines that are publicly available at stock centres. Lines that are not yet available can be rapidly generated by CRISPR/Cas9-mediated tagging (Tang, 2021).
NanoDam is thus a versatile tool that can be used as a higher throughput method to profile genome-wide binding sites of any chromatin associated protein. NanoDam can be readily adapted for use in other organisms to facilitate simpler and easier in vivoprofiling experiments, as hads been demonstrated previously for TaDa (Tang, 2021).
By combining the power of NanoDam with scRNA-seq, it was possible to identify scro and hbn as novel temporal factors in the INPs of Type II NSC lineages. It was shown that hbn and scro regulate the maintenance and transition of the middle-aged and late temporal windows. The mammalian homologues of ey (Pax6), hbn (Arx) and scro (Nkx2.1) are restricted to distinct progenitor populations in the developing mouse forebrain. This study found that scro regulates the late INP identity by repression of Ey. Interestingly, the loss of Nkx2.1 in the mouse forebrain leads to aberrant expression in ventral regions of the dorsal factor Pax6, suggesting that the repressive relationship between scro and ey may be conserved between Nkx2.1 and Pax6. Not all relationships appear to be conserved, however. It esd found that Hbn promotes progression through the middle-aged temporal stage and that maintenance of the middle-aged temporal window is regulated in part by interactions between Hbn and Grh. Arx mutant mice exhibit loss of upper layer (later-born) neurons but no change in the number of lower layer (early-born) neurons (Tang, 2021).
Intriguingly, the novel temporal factors identified in the INPs were also temporally expressed in optic lobe NSCs and the regulatory relationships between scro and Ey appeared to be conserved. This suggests that similar regulatory strategies may be shared between neural stem cells or progenitor cells in order to regulate longevity and neuronal subtype production. The remarkable conservation of the regulatory interactions of scro in two different progenitor cell types with different origins in the Drosophila brain may also be translated to the context of mammalian neurogenesis, highlighting the possibility of a more generalised regulatory network used by stem and progenitor cells to regulate cell fate, progeny fate and proliferation (Tang, 2021).
The Type II lineages in Drosophila divide in a very similar manner to the outer radial glia (oRGs) that have been attributed to the rapid evolutionary expansion of the neocortex seen in humans and other mammals. Interestingly, oRGs show a shortened cell cycle length in primates in comparison to rodent progenitors, which increase cell cycle duration as development progresses. Investigating whether oRGs use temporally expressed factors to control longevity and cell cycle dynamics at different developmental stages in order to regulate neuronal subtype generation would be important for understanding neocortex development (Tang, 2021).
There is significant heterogeneity between the Type II lineages and this study has identified differences in the regulatory relationships of hbn and scro. For example, misexpression of Ey leads to an increase in scro in all lineages except DM 2 and 3, where scro expression is reduced. To date, Hbn is the only factor identified that activates Grh in DM1, the lineage that does not normally express Grh. The heterogeneity between lineages may be a consequence of variations in combinatorial binding of temporal factors, as the NanoDam data indicate. Although INPs share temporal factors, different DM lineages display subtle to striking differences when the temporal cascade is manipulated, demonstrating the likelihood that each DM employs unique temporal cascades. Combinatorial binding would enable more complex regulatory interactions that could refine or sub-divide temporal windows in the INPs (Tang, 2021).
The Drosophila type II neuroblast lineages present an attractive model to investigate the neurogenesis and differentiation process as they adapt to a process similar to that in the human outer subventricular zone. Targeted single-cell mRNA sequencing in third instar larval brains was performed to study this process of the type II NB lineage. Combining prior knowledge, in silico analyses, and in situ validation, this multi-informatic investigation describes the molecular landscape from a single developmental snapshot. 17 markers are identified to differentiate distinct maturation stages. 30 markers are identified to specify the stem cell origin and/or cell division numbers of INPs, and at least 12 neuronal subtypes are identified. To foster future discoveries, annotated tables are provided of pairwise gene-gene correlation in single cells and MiCV, a web tool for interactively analyzing scRNA-seq datasets. Taken together, these resources advance understanding of the neural differentiation process at the molecular level (Michki, 2021).
To enable the brain's complex functions, vastly diverse neuronal types need to be rapidly generated at a very large scale during development. To reveal how neural stem cells populate the developing brain, efforts have been made to identify cell types and their lineage relationships. For instance, focuses on neuro-development in mouse, human brain tissues, and the developing human prefrontal cortex revealed intermediate stem cells (and critical genes involved) as an important mechanism for rapid cortical expansion. Underlying this rapid and diverse differentiation process is the constant change of gene-expression profiles in all cells. However, the molecular mechanisms that lead to functionally distinct neurons in the mammalian brain remain challenging to describe in detail. This is because, on the one hand, neuronal fate determination involves many genes, and, on the other hand, neural progeny cells originating from distinct lineages undergo rapid migration, which leads their intermingling nature in space (Michki, 2021).
Although they are the minority (8 stem cells per hemisphere) in the Drosophila central brain, the Drosophila type II neural lineage has a neurogenesis process analogous to the mammal's rapid cortical expansion. Compared to their mammalian counterparts, the Drosophila type II neural lineage has the advantage of being non-migrating in the larval stage. With proper labeling, type II progeny cells of the same lineage can be identified as a segregated cell cluster. Importantly, the cells' spatial relationship within a cluster serves as a considering factor when determining the age and maturation stage of these cells. The small stem cell pool and mammal-like lineage composition make the Drosophila type II neural lineage an attractive model to study the complex brain development process. In addition, many important genes and signaling pathways are conserved throughout evolution, which makes revealing the molecular mechanisms of Drosophila type II neuron differentiation a meaningful primer to study the human analogs in the outer subventricular zone (Michki, 2021).
This work used targeted single-cell transcriptome analysis to advance understanding of the Drosophila type II neuron differentiation process. After initially separating the transcriptomes of the type II neuroblast-derived cells from those labeled in the optic lobes, pseudotime analysis techniques can be used to define a maturation axis and extract putative marker genes that specify the INP, GMC, immature neuron, and mature neuron differentiation stages. Broadly expressed, not limited to the type II NB progenies, these marker genes of different maturation stages indeed form intersectional patterns that represent the spatial organization of the neurogenesis progress in the larval brain. Compared to previous antibody-based and gene-manipulation-based screening strategies, scRNA-seq data permit a high-throughput assessment of the whole gene-expression profile to rapidly identify candidate genes for functional study. For instance, in the past, Hey has been shown to mark one of the two immature neurons derived from the final cell division, and its role is exclusive as an inhibitor of Notch signaling in this immature neuron (Monastirioti, 2010). From scRNA-seq analysis, E(spl)m6-BFM, a member of the enhancer-of-split family of transcription factors, and Rbp, a rim-binding protein responsible for synaptic homeostasis and neurotransmitter release are exclusively upregulated in only the transient immature neuronal differentiation state directly after GMC division. These two marker genes can be used to guide the exploration of Hey-immature neurons in future studies. Functional knockouts of these two genes will be critical to understanding their function in newly born neurons as it pertains to their maturation and any early functional role they may play in the developing brain (Michki, 2021).
Further higher-resolution clustering of the INP and GMC cells identified transcriptomically correlated subclusters between these two stages, which supports the idea that parallel maturation transitions happen at the same developmental time point. However, scRNA-seq data alone cannot distinguish whether these parallel transitions are due to the co-existence of earlier and newly born INPs in all NB lineages or due to the intrinsic differences among NB lineages. Therefore in situ profiled the marker genes selected from the scRNA-seq selected candidates and restored their missing spatial information that indicates the maturation stage as well as the NB lineage identity. In addition, combined with prior knowledge, whether a marker gene is expressed in younger or earlier born INPs can also be speculated. The findings conclude that Sp1 is expressed in the young INPs of nearly all NB lineages, whereas TfAP-2 and Fas3 express in older INPs belonging to specific NB lineages. Interestingly, it was found that Sp1 and TfAP-2 expressed not only in neural progenitors but also in maturing neurons. These transcription factors seem to intermingle with the NB lineage-specific D/grh/ey cascades in the INP stage but eventually differentiate into completely exclusive neuron populations. Finally, higher-resolution clustering of neurons in the scRNA-seq dataset revealed that transcription factors and surface molecules are predominant markers for distinct neuronal subtypes at the third instar larval stage. This implies that most neurons of the type II NB progenies have not started to gain their differentiated functions at this stage of development (Michki, 2021).
Combining in silico scRNA-seq analysis and in situ mRNA imaging, many transcription factors and surface molecules were discovered that potentially play important roles in generating neuronal subtypes in an NB-specific, INP-specific, or function-specific manner. These discoveries helped in gaining of a comprehensive understanding of the molecular landscape along all three major neural developmental axes that define a cell's progenitor lineage identity, progenitor cell division number, and differentiation state. This model provides a general guidance for biologists to disentangle the differentiation process in complex systems beyond the Drosophila brain (Michki, 2021).
Despite its small scale and apparent simplicity, the complex interplay of molecular factors that vary along the differentiation state, lineage identity, and progenitor cell division number axes are responsible for determining the fate of each cell derived from the type II neuroblasts of Drosophila. Some of the most prominent molecular factors from the literature or identified and validated in this work are shown to occupy different domains along these three axes (see A Drosophila type II neuronal fate-specification model illustrates the complex molecular network that determines the neural differentiation process). Multi-time-point analysis and in situ validation will enable continued filling in of the blanks and develop a more complete roadmap of the type II neurogenesis process across development (Michki, 2021).
Approximately 4,000 cells that were neurons originating from 8 Drosophila type II neuroblast lineages. With low-resolution clustering, w13 molecularly distinct neural subtypes were identified. Increasing the clustering resolution just a bit higher it was possible to identify more than 20 that are still distinct. Similarly, was shown with the INPs/GMCs in the dataset, a low-resolution clustering can often mask the cellular diversity that is present in the system. As it is known that each type II neuroblast generates approximately 38 INPs throughout their developmental lifespan, the presented clustering in this paper only captures part of the INP diversity. One straightforward thought is to increase the number of sequenced single cells so that higher clustering resolution may eventually reveal even the most subtle differences between each of the hundreds of INPs in the type II system. However, as transcription factor cascades involved in INP division/maturation intertwine with those involved in NB specification and differentiation, it is expected that the INP heterogeneity can be untangled somewhat using a higher clustering resolution but still fails to provide a coherent view of the complex lineage, maturation, and differentiation landscape that this study attempted to characterize. These issues highlight the challenge of deconvoluting the INP maturation, NB lineage, and differentiation state axes and the need for a holistic, integrated approach to experimental design and subsequent bioinformatic analysis (Michki, 2021).
The data presented in this study were collected at a single developmental time point (late third instar), but it is known that type II neurogenesis precedes and continues after this stage. Repeating these scRNA-seq experiments at more developmental time points will reveal more in what order molecularly defined neural subsets are generated. Using recently developed analytical techniques to 'stitch' these multi-time-point datasets together will be advantageous to align all the cells along a unified developmental time axis. To overcome the limitation of the R9D11-Gal4 driver, which does not label neuroblasts nor the fully mature neurons, a permanent labeling strategy is required. More critically, such permanent labeling needs to be paired with technologies that provide single-lineage specification resolution, such as the introduction of single-neuroblast lineage barcoding techniques. Genetic constructs based around CRISPR-Cas9 and the Cre/Lox system have been developed for this purpose, although which exact lineage was labeled by a particular barcode was still unknown. The introduction of a spectrally unique barcode for each neuroblast lineage, in a similar vein to the recently developed Bitbow lineage tracking strategy, would be advantageous as they can provide direct in situ evidence for neuroblast lineage identity (Michki, 2021).
Finally, this work identifies several transcription factors that are specifically expressed in subsets of cells of the type II neuroblast progenies. These in silico and in situ results showed that their expressions are either constrained to particular developmental stages or in subsets of cells that are born in different orders. It would be desired to perform follow-up experiments to reveal whether these transcription factors play important roles in specifying the terminal fates of type II neuronal subtypes (Michki, 2021).
The homeobox gene homeobrain (hbn) is located in the 57B region together with two other homeobox genes, Drosophila Retinal homeobox (DRx) and orthopedia (otp) Optimal mitochondrial function determined by mitochondrial dynamics, morphology and activity is coupled to stem cell differentiation and organism development. However, the mechanisms of interaction of signaling pathways with mitochondrial morphology and activity are not completely understood. This study assessed the role of mitochondrial fusion and fission in the differentiation of neural stem cells called neuroblasts (NB) in the Drosophila brain. Depleting mitochondrial inner membrane fusion protein Opa1 and mitochondrial outer membrane fusion protein Marf in the Drosophila type II NB lineage led to mitochondrial fragmentation and loss of activity. Opa1 and Marf depletion did not affect the numbers of type II NBs but led to a decrease in differentiated progeny. Opa1 depletion decreased the mature intermediate precursor cells (INPs), ganglion mother cells (GMCs) and neurons by the decreased proliferation of the type II NBs and mature INPs. Marf depletion led to a decrease in neurons by a depletion of proliferation of GMCs. On the contrary, loss of mitochondrial fission protein Drp1 led to mitochondrial clustering but did not show defects in differentiation. Depletion of Drp1 along with Opa1 or Marf also led to mitochondrial clustering and suppressed the loss of mitochondrial activity and defects in proliferation and differentiation in the type II NB lineage. Opa1 depletion led to decreased Notch signaling in the type II NB lineage. Further, Notch signaling depletion via the canonical pathway showed mitochondrial fragmentation and loss of differentiation similar to Opa1 depletion. An increase in Notch signaling showed mitochondrial clustering similar to Drp1 mutants. Further, Drp1 mutant overexpression combined with Notch depletion showed mitochondrial fusion and drove differentiation in the lineage, suggesting that fused mitochondria can influence differentiation in the type II NB lineage. These results implicate crosstalk between proliferation, Notch signaling, mitochondrial activity and fusion as an essential step in differentiation in the type II NB lineage (Dubal, 2022).
Coordinated regulation of gene activity by transcriptional and translational mechanisms poise stem cells for a timely cell-state transition during differentiation. Although important for all stemness-to-differentiation transitions, mechanistic understanding of the fine-tuning of gene transcription is lacking due to the compensatory effect of translational control. This study used intermediate neural progenitor (INP) identity commitment to define the mechanisms that fine-tune stemness gene transcription in fly neural stem cells (neuroblasts). This study demonstrated that the transcription factor Fruitless(C) (Fru(C)) binds cis-regulatory elements of most genes uniquely transcribed in neuroblasts. Loss of fru(C) function alone has no effect on INP commitment but drives INP dedifferentiation when translational control is reduced. Fru(C) negatively regulates gene expression by promoting low-level enrichment of the repressive histone mark H3K27me3 in gene cis-regulatory regions. Identical to fru(C) loss-of-function, reducing Polycomb Repressive Complex 2 activity increases stemness gene activity. It is proposed that low-level H3K27me3 enrichment fine-tunes gene transcription in stem cells, a mechanism likely conserved from flies to humans (Rajan, 2023).
The Drosophila central brain is composed of approximately 100 paired lineages, with most lineages comprising 100-150 neurons. Most lineages have a number of important characteristics in common. Typically, neurons of a lineage stay together as a coherent cluster and project their axons into a coherent bundle visible from late embryo to adult. Neurons born during the embryonic period form the primary axon tracts (PATs) that follow stereotyped pathways in the neuropile. Apoptotic cell death removes an average of 30%-40% of primary neurons around the time of hatching. Secondary neurons generated during the larval period form secondary axon tracts (SATs) that typically fasciculate with their corresponding primary axon tract. SATs develop into the long fascicles that interconnect the different compartments of the adult brain. Structurally, this study distinguishs between three types of lineages: (1) PD lineages, characterized by distinct, spatially separate proximal and distal arborizations; (2) C lineages with arborizations distributed continuously along the entire length of their tract; and (3) D lineages that lack proximal arborizations. Arborizations of many lineages, in particular those of the PD type, are restricted to distinct neuropile compartments. It is proposed that compartments are 'scaffolded' by individual lineages, or small groups thereof. Thereby, the relatively small number of primary neurons of each primary lineage set up the compartment map in the late embryo. Compartments grow during the larval period simply by an increase in arbor volume of primary neurons. Arbors of secondary neurons form within or adjacent to the larval compartments, resulting in smaller compartment subdivisions and additional, adult specific compartments (Larsen, 2009).
The peculiar mode of generating fixed lineages of neurons from a small number of stem cell-like neuroblasts, so far found only in insects and crustaceans, has been studied in great detail since many decades. More recent genetic studies on neuroblasts in Drosophila have contributed significantly to the identification of molecular factors involved in the asymmetric distribution of cellular components, or the orientation of the mitotic spindle, during cell division. Less attention was given to the morphological characteristics of the neurons that formed part of the cell lineages derived from individual neuroblasts. This paper has addressed several aspects of the structural development of the lineages forming the Drosophila brain. Using GFP labeled clones, as well lineage specific GFP expression by Gal4 driver lines, the growth of lineages from embryo to adult were followed, focusing on cell number, neurite trajectory and neurite arborization pattern. The main interest was to identify attributes that lineages had in common, or that neurons belonging to a given lineage had in common, rather than emphasizing the diversity of neuronal phenotypes (Larsen, 2009).
Brain neuroblasts segregate from the procephalic (head) neurectoderm during early embryogenesis. They form a layer of approximately 100 cells; within this layer, neuroblasts with a specific genetic identity (i.e., pattern of expression of a certain set of transcription factors) always occupy the same position (Larsen, 2009).
Neuroblasts undergo 5-8 divisions in the embryo, producing that many ganglion mother cells; each ganglion mother cell divides once and forms two neurons or glial cells. It should be mentioned that one should expect exceptions to this general rule; for example, in the ventral nerve cord,
atypical neuroblasts like the MP cells derived from the mesectoderm divide only once. One neuroblast of the head, appears to produce only 8 neurons that all express Dilp. However, as observed for the ventral cord, the large majority of brain neuroblasts divide at intervals of 50-60 min after their segregation, and therefore should produce 10-16 labeled cells by the end of embryogenesis. The clone sizes obtained for late embryos were indeed quite uniform, but were consistently smaller than the expected value, most of them ranging between 8 and 12 cells. For example, mesodermal clones that should comprise a total of 8-16 cells (based on four mesodermal mitoses) are only two to six cells in average. The smaller than expected clone size is most likely due to the time interval required for building up a sufficient level of Flipase. For both mesodermal and brain clones, expected and observed clone size differ by about four cells, corresponding to two rounds of mitosis. Assuming a cell cycle length of about 1 h, these data point at a two-hour lag phase required for Flp build-up. In other words, heat-shocking at around 3 h (and one cannot go earlier than that, because it would interfere with gastrulation) will lead to flip-out around 5-6 h, the time when most neuroblasts, or mesodermal cells, have already divided twice (Larsen, 2009).
Aside from rather equal sizes, embryonic clones also behaved quite uniformly in regard to the pattern of axonal projection. Neurons of one lineage invariantly form a tight cluster and send axons in a single bundle that is called the primary axon tract (PAT). PATs were always directed radially away from the surface. Up until stage 15, PATs remained short and unbranched. They entered the minuscule embryonic neuropile at the location closest to their cluster of origin and stopped near the center
of the brain; many lineages located dorsally (DPL, DPM) projected towards the commissure connecting the two brain hemispheres. Only during late embryonic stages (late 16 to 17, ~15 h after fertilization) thin neurites with higher order branches became visible. The fact that longer axons (e.g., the pioneer axons establishing the major embryonic brain tracts) are not seen at an earlier stage is most likely due to the fact that the clones did not included the first born neurons. For the ventral nerve cord, most of the pioneer axons (e.g., acc, pcc, RP2) are formed by neurons that are the
first born in their lineage. Accordingly, if lineages are visualized by dye injection, they are in most cases comprised of two different types of neurons. One type has larger neurons with long axons that often project in quite different directions (like acc and pcc in the Nb1.1 lineage); these neurons are typically located deep, adjacent to the neuropile. The second type comprises smaller neurons, located more superficially in the cortex, and forming shorter and more uniform projections. The first type represents neurons born first; the second type later born neurons. Using Flip-out induced labeling of lineages, only the later born neurons are seen (Larsen, 2009).
A significant number of primary neurons undergo apoptosis at the embryo-to-larva transition. Cell death in the developing arthropod CNS has been described frequently; the current data, based on both single clone sizes as well as cell counts, put the ratio of cell death occurring at the embryo-to-larva transition at 40%. As a result of this massive amount of cell death, as well as local cell body rearrangements, the cortex thickness shrinks considerably from late embryo to early larva, while the volume of the neuropile increases due to neurite branching. The same phenomenon can be observed during the pupal phase: before onset of pupation, a stage when most neuroblasts have finished or are close to finish dividing and the number of neurons is maximal, the cortex measures 10-12 neuron diameters in thickness. In the adult brain, the cortex is very uneven in thickness. It is stretched out into a thin layer of 1-2 cell diameters thickness over most of the brain surface (VH). At certain locations, in particular in crevices in between compartments that form protrusions at the posterior and anterior brain surface (e.g., calyx, optic tubercle, antennal lobe, ventro-lateral protocerebrum), the cortex is 5-8 cell diameters in thickness. During the same period, the neuropile volume
increases dramatically. Precise neuron cell counts do not exist yet for the late larval and adult stages. Based on studies that focus on individual lineages (e.g., BLVa1-3, VH and SH) it is clear that apoptotic cell death eliminates a major fraction of the secondary neurons; whether it amounts to 40%, as in case of primary neurons during the embryo-larval transition, remains to be seen (Larsen, 2009).
The thinning of the brain cortex observed in the embryo-larval and the larval-adult transition is accompanied by rearrangement of neuronal cell bodies. These can be appreciated best when visualizing individual lineages. As might be expected, neuronal cell bodies of a given lineage form radially oriented, wedge- or column shaped clusters in the late embryo (primary lineages) or late larva (secondary lineages). In early larvae, primary neurons of a lineage are more spread out tangentially; the same happens to
secondary neurons in the adult brain cortex. That being said, cell bodies of a lineage do stay together as one cluster. There does not appear to be a large scale intermingling of cell bodies of different lineages (Larsen, 2009).
Another aspect of the architecture of the brain cortex that was addressed in this study is the delineation, or absence thereof, of lineages by glial cells. The notion appears in the literature that specialized glial boundaries separate clusters of cells that belong to different lineages. This is definitely not the case in Drosophila, as shown in this study for the cells of the engrailed-positive DPLam lineage. Cortex glia forms a meshwork of processes, called trophospongium, that wrap all neuronal cell bodies individually. The trophospongium at the boundary between cells of different lineages does not appear to be thicker, or specialized in any other way. Only at the larval stage when secondary neurons are being generated, clusters of newly born ganglion mother cells and neurons, located at the surface around the neuroblasts, are enclosed as a group (a sublineage) by cortex glia; as these cells are pushed away from the neuroblast by subsequent divisions, they
become individually wrapped by cortex glia (Larsen, 2009).
Anatomical work on insect brains, employing methods such as Golgi silver impregnation, dye backfilling and injection, or antibody labeling, have visualized the arborization patterns of a large number of individual neurons that vary enormously in size and shape. They include giant neurons with arborizations reaching throughout most compartments, as well small neurons with arbors restricted to fractions of a single compartment. To date, no systematic effort was undertaken to investigate how parameters like cell size or cell shape are represented numerically (are there 90% of giant neurons and 10% of dwarfs, or vice versa?) or related to development (are giants always born
earlier than dwarfs? Do they come from different lineages?). This paper does not provide a definitive answer to these questions, but presents a step along the way that will, hopefully soon, arrive at these answers (Larsen, 2009).
Neurons of most of the brain lineages visualized by clones or specific Gal4 drivers share a common projection pattern and, most likely, terminal arborization pattern. Three main types of lineages are defined, based on their geometry: PD lineages with well defined proximal and distal arborizations (e.g., MB lineages, BAla1 antennal projection neurons, DALv2 ellipsoid body neurons), C lineages where proximal and distal projections blend into each other (e.g., DPLam), and D lineages which lack proximal arborizations (e.g., BAla3). At both larval and adult stages, the arborization of most lineages is restricted to a minor fraction of the neuropile volume (5%-20%). Many PD lineages and some C lineages outline discrete compartments; aside from the well studied calyx and lobes of the mushroom body, defined by the four MB lineages, one can point at the BA lineages that generate the antennal projection neurons, the primary DALv2 linage whose proximal arbors are restricted to the larval BC compartment, many of the primary BL lineages that outline the BPL compartment, or the BAla3 lineage that is mostly restricted to the BPM. Proximal arborizations of the secondary DPMpm, DPMm, and CM4 lineages define the protocerebral bridge; distal arborizations produce the fan-shaped body and the noduli. Proximal arbors of the secondary DALv2 define the 'bulbs' (formerly called 'lateral triangle'), which represent the input domain of the ellipsoid body. Distal secondary DALv2 neurons generate the ellipsoid body. It is proposed that lineages such as MB, BAla/Bald, DPMm/DPMpm/CM4, or DALv2 act to 'scaffold' these compartments. According to this hypothesis, each compartment, A', has its own 'scaffolding lineage', A (or set of scaffolding lineages). A 'scaffolding lineage' would then be defined in
the following manner: (1) during development, the outgrowth of neurites from a lineage A actually creates the compartment A'. If A is deleted, A' also does not form. This has been shown experimentally for the calyx, the compartment scaffolded by the four MB lineages. (2) The arborization of lineage A forms a dense matrix of terminal axons on which synapses of A neurons themselves, as well as extrinsic neurons that enter compartment A' from the outside are made. Again, the calyx provides an example in this case: electron microscopic investigations have shown that well over two thirds of the postsynaptic terminal neurites belong to neurons of the MB lineages (Larsen, 2009).
The data suggest that a lineage-directed approach may facilitate the analysis of Drosophila brain development and structure considerably. This requires, first and foremost, to identify suitable Gal4 driver lines whose expression is restricted to one or a few lineages, and which then can be used to label, ablate, activate, or otherwise manipulate this lineage. Large scale screens are underway to attain this goal. Lineage-specific driver lines will also be crucial for the next step of the analysis, which looks at individual neurons within lineages. Published data suggest that many lineages can be subdivided 'vertically' into two hemilineages, and 'horizontally' into several sublineages. Neuroblasts produce series of ganglion mother cells (GMCs), each of which divides into an 'a' and a 'b' daughter cell. It has been shown for many lineages of the thoracic ganglia that all neurons of the a-hemilineage share properties that are different from neurons of the b-hemilineage. In some cases, a and b-hemilineages produce different SATs; or one complete hemilineage undergoes apoptosis, whereas the other one survives. Preliminary data suggest that hemilineages also exist for the brain (Larsen, 2009).
Sublineages are groups of neurons born during a defined time interval. For example, the primary neurons represent one significant sublineage for each (hemi)lineage; most likely the primary neurons of most lineages can be further subdivided into smaller subgroups. It has been shown that the lineages scaffolding the fan-shaped body comprise multiple sublineages with different distal arborizations. The fan-shaped body is subdivided into six horizontal layers and eight vertical columns. All neurons
of a given lineage (e.g., DPMpm) project to two distinct columns. Within a given lineage, neurons born at different times form sublineages that target different layers within the columns. Preliminary data shows that DALv2 forms sublineages whose distal arbors define the discrete layers within the ellipsoid body. Other lineages may form more overlapping projections; for example, each neuron of one of the MB lineages forms dendritic arbors that spread throughout most of the neuropile volume occupied by the lineage as a whole (Larsen, 2009).
The neurons of the primary lineages formed during embryogenesis form primary axon tracts (PATs) with invariant and characteristic trajectories. Note that, following a widely upheld convention, the unbranched processes that initially grow out from neurons are called 'axons,' irrespective of the fact that eventually, they will form terminal side branches that carry postsynaptic sites, presynaptic sites, or both. For some lineages, PATs can be still recognized in the differentiated larval neuropile. That is particularly
true for lineages that form long PATs, like the antenno-protocerebral tract, central anterior protocerebral tract (CAPT), or various commissural tracts. In other cases, the individual axons of one lineage may disperse to a certain extent and form a loose bundle. Once secondary axon tracts (SATs) form, they always extend in close proximity to the PATs of the corresponding lineage. This confirms that, even though several days pass between the time when it has finished producing its primary lineage and starts forming its secondary lineage, a neuroblast remains stationary, in close contact with the primary neurons. As a result, secondary neurons are born in contact with their older, primary siblings; the first primary axons they encounter when growing out their
own axons are the ones belonging to these siblings (Larsen, 2009).
In the neuropile, SATs of several lineages form thicker bundles, or fascicles. The term fascicle is used because according to convention it denotes a bundle of nerve fibers with different directionality and endings. In a previous characterization of secondary lineages, the most prominent fascicles have been characterized. This study shows that the larval fascicles, formed by discrete sets of SATs, persist throughout metamorphosis and become the brain fascicles of the adult. In adult brain preparations labeled with markers for synapses, the fascicles (formed by long axons lacking synapses) stand out as signal-negative spaces (Larsen, 2009).
Being able to follow fascicles from the larval period onward will help to unravel the connectivity of the adult brain. At present, unlike for vertebrate brains, knowledge of the 'macro-connectivity' (i.e., fiber bundles connecting different brain compartments)
of the insect brain is very rudimentary. Only about a few fiber bundles are known, such as the antenno-protocerebral tract connecting the antennal compartment with the calyx. Other tracts have been tentatively named on section-based maps of the adult brain, but the beginning and ending of these tracts largely remains unclear, and the tract names suggested are not in wide use. The analysis of the secondary axon tracts will allow for a new and systematic effort to unravel macro-connectivity of the brain (Larsen, 2009).
One of the key characteristics of neural development in Drosophila (and probably insects in general) is that neurons of a lineage form a relatively coherent unit, where somata, axons and major parts of their arborizations stay together and define discrete modules of the brain neuropile. In the few cases where experimental studies were done, removal of a particular lineage leads to the absence of the corresponding module; there is little or no regulation. How widespread are these insect-type neural lineages in the animal kingdom? Do they exist in vertebrates (Larsen, 2009)?
The second question can be answered in the negative. A number of fate mapping studies where the birth and migration of neurons was followed were done by the infection of progenitors with reporter-construct carrying viruses or mitotic recombination. These experiments indicate that clones of cells derived from individual neural progenitors do not form structural modules where cell bodies all adhere to each other, and axons form coherent bundles. Vertebrate neural progenitors ('neural stem cells') divide within the ventricular layer, or, in the forebrain, the subventricular zone of the embryonic neural tube. Subsequently, guided by radial glia (specialized neuroepithelial cells that later become astrocytes, neurons migrate preferentially radially. In a mouse study where labeled stem cells were integrated into the 10.5 day anterior neural tube (presumptive forebrain), these cells were found to undergo 9-11 rounds of mitosis every 10 h, producing clones of approximately 600 pyramidal cells. (One might point out that this figure lies in the same ballpark as the number of neurons produced by one insect neuroblast. In Drosophila, most lineages consist of 150-200 neurons; some lineages have more than 500 neurons. The neurons of one clone were relatively close to each other, but left in between them large spaces filled with unlabeled neurons, indicating that cells of many neighboring clones intermingle. More importantly, the dendritic or axonal projections
of a clone did not form coherent bundles. Aside from the radial clones of pyramidal cells, clones of tangentially migrating neurons were found. These clones correspond to the interneurons, which are born in a restricted domain of the ventral forebrain (eminence), from where they spread out tangentially to populate the different areas of the cortex (Larsen, 2009).
One can conclude that vertebrate neural lineages, at least up to a certain point in development, appear 'open': Cell bodies of numerous lineages intermingle within a given volume of the brain neuropile, and projections of members of a given lineage do not form distinct bundles or compartments. Outside arthropods and vertebrates, little is known about the relationship between neural architecture and neural development. In various protostome taxa, neuronal cell bodies in the cortex of the central nervous system
are clustered around axon bundles (e.g., plathelminthes); whether these clusters of neurons correspond to lineages remains to be shown (Larsen, 2009).
The fact that the Drosophila nervous system is composed of structural modules that are in many cases defined by discrete lineages offers the exciting possibility of getting closer at the link between genes and behavior. In a developmental sense, lineages represent 'units of gene expression'. The expression pattern of more than fifty transcription factors in specific embryonic neuroblasts has been described. In the embryo, a given transcription factor becomes active in one, or a small number of, neuroblasts; a particular neuroblast thereby acquires a 'genetic address,' consisting of specific sets of transcription factors. It is thought that this genetic address will essentially be involved in shaping the morphology and function of that lineage. Lineages also represent to some extent structural modules of the brain. It stands to reason that in many cases, a lineage with its highly restricted
dendritic arborization and axonal pathway, will be involved in a single or a limited number of behaviors, or, probably more accurately, 'behavioral subroutines'. If that is indeed correct, one can manipulate the structural module scaffolded by a given lineage, and thereby address its function, and what aspects of that function is controlled by a given gene expressed in the lineage. For example, completely removing that lineage by driving a cell death-inducing gene, using a lineage specific promoter, would eliminate
the module. Behavioral assays may show, in most cases, the deterioration of certain behavioral subroutines. Using the same driver to modify the expression of genes specifically found in a particular lineage, one might be able to manipulate the genes one at a time, to find that one gene may be simply involved in increasing the cell number in that lineage, or the density of synapses in the scaffolded compartment, etc. By systematically following this approach for each one of the ~100 lineages of the brain the expectation is that a much better understanding of how genes control behavior will be achieved (Larsen, 2009).
Awasaki, T., Kao, C. F., Lee, Y. J., Yang, C. P., Huang, Y., Pfeiffer, B. D., Luan, H., Jing, X., Huang, Y. F., He, Y., Schroeder, M. D., Kuzin, A., Brody, T., Zugates, C. T., Odenwald, W. F. and Lee, T. (2014). Making Drosophila lineage-restricted drivers via patterned recombination in neuroblasts. Nat Neurosci 17(4): 631-7. PubMed ID: 24561995
The Drosophila cerebrum originates from about 100 neuroblasts per hemisphere, with each neuroblast producing a characteristic set of neurons. Neurons from a neuroblast are often so diverse that many neuron types remain unexplored. New genetic tools were developed in this study that target neuroblasts and their diverse descendants, increasing the ability to study fly brain structure and development. Common enhancer-based drivers label neurons on the basis of terminal identities rather than origins, which provides limited labeling in the heterogeneous neuronal lineages. This study successfully converted conventional drivers that are temporarily expressed in neuroblasts, into drivers expressed in all subsequent neuroblast progeny. One technique involves immortalizing GAL4 expression in neuroblasts and their descendants. Another depends on loss of the GAL4 repressor, GAL80, from neuroblasts during early neurogenesis. Furthermore, this study expanded the diversity of MARCM-based reagents and established another site-specific mitotic recombination system. These transgenic tools can be combined to map individual neurons in specific lineages of various genotypes (Awasaki, 2014).
This technique report describes engineering of lineage-restricted drivers and
demonstrates their power in cell lineage analysis of Drosophila brains. As opposed
to non-selective clonal analysis with uncontrollable biases, lineage-restricted
drivers allowed recovery of clean isolated clones consistently from the same NB
lineage(s), greatly facilitating the annotation of related subclones to reveal the story
of each complex lineage. Furthermore, lineage-restricted drivers, derived through
irreversible activation of a ubiquitous transgene in the progenitors, ensure coverage
of neuronal offspring without internal gaps, which is critical for comprehensive
single-neuron mapping of heterogeneous lineages. However, it is important to note
that a given driver may not cover the lineage(s) of interest from the beginning of
neurogenesis. In practice, it is challenging to determine the exact coverage of a
particular driver, in terms of the frequency of hits and timing of onset in a given NB,
prior to clonal analysis among its potential targets (Awasaki, 2014).
Twin-spot MARCM was used with a lineage-restricted driver to efficiently
map the post-embryonic VLPl4 lineage. The gap-free coverage is supported
by the recovery of only four unpaired NB clones uniformly induced around the
beginning of larval neurogenesis. Examining NB clones of
reducing sizes was particularly informative. Notably, distinct parts of neurite
elaborations disappeared in sequence as the later-derived VLPl4 NB clones reduced
in size. This indicates the presence of diverse classes of neuronal offspring that
innervate distinct neuropils and arise in an invariant sequence. The rough cell numbers for different neuron classes were further deduced by counting the cell
bodies of NB clones that were induced at transition points when the production of
one neuron class stops and another neuron class starts. Labeling the NB clones as
well as the preceding GMC clones in distinct colors as twin spots provides
immediate insights into the identity of neuron(s) made during clone induction.
However, the chance in obtaining NB clones drops sharply during mid-larval
development possibly due to rapid cell cycles. Complete single-neuron lineage
mapping inevitably requires systematic follow-up analyses of single-cell clones from
dividing GMCs in serial temporal windows. Use of lineage-restricted drivers to
target specific lineages of interest is further essential for efficient recovery and
assignment of isolated single-neuron clones. It is emphasized that the ability
to map single neurons in four AL lineages simultaneously with R44F03-primed dual
independent MARCMs is unprecedented. Concurrent mapping of
multiple lineages unambiguously reveals the neuron types made by distinct lineages
at the same time. Notably, neighboring lineages appear autonomously controlled
and even end at different time points (hours apart) after puparium formation. Four additional neuron types were uncovered in the previously mapped lAL
lineage. All these advances in neuronal lineage analysis were not possible without
lineage-restricted drivers (Awasaki, 2014).
From mapping of the VLPl4 lineage, additional phenomena were observed about
the R13C01^dpn lineage-restricted driver. First, it uncovers certain lineages at high
frequencies but may hit many additional lineages sporadically. Second, each of the
preferred lineages shows a characteristic onset time at which progeny neurons
begin to be labeled. The R44F03^dpn driver exhibits similar phenomena but targets
a distinct, though partially overlapping, subset of NB lineages. These observations
demonstrate the spatiotemporal specificity characteristic of each lineage-restricted
driver in the coverage of the heterogeneous dynamic pool of neural progenitors.
About 5% of GAL4s screened exhibit significant activities in various sets of 5 to 20
NBs, suggesting combinatorial specification of NB fates. Mapping them onto specific
NB (sub)lineages requires extensive efforts (Awasaki, 2014).
The recent identification of stereotyped but complex NB clones underscores the
importance in mapping individual neurons made by one NB, to fully understand fly
brain anatomy and development. Twin-spot MARCM with lineage-restricted drivers
offers the ability to focus on a specific subset of NB lineages for detailed sublineage
analysis. Characterizing diverse lineage-restricted drivers may
ultimately allow coverage of the entire Drosophila brain for single-cell-resolution cell
lineage analysis. It would further provide genetic handles for targeted molecular
studies in specific NBs and/or their progenies. As to dual independent MARCM, the possibility is
envisioned of manipulating gene functions in specific NBs, targeted by
KDRT-MARCM, followed by detailed phenotypic analysis with twin-spot MARCM. Due to perdurance of gene expression, creating mutant NBs is often
necessary for eliminating gene functions from those transient-existing GMCs, which
unfortunately clouds the determination of particular GMCs' fates. Dual independent
MARCM resolves this dilemma by restoring single-GMC phenotypic analysis in
mutant whole NB clones. Taken together, the engineered lineage-restricted drivers
help in the realization of a new level of precision in targeted clonal analysis and should
enhance the lineage-based research on fly brain formation and function (Awasaki, 2014).
The central complex of Drosophila melanogaster plays important functions in various behaviors, such as visual and olfactory memory, visual orientation, sleep, and movement control. However little is known about the genes regulating the development of the central complex. This study reports that a mutant gene affecting central complex morphology, cbd (central brain defect), was mapped to ten-a, a type II trans-membrane protein coding gene. Down-regulation of ten-a in pan-neural cells contributed to abnormal morphology of central complex. Over-expression of ten-a by C767-Gal4 was able to partially restore the abnormal central complex morphology in the cbd mutant. Tracking the development of FB primordia revealed that the C767-Gal4 labeled interhemispheric junction, that separated fan-shaped body precursors at the larval stage, withdrew to allow the fusion of the precursors. While the C767-Gal4 labeled structure did not withdraw properly and detached from FB primordia, the two fan-shaped body precursors failed to fuse in the cbd mutant. It is proposed that the withdrawal of the C767-Gal4 labeled structure is related to the formation of the fan-shaped body. These result revealed the function of ten-a in central brain development, and possible cellular mechanism underlying Drosophila fan-shaped body formation (Cheng, 2013).
The central complex is an interconnected neuropil structure across and along the sagittal mid-section of the fly brain and includes the protocerebral bridge (PB), the fan-shaped body (FB), the paired nodule (NO), and the ellipsoid body (EB). It is involved in multi-modal behavioral control, such as locomotion, visual pattern memory and spatial orientation. The development of the central complex can be traced back to the larval stage. Lineage analysis has revealed the neurons that contribute to the central complex, but the molecular and cellular mechanism of central complex formation is not fully understood (Cheng, 2013).
In the 1980s, Martin Heisenberg and coworkers generated a series of structural mutants, in which the morphology of adult central brain structures like mushroom bodies and the central complex were destroyed. Among these mutants, mbm (mushroom body miniature), ceb (central brain deranged) and nob (no-bridge) have been identified. mbm was found to be a transcription factor, a nucleic acid-binding zinc finger protein, while ceb was reported to encode Neuroglian, a cell adhesion molecule that is crucial for axonal development, synapse formation and female receptivity. As to nob, it interacted with drl at the interhemispheric junction to affect the formation of protocerebral bridge. Another mutant type is central body defect (cbd), of which the most typical phenotype is that the fan-shaped body and the ellipsoid body are fragmented in the middle, or some fusion of the fan-shaped body and the ellipsoid body. So far, the molecular basis of most structural mutants is unclear (Cheng, 2013).
Ten-a belongs to a large protein family, Teneurin, which contains an N-terminal intracellular domain, a single transmembrane domain, eight EGF-like domains, a 6-blade β-propeller TolB-like domain, and 26 YD repeats. From invertebrates to vertebrates, Teneurins function as signaling molecules at the cell surface as type II transmembrane receptors, while the intracellular domain cleaved from membrane works as a transcription regulator and carboxyl terminus functions as a bioactive peptide. The Teneurin family members are thought to be important for establishment and maintenance of neuronal connections, neurite outgrowth and axon guidance. Recent reports showed that two Drosophila Teneurin members, Ten-a and Ten-m, are crucial for proper synaptic matching and the maintenance of neuromuscular junction. Although Teneurin may play a role in mammalian brain functio, detailed study is still largely lacking (Cheng, 2013).
This study reports that the Drosophila structural mutant gene cbd, the most typical phenotype of which is the fragmented fan-shaped body and ellipsoid body, is ten-a. The cbd mutation disrupts the formation of the FB, by preventing the merging of the two FB parts. This defect was rescued by over-expression of ten-a in a C767-Gal4 labeled structure which separated the FB parts but later disappeared to allow the merging of the two FB primordia. These results might reveal the molecular and cellular mechanism of Drosophila central complex development (Cheng, 2013).
This study found that the Drosophila central brain morphological mutant cbd is actually ten-a, a member of the teneurin family. Ten-a is required for fusion of the fan-shaped body precursors, before the formation of the complete normal FB. Mutation in ten-a leads to the failure of the two FB precursors to merge and consequently to the deranged fan-shaped body in adult flies (Cheng, 2013).
Aside from the FB morphological defect itself, ten-a mutation might cause other abnormalities that contribute to the morphological defect. For example, Ten-a might affect the projections and contra-lateral crossing of FB neurons resulting from lineages of FBP1 and FBP2, which contribute to two staves of the fan-shaped body, consequently led to a cleaved fan-shaped body. Nevertheless, the generation and projection of large field ExFl neurons labeled by NP6510-Gal4 or C205-Gal4 are not affected when ten-a mutated, which suggested that ten-a mainly produce the morphological defect by exerting its effect on FBP1 and FBP2 neuron arborizations. Actually, based on the morphological observation in cbd KS171 flies and the rescue results, it was postulated that the interhemispheric structure C767-Gal4 labeled was related to FB primordial fusion. But it seemed to have no effect on axonal projections and terminal arborization of F1 and F5 neurons (Cheng, 2013).
ten-a knockdown results showed that the neuronal ten-a was required for the central complex formation. Further rescue experiments suggested that neither neurons nor glial cells alone were sufficient for normal central complex formation. After screening, one Gal4 line was found finally. C767-Gal4 could be used to rescue the cbd mutant phenotype significantly. To identify the cell types labeled by C767-Gal4, neuron specific marker ELAV or glial cell specific marker REPO were used to co-stain with C767-Gal4 labeling cells. The results showed that nlsGFP driven by C767-Gal4 was co-localized with both neuronal and glial markers from larval to early pupal stages. Since previous studies showed some adhesion molecules were expressed both in neurons and glia for mediating the fasciculation of axon bundles, axon guidance or targeting, it is suggested that the rescue results by C767-Gal4 might just attribute to that the Gal4 expressed both in neurons and glial cells. That is to say, only when ten-a functions in certain neurons and glial cells together, the FB precursors could merge normally. However, which neurons and glial cells were required for the partial rescue could not be identified from current results. To solve this problem, more Gal4 lines which can rescue the cbd mutant phenotype are needed. Then, dependent on the expression patterns of these Gal4 lines, the neurons and glial cells which ten-a functions in may be identified (Cheng, 2013).
If Ten-a functions in C767-Gal4 labeled cells to influence the merging of FB primordia, what is its working partner for the arborization of FB neurons? As a Drosophila homolog of vertebrate Teneurin, Ten-a has been reported to be involved in embryo development, especially in the central nervous system. Ten-a, as well as its homologue Ten-m, was recently found to be required for synaptic matching between olfactory receptor neurons and corresponding projection neurons. Ten-a and Ten-m were also important for establishing the correct connection in the larval neuromuscular junction. In the current work, lack of normal Ten-a function led to failure in merging of FB precursors. It is possible to assume that Ten-a itself mediates homophilic interaction between neurons and glial cells to regulate the fusion of the central complex, such as Nrg, which is expressed on both neurons and glial cells and interacts to control axonal sprouting and dendrite branching. Meanwhile, Ten-a may interact with other molecules such as Ten-m, or other membrane proteins that function in heterophilic way at the cell surface. Further molecular and cellular experiments are needed to elaborate this important issue (Cheng, 2013).
Vertebrate Teneurins have been suggested to be related to mental diseases, and the discovery of Ten-a function in Drosophila brain development seems to support the hypothesis. Neuroglian (Nrg), whose vertebrate homologue L1-CAM has been implicated in neurological disorders, is also required for development of normal brain morphology in Drosophila. Considering that both Nrg and Ten-a are type-II transmembrane proteins with extracellular EGF repeats and also function in glial cells for brain development, it is possible that Teneurins in vertebrates also affect brain development, and probably synapse formation, as vertebrate Nrg does (Cheng, 2013).
In summary, this work elucidates the function of ten-a in development of the Drosophila central brain, and the cellular mechanism underlying FB formation (Cheng, 2013).
Environmental factors such as the availability of oxygen are instructive cues that regulate stem cell maintenance and differentiation. This study used a genetically encoded biosensor to monitor the hypoxic state of neural cells in the larval brain of Drosophila. The biosensor reveals brain compartment and cell-type specific levels of hypoxia. The values correlate with differential tracheolation that is observed throughout development between the central brain and the optic lobe. Neural stem cells in both compartments show the strongest hypoxia response while intermediate progenitors, neurons and glial cells reveal weaker responses. The distance between a cell and the next closest tracheole is a good predictor of the hypoxic state of that cell. This study indicates that oxygen availability appears to be the major factor controlling the hypoxia response in the developing Drosophila brain and that cell intrinsic and cell-type specific factors contribute to modulate the response in an unexpected manner (Baccino-Calace, 2020).
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