InteractiveFly: GeneBrief

Chronologically inappropriate morphogenesis: Biological Overview | References

BIOLOGICAL OVERVIEW

Many neural progenitors, including Drosophila mushroom body (MB) and projection neuron (PN) neuroblasts, sequentially give rise to different subtypes of neurons throughout development. A novel BTB-zinc finger protein, named Chinmo (Chronologically inappropriate morphogenesis), governs neuronal temporal identity during postembryonic development of the Drosophila brain. In both MB and PN lineages, loss of Chinmo autonomously causes early-born neurons to adopt the fates of late-born neurons from the same lineages. Interestingly, primarily due to a posttranscriptional control, MB neurons born at early developmental stages contain more abundant Chinmo than their later-born siblings. Further, the temporal identity of MB progeny can be transformed toward earlier or later fates by reducing or increasing Chinmo levels, respectively. Taken together, it is suggested that a temporal gradient of Chinmo (Chinmo^high --> Chinmo^low) helps specify distinct birth order-dependent cell fates in an extended neuronal lineage (Zhu, 2006).

Neural progenitors often generate distinct subtypes of neurons in an invariant temporal sequence during development. For example, cortical progenitors produce neurons that will occupy different layers of the neocortex in a stereotyped temporal order. Orderly generation of distinct cell types is also observed in the retina, spinal cord, and hindbrain. However, the molecular mechanisms governing the specification of neuronal cell fates based on birth order/timing, especially in vertebrates, remain largely unknown (Zhu, 2006).

Several studies suggest that the sequential production of different neuronal subtypes reflects temporal changes in neural progenitors. Heterochronic transplant or coculture experiments have yielded evidence supporting two distinct models elaborating such temporal changes. In the 'progressive restriction model,' neural progenitors progressively lose their intrinsic capabilities to produce different types of cells: young progenitors generate specific progeny in response to changes in extrinsic cues, and older progenitors appear intrinsically limited to generate only later-born cell fates. Evidence for the progressive restriction model is mainly derived from heterochronic transplantation experiments, in which early cortical progenitors can develop into any later-born cell fates if transplanted into an older cortex, but not the converse. Analogous studies, however, reveal a different scenario for retina progenitor cells (retinoblasts). Heterochronic coculture or transplant experiments show that the type of cells that the donor retinoblasts generate is independent of the developmental age of the host environment. These observations have led to proposal of the 'competence model', which suggests that progenitor cells pass through a series of distinct competence states to produce different types of progeny sequentially. Although progenitors are more influenced by the environment in the progressive restriction model, both models acknowledge intrinsic differences in progenitor cells of different ages (Zhu, 2006 and references therein).

The Drosophila embryonic ventral nerve cord (VNC) has been an informative model system for studying the specification of neuronal temporal identity. In each hemisegment of the Drosophila VNC, 30 neural progenitors, neuroblasts (Nbs), undergo a series of asymmetric divisions, with each division generating a variety of distinct neurons and/or glia in a fixed temporal sequence. In many lineages, individual Nbs and their progeny can be uniquely identified based on their spatial positions and expression of specific cell markers. Interestingly, temporal cell fate specification in most embryonic Nb lineages appears to be governed by a common mechanism involving the sequential expression of a set of transcription factors, Hunchback (Hb) --> Kruppel (Kr) --> POU domain transcription factors (Pdm) --> Castor (Cas). These transcription factors are transiently expressed in the Nbs in the above sequential order, and the temporal window of this expression for each transcriptional factor averages about one-cell-cycle. These factors are inherited by the postmitotic cells, which are born within these temporal gene expression windows, and in the daughter cells they act to specify temporal identity (Brody, 2002 and Isshiki, 2001). Together, these suggest that intrinsic changes in Nb gene expression at different developmental stages play an important role in specifying the temporal identities of Nb progeny (Zhu, 2006 and references therein).

Cellular mechanisms governing temporal cell fate specification are also critical during postembryonic development of the Drosophila brain. By the unparalleled ability to genetically label single neurons at specific developmental time points, MARCM (mosaic analysis with a repressible cell marker) has been used to demonstrate the presence of stereotyped neuronal temporal identities in a number of Drosophila postembryonic neuronal lineages, including the mushroom body (MB) and projection neuron (PN) lineages. In a MARCM-based screen, novel BTB-zinc finger protein, named Chinmo (Chronologically inappropriate morphogenesis), was identified based on defects in neuronal development in the Drosophila olfactory learning/memory center, the MBs. Chinmo's loss-of-function phenotypes were examined in multiple postembryonic neuronal lineages; mutant neurons born at early developmental stages often adopted the fates of late-born neurons of the same lineage. Interestingly, possibly due to differential translation of chinmo messages, early-born neurons contain more Chinmo than their later-born siblings; and a reduction or an increase in the Chinmo levels can accelerate or delay fate transitions in MB neuronal temporal identity. Taken together, a novel mechanism is suggested for the specification of neuronal temporal identity: postmitotic neurons born at early developmental stages contain more abundant Chinmo than later-born neurons in the same lineage, and such temporal gradients of Chinmo (Chinmo^high --> Chinmo^low) specify temporal cell fates in an extended neuronal lineage (Zhu, 2006).

In Drosophila, most Nbs repeatedly undergo asymmetric divisions to produce multiple rounds of two postmitotic neurons following the transient derivation of ganglion mother cells (GMCs). In an extended multicellular lineage, MARCM permits labeling of single-cell/two-cell clones that consist of the progeny born shortly after induction of mitotic recombination. By examining single-cell/two-cell clones generated at different developmental stages, it has been demonstrated that postembryonic development of the Drosophila MBs involves sequential generation of four distinct subtypes of neurons (γ → α′/β′ → pioneer α/β → α/β (Zhu, 2006).

To elucidate the molecular mechanisms underlying temporal cell fate specification, a screen was performed for mutations that affect sequential generation of distinct MB neurons in mosaic organisms. By using MARCM with a ubiquitous MB GAL4 driver (GAL4-OK107), one can generate and selectively visualize clones of MB neurons that are homozygous mutant for a specific chromosome arm within an otherwise heterozygous organism. A mature MB Nb clone, induced shortly after larval hatching (ALH), normally generates progeny that populate all the five MB lobes. The γ lobe (weakly labeled by the mAb 1D4) is derived from MB neurons born before the midthird-instar stage, the α′ and β′ lobes (negative for the mAb 1D4) consist of projections from late larval-born MB neurons, and the α and β lobes (strongly labeled with the mAb 1D4) are pupal-born MB neurons' derivatives. It was reasoned that mutations in genes autonomously controlling MB neuron temporal identity would cause predictable changes in the cellular composition of Nb clones and that one should be able to identify such abnormalities via analysis of subtype-specific axon projections. For example, a mutation in a gene required for early temporal fates might lack γ neurons. Conversely, mutations in genes mediating the acquisition of late-born fates might show defects in α/β production. 3500 mutagenized chromosome arms were screened and recovered one such MB neuronal temporal identity mutant, l(2L)MB523, was found whose full-sized Nb clones specifically lack larval-born MB γ and α′/β′ neurons (Zhu, 2006).

In contrast with wild-type controls, MB Nb clones homozygous for l(2L)MB523 generated at newly hatched larval (NHL) stage contain only one vertical and one horizontal axon bundle despite presence of many cell bodies. Colocalization was observed of l(2L)MB523 mutant axon bundles with the strong 1D4-positive α and β lobes, and mutant Nb clones contain ectopic fascicles of axons that also have high affinity for the mAb 1D4. These results suggest that most of the axons in the l(2L)MB523 mutant Nb clones likely differentiate as the α/β type of MB projections. In addition, there appears to be far more projections of the pioneer α/β type, as evidenced by prominent neurite staining on the dorsal/upper portion of the proximal two thirds of the mutant β lobe. Pioneer α/β neurons are normally born before α/β neurons (born within 6 hr before pupal formation [BPF]). Unlike other MB neurons, each pioneer α/β neuron extends only one primary dendrite into the calyx, and these dendrites, even in cells with different clonal origins, analogously project to the anterodorsal side of the calyx. Besides, their medial axon projections selectively extend along the upper surface of the β lobe but fail to reach the lobe terminus (Zhu, 2006).

Furthermore, in contrast with GAL4-OK107-labeled clones that have comparable sizes irrespective of genotypes, GAL4-201Y and GAL4-NP21 labeled much fewer cell bodies in l(2L)MB523 mutant Nb clones. Given that both GAL4-201Y and GAL4-NP21 selectively label γ neurons, this observation further suggests that early-born MB neurons in l(2L)MB523 clones have acquired later-born cell fates (Zhu, 2006).

To map l(2L)MB523, a lethal mutation was identified within the 22A6–22B1 cytogenomic region, based on a series of complementation tests. It was subsequently found that l(2L)MB523 belongs to the complementation group already consisting of one P element insertion, l(2)04111^K13009, and one EMS mutation, l(2)04111^M33. To determine which gene is affected in the l(2)04111 complementation group, the lethal P insertion line was characterized. Viable revertants were obtained following induction of P element excision, ascribing the lethality to disruption of an essential gene by a P element in l(2)04111^K13009. Then the P element's insertion site was determined by inverse PCR, and it was found that one P element is inserted in the first exon of the gene CG31666 in l(2)04111^K13009. CG31666 encodes a predicted BTB-zinc finger protein of 604 amino acids, with one BTB domain (amino acid 22–128) near the N terminus and two C₂H₂-type zinc fingers (amino acid 517–540 and 545–568) in the C-terminal region. Sequencing of CG31666's predicted exons and their splicing junctions further allowed identificaiton of a missense mutation in l(2)04111^M33, converting an evolutionally conserved aromatic amino acid (Phe88) to Isoleucine in the BTB domain. This missense mutation apparently blocks CG31666 gene's function because the same mutation abolished ectopic CG31666's gain-of-function phenotypes (Zhu, 2006).

To examine whether CG31666 is indeed expressed in the developing MBs and whether its expression is compromised by the unidentified l(2L)MB523 mutation, a peptide antibody was generated that specifically recognizes CG31666. Immunostaining with the anti-CG31666 antibody revealed nuclear localization of CG31666 in wild-type MB neurons at the mid-third-instar stage. In contrast, no CG31666 immunoreactivity was detected in l(2L)MB523 mutant MB clones. These results demonstrate CG31666 expression in the larval MBs and indicate the l(2L)MB523 as likely a protein null allele of CG31666 (Zhu, 2006).

This study shows that Chinmo levels in MB Nb progeny are predictive of daughter cells' temporal fates. Examinations and manipulations of Chinmo levels in vivo reveal how Chinmo^high → Chinmo^low may help govern the γ → α′/β′ → pioneer α/β → α/β order of MB neurogenesis. First, prospective γ neurons contain more abundant Chinmo than prospective α′/β′ neurons, and Chinmo expression apparently drops to zero in prospective α/β neurons. Second, MB Nbs repeatedly produce prospective γ neurons during the first 3 days of larval development; and, interestingly, gradients of Chinmo also exist among the sequentially derived prospective γ neurons. Third, most of the subsequently derived progeny likely become null for Chinmo after removal of chinmo from a young MB Nb, which explains why most of the larval-born MB neurons adopt the Chinmo null temporal fate and develop into pioneer α/β neurons in chinmo mutant MB Nb clones. Fourth, possibly due to perdurance of Chinmo after clone induction, most of single-cell clones of chinmo mutant MB neurons adopt their next temporal fates, reflecting likely presence of different residual Chinmo in the single-cell clones that are generated at different developmental stages. Chinmo perdurance may result from transcription of chinmo before the GMC → postmitotic neuron division, as suggested by presence of abundant chinmo transcripts in the larval MB Nbs. Fifth, MB Nbs precociously produce late-type neurons in a chinmo¹/+ heterozygous background but prolong the production of early-type neurons in the organisms that carry extra copies of chinmo. Such gene dosage affects the absolute amounts of Chinmo protein but probably leave the gradients largely unaltered, as suggested by their subtle phenotypes in shifting the timing of temporal cell fate transition. Interestingly, if Chinmo protein expression could be maintained at a high level through development (e.g., with GAL4-OK107-mediated induction of UAS-chinmo-3′UTR, MB progenitors appear remaining young and continuously yield the first-derived subtype of MB neurons until the end of neurogenesis. Taken together, the specification of distinct MB temporal cell fates on the basis of Chinmo's levels of expression suggests a novel mechanism by which time/birth order-dependent cell fates are determined (Zhu, 2006).

Posttranscriptional regulation is potentially used to control gradient expression of chinmo. Although changing chinmo's gene dosage might modulate the absolute amounts of Chinmo protein, multiple independent lines of evidence do support the notion that gradients of Chinmo are primarily derived from differential translational control in postmitotic neurons. Remarkably, such differential translation phenomena can later be recapitulated and used to tell the temporal identity of individual postmitotic neurons. The fact that these characteristics are stably inherited by every postmitotic neuron is in great contrast to the mechanism thought to operate in the embryonic CNS. In the embryo the regulatory networks governing the successive Hb → Kr → Pdm → Cas expression are likely to operate only transiently during neurogenesis. This argues against the possibility that Chinmo gradients are directly generated by some timing machinery. Instead, postmitotic cells likely acquire the ability to synthesize specific amounts of Chinmo based on birth order/timing following their initial temporal cell fate specification. Levels of Chinmo subsequently govern birth order/timing-dependent subtype-specific terminal differentiation in individual postmitotic neurons. Interestingly, Chinmo is also selectively required for proper specification of early temporal cell fates, at least, in the anterodorsal lineage of PNs. Taken together, these Chinmo studies so far suggest that temporal cell fate specification may generally take place step by step, and that a temporal gradient of Chinmo is possibly the intermediate master for specifying distinct birth order/timing-dependent neuronal morphologies (and, likely, subtype-specific molecular features as well) (Zhu, 2006).

Multiple mechanisms may act independently and/or in concert to simultaneously and/or sequentially govern neuronal temporal identity. For instance, a non-Chinmo mechanism is apparently required for proper specification of distinct subtypes of MB α/β neurons; and gradients of Chinmo may operate collaboratively with additional mechanisms to increase neuronal diversity in the PN lineages. It is speculated that Hb → Kr → Pdm → Cas and gradients of Chinmo may complement each other and, respectively, mediate quickly changing temporal cell fates and control slowly progressing cell fate transitions. Interestingly, Chinmo is also enriched but not uniformly expressed in the developing embryonic nervous system, suggesting its possible involvement in regulating cell fate transitions during embryonic neurogenesis as well. In sum, systematic analysis of Chinmo functions promises to shed new light on how numerous distinct neuron types can be derived from a limited number of progenitors during development of the complex nervous system (Zhu, 2006).

Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. This study shows that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity (Maurange, 2008).

Initially investigated was whether distinct temporal subsets of neurons are generated throughout the larval CNS. Chinmo and Broad Complex (Br-C), two BTB-zinc finger proteins known to be expressed in the postembryonic CNS, are distributed in complementary patterns in the central brain, thoracic, and abdominal neuromeres at the larval/prepupal transition stage at 96 hr (timings are relative to larval hatching at 0 hr). Chinmo is expressed by early-born neurons located in a deep layer, whereas Br-C marks later-born neurons in a largely nonoverlapping and more superficial layer. The deep Chinmo⁺ layer comprises most/all neurons born in the embryo plus an early subset of those generated postembryonically. Thoracic postembryonic neuroblasts undergo the Chinmo --> Br-C switch at ~60 hr such that they have each generated an average of 15 Chinmo⁺ cells expressing little or no Br-C and 39 Chinmo^- Br-C⁺ cells by 96 hr. The Chinmo⁺ and Br-C⁺ neuronal identities can be recognized as distinct cell populations on the basis of an ~2-fold difference in cell-body volume. This equates to an average cell-body diameter for Chinmo⁺ neurons of 4.5 μm, compared to only 3.6 μm for Br-C⁺ neurons. Plotting cell diameter versus deep-to-superficial position within postembryonic neuroblast clones reveals an abrupt decrease in neuronal size at the Chinmo --> Br-C transition. Together, these results provide evidence that most, if not all, postembryonic neuroblasts sequentially generate at least two different populations of neurons. First they generate large Chinmo⁺ neurons and then they switch to producing smaller Br-C⁺ neurons (Maurange, 2008).

To begin dissecting the neuronal switching mechanism, the functions of Chinmo and Br-C were investigated, but neither factor was found to be required for the transition in cell identity and cell size. Next it was asked whether a temporal transcription factor series related to the embryonic Hb --> Kr --> Pdm --> Cas sequence might be involved. Cas is known to be expressed in the larval CNS (Almeida, 2005), and this study shows that many different thoracic neuroblasts progress through a transient Cas⁺ phase during the 30-50 hr time window. Thoracic neuroblasts transiently express another member of the embryonic temporal series, Svp, during a somewhat later time window, from ~40 to ~60 hr. These results indicate that postembryonic Cas and Svp bursts are observed in many, but probably not all, thoracic progenitors and that their timing varies from neuroblast to neuroblast (Maurange, 2008).

To determine Svp function, thoracic neuroblast clones were generated homozygous for svp^e22, an amorphic allele. In ~53% of svp^e22 neuroblast clones induced in the early larva (at 12-36 hr), the Br-C⁺ neuronal identity is completely absent, all neurons express Chinmo, and there is no sharp decrease in neuronal size. The proportion of lineages failing to generate Br-C⁺ neurons rises to ~70% when clones are induced in the embryo and falls to only ~7% with late-larval (65-75 hr) induction. This is consistent with a previous finding that Svp bursts are asynchronous from neuroblast to neuroblast. The expression and clonal analyses together demonstrate that a progenitor-specific burst of Svp is required in many lineages for the switch from large Chinmo⁺ to small Br-C⁺ neurons (Maurange, 2008).

Thoracic neuroblast lineages homozygous for a strong cas allele, cas²⁴, show no obvious defects in the Chinmo --> Br-C transition when induced at 12-36 hr. However, since Cas is expressed in many postembryonic neuroblasts before their first larval division, it can only be removed by inducing clones during embryonic neurogenesis. Such cas²⁴ clones generate supernumerary Chinmo⁺ neurons and completely lack Br-C⁺ neurons at 96 hr, although this switching phenotype is restricted to only ~16% of thoracic neuroblasts. Constitutively expressing Cas blocks the Chinmo --> Br-C switch in a similar manner, with a frequency dependent upon whether thoracic UAS-cas clones are induced during embryogenesis (~47%), at early-larval (~10%) or at late-larval (0%) stages. This indicates that the response to Cas misexpression decreases as neuroblasts age. Together, the expression and loss- and gain-of-function analyses demonstrate that Chinmo and Br-C are negative and positive targets, respectively, of Cas and Svp. They also strongly suggest that progression through transient Cas⁺ and Svp⁺ states permits many postembryonic neuroblasts to switch from generating large to small neurons (Maurange, 2008).

To investigate whether Cas and Svp regulate neural proliferation as well as neuronal fates, the effector mechanism ending neurogenesis in the central brain and thorax was identified. In these regions, most neuroblasts cease dividing in the pupa at ~120 hr. Correspondingly, neurogenesis in all regions of the wild-type CNS ceases before the adult fly ecloses such that no adult neuroblasts are detected. In contrast to the central abdomen, blocking cell death by removing Reaper, Hid, and/or Grim (RHG) activity in the central brain and thorax does not prevent or delay pupal neuroblast disappearance. However, time-lapse movies of individual thoracic neuroblasts at ~120 hr reveal an atypical mitosis that is much slower than at ~96 hr, producing two daughters of almost equal size. This is largely accounted for by a reduction in the average diameter of neuroblasts from 10.4 μm at 96 hr to 7 μm at 120 hr, as GMC size does not vary significantly during this time window. The end of this atypical progenitor mitosis temporally correlates with reduced numbers of Mira⁺ cells and disappearance of the M phase marker phosphorylated-Histone H3 (PH3), indicating that it marks the terminal division of the neuroblast (Maurange, 2008).

Next whether late changes in basal complex components might underlie loss of neuroblast self-renewal was addressed. At 120 hr, it was found that Mira becomes delocalized from the cortex to the cytoplasm and nucleus of many interphase neuroblasts. In metaphase neuroblasts, Mira fails to localize to the basal side of the cortex, although it does selectively partition into one daughter during telophase. This late basal restoration resembles the 'telophase rescue' associated with several apical complex mutations. Pros, the Mira-binding transcription factor and GMC-determinant, is not detectable in the neuroblast nucleus at 96 hr, but at 120 hr a burst of Pros was observe in the nucleus of many Mira⁺ cells of intermediate size indicative of neuroblasts in the interphase preceding the terminal mitosis. Clones lacking Pros activity contain multiple Mira⁺ neuroblast-like cells. It was found that they do not respect the ~120 hr proliferation endpoint and even retain numerous dividing Mira⁺ progenitors into adulthood. In addition, GAL80^ts induction was used to induce transiently the expression of a YFP-Pros fusion protein well before the normal ~120 hr endpoint. Under these conditions, YFP-Pros can be observed in the nucleus of neuroblasts, most Mira⁺ progenitors disappear prematurely, and neural proliferation ceases much earlier than normal. Together, these results provide evidence that most neuroblasts terminate activity in the pupa via a nuclear burst of Pros that induces cell-cycle exit. These progenitors are referred to as type I neuroblasts to distinguish them from the much smaller population of type II neuroblasts that terminate via RHG-dependent apoptosis (Maurange, 2008).

Whether the postembryonic pulses of Cas and Svp in type I neuroblasts are implicated in scheduling their subsequent cell-cycle exit was tested. Remarkably, it was observed that many svp^e22 clones induced at early-larval stages retain a single Mira⁺ neuroblast at 7 days into adulthood. The persistent adult neuroblasts in a proportion of these clones also express the M phase marker, PH3, indicating that they remain engaged in the cell cycle and, accordingly, they generate approximately twice the normal number of cells by 3 days into adulthood. Furthermore, superficial last-born neurons in these over proliferating svp^e22 adult clones are Chinmo⁺ Br-C^- indicating a blocked Chinmo --> Br-C transition. Similar phenotypes are obtained in some UAS-cas and cas²⁴ clones. This analysis demonstrates that stalling the temporal series not only inhibits the late-larval switch to Br-C⁺ neuronal identity but also prevents the pupal cell-cycle exit of type I neuroblasts (Maurange, 2008).

To test the regulatory relationship between Pros and the temporal series, svp^e22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svp^e22 GMCs express nuclear Pros as normal. This likely accounts for why adult clones lacking Svp retain only a single neuroblast, whereas those lacking Pros contain multiple neuroblast-like progenitors. Importantly, these results demonstrate that nuclear Pros acts downstream of the temporal series in type I neuroblasts. Together, the genetic and expression analyses of Svp and Pros show that the temporal series triggers a burst of nuclear Pros in type I neuroblasts, thus inducing their cell-cycle exit (Maurange, 2008).

To determine how the temporal series is linked to the cessation of progenitor divisions, two transcription factors expressed in neuroblasts in a temporally restricted manner were examined. Dichaete (D), a member of the SoxB family, is dynamically expressed in the early embryo and is required for neuroblast formation. Consistent with the previous studies, it was observed that most or all embryonic neuroblasts progress through a transient D⁺ phase, but those in the lateral column of the ventral nerve cord initiate expression after their medial and intermediate counterparts. D subsequently becomes repressed in ~85% of neuroblasts during late-embryonic and postembryonic stages. Grainyhead (Grh) is first activated in neuroblasts in the late embryo and is required for regulating their mitotic activity during larval stages (Almeida, 2005, Bray, 1989, Brody, 2000, Cenci, 2005). Blocking early temporal series progression in the embryo, either by persistent Hb or loss of Cas activity, prevents most neuroblasts from downregulating D and also from activating Grh at late-embryonic and postembryonic stages. As forcing premature Cas expression leads to precocious D repression and Grh activation, both factors are likely to be regulated by Cas rather than by a later member of the temporal series. These results demonstrate that transient embryonic Cas activity permanently switches the expression of Grh on and D off. They also identify Grh and D as positive and negative targets, respectively, of the temporal series in many neuroblasts (Maurange, 2008).

Loss of Grh activity in thoracic neuroblasts (here defined as type I neuroblasts) leads to their reduced cell-cycle speed and disappearance during larval stages. At 96 hr, it was observed that 65% of grh³⁷⁰ type I neuroblasts are smaller than normal (~6.4 μm in diameter), delocalize Mira from the cortex to the cytoplasm and nucleus, and strongly express Pros in the nucleus. These events, reminiscent of the 120 hr terminal cell cycle of wild-type progenitors, show that Grh is required to prevent the premature cell-cycle exit of type I neuroblasts. Given this finding, and that Cas is required to activate Grh, the question arises as to how some neuroblasts lacking embryonic Cas activity are able to continue dividing into adulthood. However, cas²⁴ neuroblasts persisting in adults all retain Grh, suggesting that they may derive from clones that lacked only the last of the two Cas pulses observed in some embryonic neuroblasts, perhaps retaining enough Cas activity to support Grh activation but not later progression of the temporal series (Maurange, 2008).

To determine if late temporal series inputs, after embryonic Cas, are also required to maintain the long-lasting postembryonic expression of Grh, svp^e22 clones were induced in early larvae. Although type I neuroblasts in these mutant clones have a stalled temporal series, they retain postembryonic Grh expression through to adult stages. Thus, two sequential inputs from the temporal series are required for type I neuroblasts to undergo timely Pros-dependent cell-cycle exit. First, embryonic Cas activity switches on sustained Grh expression, inhibiting premature nuclear Pros and permitting continued mitotic activity. Second, a late postembryonic input, requiring Svp, counteracts this activity of Grh by triggering a pupal burst of nuclear Pros (Maurange, 2008).

Despite undergoing premature cell-cycle exit, it was noticed that grh mutant neuroblasts can still generate both Chinmo⁺ and Br-C⁺ neurons. Thus, Grh is not required for the neuronal Chinmo --> Br-C switch. Conversely, neither Chinmo nor Br-C appears to be required postembryonically for neuroblast cell-cycle exit. In summary, the properties of both neuroblasts and neurons are regulated by downstream targets of the temporal series (Maurange, 2008).

Next whether the temporal series and its targets also function in type II neuroblasts, which terminate via RHG-dependent apoptosis rather than Pros-dependent cell-cycle exit, was tested. Focus was placed on one identified type II neuroblast in the central abdomen, called dl, which undergoes apoptosis at 70-75 hr and produces only a small postembryonic lineage of ~10 neurons. It was observed that the dl neuroblast expresses bursts of Cas (~45 to ~60 hr) and Svp (~62 to ~65 hr) and sequentially generates Chinmo⁺ (~7 deep) and Br-C⁺ (~3 superficial) neurons. Loss of Cas or Svp activity, or prevention of temporal series progression in several other ways all lead to a blocked Chinmo --> Br-C transition, a failure to die at 70-75 hr, and the subsequent generation of many supernumerary progeny. These results show that the temporal series performs similar functions in type I and type II neuroblast lineages, regulating both the Chinmo --> Br-C neuronal switch and the cessation of progenitor activity. Next, the mechanism linking the temporal series to RHG-dependent death of type II neuroblasts was tested. As for type I neuroblasts, dl progenitors in cas²⁴ clones, induced in the embryo, fail to activate Grh and repress D. Moreover, if grh activity is reduced, or if D is continuously misexpressed, dl progenitors persist long after 75 hr. Therefore, both of these early Cas-dependent events are essential for subsequent type-II neuroblast apoptosis. However, in contrast to members of the temporal series themselves, persistent misexpression of their target D does not block the Chinmo --> Br-C switch in the dl lineage. Thus, the D^- Grh⁺ state of type I and type II neuroblasts, installed via an embryonic Cas pulse, appears to be necessary for progenitor termination but not for Chinmo --> Br-C switching. Nevertheless, dl neuroblasts stalled at a postembryonic stage in svp^e22 and UAS-cas clones retain the D^- Grh⁺ code yet still fail to undergo apoptosis. Therefore, as with type I cell-cycle exit, timely type II apoptosis requires both embryonic Cas-dependent and postembryonic Svp-dependent inputs from the temporal series (Maurange, 2008).

Finally, the regulatory relationship between the temporal series and AbdA, a Hox protein transiently expressed in postembryonic type II (but not type I) neuroblasts and required for their apoptosis was dissected. dl neuroblasts lacking postembryonic Svp activity or persistently expressing Cas still retain AbdA expression yet do not die. This suggests that AbdA is unable to kill neuroblasts unless they progress, in a Svp-dependent manner, to a late Cas^- temporal state. To test this prediction directly, use was made of the previous finding that ectopic AbdA is sufficient to induce neuroblast apoptosis, albeit only within a late time window. Constitutive AbdA-induced apoptosis is efficiently suppressed by persistent Cas, not only in type II but also in type I neuroblasts. This result demonstrates that, in order to terminate, type II neuroblasts must progress to a late Cas^- state, thus acquiring a D^- Grh⁺ Cas^- AbdA⁺ code. It also suggests that AbdA is sufficient to intercept progression of the temporal series in type I neuroblasts, inducing an early type II-like termination (Maurange, 2008).

This study has found that the Drosophila CNS contains two distinct types of self-renewing progenitors: type I neuroblasts terminate divisions by cell-cycle withdrawal and type II neuroblasts via apoptosis. Despite these different exit strategies, both progenitor types use a similar molecular timer, the temporal series, to shut down proliferation and thus prevent CNS overgrowth. These findings demonstrate that the temporal series does considerably more than just modifying neurons; it also has multiple inputs into neural proliferation. The identification and analysis of several pan-lineage targets of the temporal series also begins to shed light on the mechanism by which developmental age modifies the properties of neuroblasts and neurons. Two targets, Chinmo and Br-C, are part of a downstream pathway temporally regulating the size and identity of neurons. Two other temporal series targets, Grh and D, function in neuroblasts to regulate Prospero/RHG activity, thereby setting the time at which proliferation ends. The temporal series regulates both cell proliferation and cell identity; a feedforward mechanism is proposed for generating combinatorial transcription factor codes during progenitor aging (Maurange, 2008).

This study has found that the temporal series regulates a widespread postembryonic switch in neuronal identity. Most, if not all, type I and type II neuroblasts first generate a deep layer of large Chinmo⁺ neurons and then switch to producing a superficial layer of small Br-C⁺ neurons. Two lines of evidence argue that this postembryonic neuronal switch is likely to be regulated by a continuation of the same temporal series controlling early/late neuronal identities in the embryo. First, the postembryonic Chinmo --> Br-C neuronal switch is promoted by the transient redeployment of two known components of the embryonic temporal series, Cas and Svp. Second, this switch remains inhibitable by misexpression of the other embryonic temporal factors such as Hb. Since both Cas and Svp are expressed somewhat earlier than the neuronal-size transition, it is likely that they promote bursts of later, as yet unknown, members of the temporal series that more directly regulate Chinmo and Br-C. Although neuronal functions for both BTB zinc-finger targets have yet to be characterized, a progressive early-to-late decrease in postmitotic Chinmo levels is known to regulate the temporal identities of mushroom-body neurons (Zhu, 2006). The current results now suggest that this postmitotic gradient mechanism may be linked to, rather than independent from, the temporal series (Maurange, 2008).

Type I neuroblasts in clones lacking postembryonic Cas/Svp activity or retaining an early temporal factor, fail to express nuclear Pros during pupal stages and thus continue dividing long into adulthood. These overproliferating adult clones each contain only a single neuroblast, sharply contrasting with adult clones lacking Brat or Pros, in which there are multiple neuroblast-like progenitors (Bello, 2006). Hence, manipulations of the temporal series and its progenitor targets offer the prospect of immortalizing neural precursors in a controlled manner, without disrupting their self-renewing asymmetric divisions (Maurange, 2008).

This study demonstrates that type I and type II neuroblasts must progress through at least two critical phases of the temporal series in order to acquire the D^- Grh⁺ Cas^- combinatorial transcription factor code that precedes Pros/RHG activation. The early phase corresponds to embryonic Cas activity switching neuroblasts from D⁺ Grh^- to D^- Grh⁺ status. The equally essential, but less well-defined, late postembryonic phase of the temporal series requires transition to a Cas^- state and a late Svp burst. For type I neuroblasts, Grh and a late Cas^- temporal identity are both required for timely expression of nuclear Pros and subsequent cell-cycle withdrawal. For type II neuroblasts, these two inputs are also necessary for RHG-dependent apoptosis, with the additional requirement that D must remain repressed. Although the temporal series and its targets are similarly expressed in type I and type II neuroblasts, only the latter progenitors undergo a larval burst of AbdA. This AbdA expression is likely to be the final event required to convert the D^- Grh⁺ Cas^- state, installed by the temporal series, into the D^- Grh⁺ Cas^- AbdA⁺ combinatorial code for RHG-dependent apoptosis. This code prevents type II neuroblasts in the abdomen from reaching the end of the temporal series and accounts for why they generate fewer progeny and terminate earlier than their type I counterparts in the central brain and thorax (Maurange, 2008).

The data in this study support an indirect feedforward model for neuroblast aging. Key to this model is the finding that, although members of the temporal series are only expressed very transiently, some of their targets can be activated or repressed in a sustained manner, as observed for Chinmo/Br-C in neurons and also for Grh/D in neuroblasts. In principle, this indirect feedforward allows aging progenitors to acquire step-wise the combinatorial transcription factor codes modulating cell-cycle speed, growth-factor dependence, competence states, and neural potential. Like Drosophila neuroblasts, isolated mammalian cortical progenitors can sequentially generate neuronal fates in the correct in vivo order (Shen, 2006). These studies suggest that it will be important to investigate whether the transcription factors controlling this process also regulate cortical proliferation and whether their targets include BTB-zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some insect/mammalian parallels seem likely, since it is known that Sox2 downregulation and Prox1 upregulation can both promote the cell-cycle exit of certain types of vertebrate neural progenitors (Dyer, 2003, Graham, 2003). Thus, although insect and mammalian neural progenitors do not appear to use the same sequence of temporal transcription factors, at least some of the more downstream components identified in this study might be functionally conserved (Maurange, 2008).

chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila

The Drosophila STAT transcription factor Stat92E regulates diverse functions, including organ development and stem cell self-renewal. However, the Stat92E functional effectors that mediate these processes are largely unknown. This study shows that chinmo is a cell-autonomous, downstream mediator of Stat92E that shares numerous functions with this protein. Loss of either gene results in malformed eyes and head capsules due to defects in eye progenitor cells. Hyperactivation of Stat92E or misexpression of Chinmo results in blood cell tumors. Both proteins are expressed in germline (GSCs) and cyst stem cells (CySCs) in the testis. While Stat92E is required for the self-renewal of both populations, chinmo is only required in CySCs, indicating that Stat92E regulates self-renewal in different stem cells through independent effectors. Like hyperactivated Stat92E, Chinmo misexpression in CySCs is sufficient to maintain GSCs nonautonomously. Chinmo is therefore a key effector of JAK/STAT signaling in a variety of developmental and pathological contexts (Flaherty, 2010).

This study has revealed important information about chinmo and its role in Stat92E-dependent biological processes, including eye development, hematopoeisis, and stem cell self-renewal. chinmo was identified as a cell-autonomously induced downstream mediator of JAK/STAT activity that shares loss- and gain-of-function phenotypes with Stat92E in several tissues. Although chinmo was originally identified in a screen for genes required for temporal identity of mushroom body neurons, no factors that regulate its expression had been identified. The fact that chinmo and Stat92E exhibit a high degree of functional overlap suggests that chinmo performs multiple Stat92E-dependent functions, including growth of the eye disc, formation of melanotic tumors, proliferation of mature hemocytes, self-renewal of adult stem cells, and repression of Ser. Furthermore, the results raise the interesting hypothesis that the JAK/STAT pathway is also required for the temporal identity of neurons in the mushroom body. It was also shown that Chinmo, like stabilized Stat92E, is expressed in GSCs and in CySCs in the testis. However, unlike Stat92E, Chinmo is required intrinsically only for the self-renewal of CySCs and not of GSCs. These data clearly indicate that Stat92E acts through distinct effector genes in these stem cells to promote cell-autonomous self-renewal. Finally misexpression of chinmo in CySCs results in the expansion of GSCs and CySCs, a phenotype also observed with misexpression of hop^Tum-l or of zfh1 in somatic cells. This provides additional evidence for the coordination of self-renewal and differentiation between adjacent GSCs and CySCs (Flaherty, 2010).

The BTB domain mediates protein-protein interactions, including dimerization, recruitment of transcriptional repressors to DNA, and protein degradation by acting as adaptors for Cul-3 E3 ubiquitin ligases. In the antenna, Ser was found to be cell-autonomously repressed by both Stat92E and Chinmo (this study; Flaherty, 2009). These data suggest that Chinmo might act as a transcriptional repressor, at least in the antennal disc, and that Ser might be one of its transcriptional targets. It should be stressed that these results do not rule out the possibility that Chinmo, through its BTB domain, can also act as an adaptor for Cul-3 and promote protein degradation. In fact, recent work has revealed that even BTB-ZF transcription factors like promyelocytic leukemia zinc finger (PLZF) can interact with Cul-3. Whereas the role of the BTB domain in PLZF-dependent transcriptional repression has been well documented, the physiological role of the PLZF-Cul-3 interaction and the proteins it modifies are as yet unknown. Future experiments will be needed to determine if Chinmo can act both as a transcriptional repressor per se and as an adaptor for E3 ligases. In either scenario, factors modified by Chinmo, in addition to Ser, will need to be identified and characterized (Flaherty, 2010).

This study shows that Chinmo, like Zfh1, is essential for the self-renewal of CySCs in the Drosophila testis. Furthermore, sustained expression of Chinmo in somatic cells, like that of Zfh1, is sufficient to induce the expansion of GSCs and CySCs. In addition, chinmo and zfh1 do not regulate each other's expression. chinmo and zfh1 both appear to act downstream of Stat92E to maintain CySCs, which raises the possibility that these factors function either in an epistatic or parallel manner in the somatic lineage (this study; Leatherman, 2008). chinmo does not act through zfh1, but it was not possible to determine if the reciprocal was true. However, it is hypothesized that if zfh1 is upstream of chinmo then CySCs lacking either of these factors should differentiate at the same time point. In fact, CySCs lacking zfh1 differentiate faster than those lacking chinmo, suggesting that zfh1 may not reside upstream of chinmo. Despite the unresolved genetic relationship between zfh1 and chinmo, the data are consistent with a model in which they function in a parallel pathway in the self-renewal of CySCs and the expansion of GSCs and CySCs (Flaherty, 2010).

Zfh1 has been shown to act as a transcriptional repressor (see Leatherman, 2008). The current data suggest that Chinmo may inhibit transcription directly or by post-translational modification of factors that silence genes. Zfh1 is expressed highly in CySCs and at low levels in early cyst cells. It is not expressed in late cyst cells. In contrast, Chinmo is expressed at high and comparable levels in CySCs and early cyst cells, but not in late cyst cells. Taking into account all of these results, two models are proposed to explain the function of Chinmo in the somatic lineage of the testis. The first hypothesizes that Zfh1 and Chinmo regulate distinct downstream effectors, all of which are required for the maintenance of CySCs. In this model, early cyst cells, which express high levels of Chinmo but low levels of Zfh1, can become late cyst cells only when Chinmo expression is sufficiently decreased there, allowing for full cyst cell differentiation and the complete development of mature germ cells. In the second model, Chinmo and Zfh1 regulate different genes critical for CySC self-renewal, but in contrast to the first, Chinmo only has a function in CySCs and not in early cyst cells. In this second model, the existence is invoked of cofactors expressed only in CySCs that act in concert with Chinmo, thereby restricting Chinmo function only to CySCs (Flaherty, 2010).

Activation of the JAK/STAT signaling pathway in CySCs is sufficient to promote self-renewal of both CySCs and GSCs, indicating that CySCs can influence GSC maintenance (Leatherman, 2008). Although the mechanisms by which CySCs regulate GSC self-renewal have not yet been elucidated at the molecular level, two models have been proposed. Activated Stat92E, through its functional effectors, could (1) block CySC differentiation intrinsically, thus also inhibiting GSC differentiation at the same time, or (2) send one or more non-cell-autonomous signals from CySCs to GSCs, thus promoting GSC self-renewal. Either model would result in an increase in the number of CySCs and GSCs. In the first model, sustained activation of JAK/STAT signaling in CySCs allows these cells to continue proliferating. Therefore, they accumulate outside of the niche. Previous studies have shown that the mitoses of GSCs and CySCs must be linked in order to have the appropriate number of GSCs, and their progeny always encapsulated by two CySCs/cyst cells. In the second model, the self-renewal of GSCs depends on two independent signals: one is Upd sent from the niche, which activates Stat92E in adjacent GSCs, and the other is an unknown factor presumed to come from the neighboring CySCs. A similar situation occurs in female flies, where two signals are required for GSC maintenance. First, cap cells, which form the ovarian niche, produce Decapentaplegic (Dpp), which acts on GSCs by inhibiting the bam gene. Second, an Upd cytokine, produced by ovarian somatic support cells adjacent to the niche, acts on cap cells to increase Dpp production, thus influencing GSC self-renewal in a nonautonomous manner. It is currently unknown whether the nonautonomous signal from CySCs to GSCs in the testis is a secreted factor (Flaherty, 2010).

The results indicate that Chinmo has an important role in the self-renewal of CySCs, in the inhibition of CySC differentiation, and in the transduction of the nonautonomous signal from CySCs to GSCs. Furthermore, this expansion requires the BTB and ZF domains in Chinmo, suggesting that the molecular function of Chinmo in this process may be transcriptional repression and/or protein degradation. The fact that somatic misexpression of Chinmo, like that of Zfh1, can promote GSC self-renewal/expansion indicates that at least three factors play important roles in regulating stem cell self-renewal in a nonautonomous manner: activated Stat92E, Zfh1, and Chinmo. These observations raise many issues, such as the importance of JAK/STAT pathway activity in the soma, whether chinmo can bypass the requirement for Stat92E in CySCs, and the mechanism of nonautonomous self-renewal between adjacent stem cell populations. These issues need to be addressed at the molecular level in the future (Flaherty, 2010).

A protein BLAST search against the nonredundant mouse database identified mZFP509 as a potential Chinmo ortholog. mZFP509 is a 757 amino acid protein that has the same overall structure as Chinmo: an N-terminal BTB domain located between residues 20-120 separated from two C-terminal C₂H₂ zinc fingers by a stretch of ~400 amino acids. mZFP509 is 27% identical to Chinmo (with 36% identity in the BTB domain and 31% identity the ZF region) and is 83.7% identical to hZFP509. Microarray studies indicate that mzfp509 is enriched in normal and cancer stem cells. mzfp509 transcripts are present in mESCs but are substantially reduced during their differentiation. They are also significantly increased in PU.1-deficient preleukemic hematopoietic stem cells and normal mammary stem cells. These data suggest that ZFP509 and Chinmo are orthologs and that what is discovered about Chinmo in Drosophila may have a high probability of holding true for its mammalian counterpart. For example, blocking hZFP509 function may have therapeutic value in inhibiting cancer stem cells, thus offering better outcomes for human patients (Flaherty, 2010).

BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila

During tumorigenesis, pathways that promote the epithelial-to-mesenchymal transition (EMT) can both facilitate metastasis and endow tumor cells with cancer stem cell properties. To gain a greater understanding of how these properties are interlinked in cancers, Drosophila epithelial tumor models were used, that are driven by orthologues of human oncogenes (activated alleles of Ras and Notch) in cooperation with the loss of the cell polarity regulator, scribbled (scrib). Within these tumors, both invasive, mesenchymal-like cell morphology and continual tumor overgrowth, are dependent upon Jun N-terminal kinase (JNK) activity. To identify JNK-dependent changes within the tumors a comparative microarray analysis was used to define a JNK gene signature common to both Ras and Notch-driven tumors. Amongst the JNK-dependent changes was a significant enrichment for BTB-Zinc Finger (ZF) domain genes, including chronologically inappropriate morphogenesis (chinmo). chinmo was upregulated by JNK within the tumors, and overexpression of chinmo with either RasV12 or Nintra was sufficient to promote JNK-independent epithelial tumor formation in the eye/antennal disc, and, in cooperation with RasV12, promote tumor formation in the adult midgut epithelium. Chinmo primes cells for oncogene-mediated transformation through blocking differentiation in the eye disc, and promoting an escargot-expressing stem or enteroblast cell state in the adult midgut. BTB-ZF genes are also required for Ras and Notch-driven overgrowth of scrib mutant tissue, since, although loss of chinmo alone did not significantly impede tumor development, when loss of chinmo was combined with loss of a functionally related BTB-ZF gene, abrupt, tumor overgrowth was significantly reduced. abrupt is not a JNK-induced gene, however, Abrupt is present in JNK-positive tumor cells, consistent with a JNK-associated oncogenic role. As some mammalian BTB-ZF proteins are also highly oncogenic, this work suggests that EMT-promoting signals in human cancers could similarly utilize networks of these proteins to promote cancer stem cell states (Doggett, 2015).

This report has defined the transcriptional changes induced by JNK signaling within both scrib>RasACT and scrib>NACT tumors by carrying out comparative microarray expression arrays. This analysis that JNK exerts a profound effect upon the transcriptional profile of both Ras and Notch-driven tumor types. The expression of nearly 1000 genes was altered by the expression of bskDN in either Ras or Notch-driven tumors, and less than half of these changes were shared between the two tumor types, indicating that JNK signaling elicits unique tumorigenic expression profiles depending upon the cooperating oncogenic signal. Nevertheless, of the 399 JNK-regulated probe sets shared between Ras and Notch-driven tumors, it is hypothesized that these had the potential to provide key insights into JNK's oncogenic activity, and to prioritize these targets, it was considered that the expression of the critical oncogenic regulators would not just be altered by bskDN, but would be normalized to close to wild type levels. This subset of the 399 probe set was identified by comparing the expression profile of each genotype back to control tissue, thereby producing a more focussed JNK signature of 103 genes. Notably, this included previously characterized targets of JNK in the tumors, such as Mmp1,cherand Pax, thereby providing validation of the approach. Also amongst these candidates were 4 BTB-ZF genes; two of which were upregulated by JNK in the tumors (chinmo and fru), and two downregulated (br and ttk) (Doggett, 2015).

Focussing upon chinmo, chinmo overexpression was shown to be sufficient to prime epithelial cells for cooperation with RasACT in both the eye antennal disc and in the adult midgut epithelium, and that chinmo is required for cooperative RasACTor NACT-driven tumor overgrowth, although its function was only exposed when its knockdown was combined with knockdown of a functionally similar BTB-ZF transcription factor, abrupt. This family of proteins is highly oncogenic in Drosophila, since previous work has shown that ab overexpression can cooperate with loss of scrib to promote neoplastic overgrowth, and in these studies, it was also shown that overexpression of a fru isoform normally expressed in the eye disc is capable of promoting cooperation with RasACT and NACT in the eye-antennal disc, in a similar manner to chinmo overexpression. Thus, whether fru also plays a role in driving Ras or Notch-driven tumorigenesis warrants further investigation. Indeed, a deeper understanding of the oncogenic activity of these genes is likely to be highly relevant to human tumors, since of the over 40 human BTB-ZF family members, many are implicated in both haematopoietic and epithelial cancers, functioning as either oncogenes (eg., Bcl6, BTB7) or tumor suppressors (eg., PLZF, HIC1). Furthermore, over-expression of BTB7, can also cooperate with activated Ras in transforming primary cells, and its loss makes MEFs refractory to transformation by various key oncogenes such as Myc, H-rasV12 and T-Ag, suggesting that cooperating mechanisms between BTB-ZF proteins and additional oncogenic stimuli might be conserved (Doggett, 2015).

JNK signaling in Drosophila tumors is known to promote tumor overgrowth through both the STAT and Hippo pathways. Deregulation of the STAT pathway was evident in the arrays through the upregulation of Upd ligands by JNK in both Ras and Notch-driven tumors. In contrast, although cher was identified in the arrays as being upregulated in both tumor types and previous studies have shown that cher is partly required for the deregulation of the Hippo pathway in scrib>RasACT tumors, more direct evidence for Hippo pathway deregulation amongst the JNK signature genes was lacking. In part, this could be due to JNK regulating the pathway through post-transcriptional mechanisms involving direct phosphorylation of pathway components. However, the failure to identify known Hippo pathway target genes, and proliferation response genes in general, may simply highlight limitations in the sensitivity of the array assay and the cut-offs used for determining significance, despite its obvious success in correctly identifying many known JNK targets (Doggett, 2015).

Whether tumor overgrowth through STAT and Yki activity is somehow associated with a stem cell or progenitor-like state remains uncertain. Although imaginal discs exhibit developmental plasticity and regeneration potential, and JNK signaling is required for both of these stem-like properties, there is no positive evidence for the existence of a population of asymmetrically dividing stem cells within imaginal discs. Instead, symmetrical divisions of progenitor cells may be the means by which imaginal discs can rapidly generate enough cells to form the differentiated structures of the adult fly. To date, progenitor cells have only been characterized in the eye disc neuroepithelium. These cells have a pseudostratified columnar epithelial morphology and express the MEIS family transcription factor, Hth, which is downregulated as cells initiate differentiation and begin expressing Dac and Eya. Interestingly, they also require Yki for their proliferation, and can be induced to overproliferate in response to increased STAT activity. However, analysis of cell fate markers indicated that tumor overgrowth was not likley to be solely due to the overproliferation of these undifferentiated progenitor cells. Although scrib>RasACT/NACT tumors, were characterized by the failure to transition to Dac/Eya expression in the eye disc, blocking JNK in scrib > RasACT/NACT tumors did not restore tumor cell differentiation, despite overgrowth being curtailed, and Hth expression was not maintained in the tumors in a JNK-dependent manner. Nevertheless, a JNK-induced gene such as chinmo is likely to be associated with promoting a progenitor-like state, since it is a potential STAT target gene required for adult eye development that is expressed in eye disc progenitor cells in response to increased Upd activity and its overexpression alone is sufficient to block Dac/Eya expression. Furthermore, chinmo is also required for cyst stem cell maintenance in the Drosophila testis, and the current work has shown that chinmo overexpression promotes increased numbers of esgGFP expressing stem cells or enteroblasts in the adult midgut. As the BTB-ZF protein Ab is also highly oncogenic and expressed in the eye disc progenitor cells, it is hypothesize that the JNK-induced expression ofchinmo in scrib>RasACT/NACT tumors could cooperate with Ab to maintain a progenitor-like cell state in the eye disc, and that this is required for scrib->RasACT/NACT tumor overgrowth. However, although Ab was expressed in chinmo-expressing, JNK positive tumor cells, Ab does not appear to be a JNK-induced gene. What JNK-independent mechanisms control ab expression will therefore require further analysis (Doggett, 2015).

Interestingly, previous studies have observed that ab overexpression in eye disc clones upregulates chinmo expression and although the effect of chinmo expression upon ab is yet to be described, the data at least suggest that the control of their expression is interlinked in a yet to be defined manner (Doggett, 2015).

Consistent with Chinmo being important for scrib->RasACT/NACTv tumor overgrowth, chinmo overexpression itself is also highly oncogenic. Over-expression of chinmo with RasACT or NACT drives tumorigenesis in the eye-antennal disc, and also resulted in enlarged brain lobes, presumably due to the generation of overexpressing clones within the neuroepithelium of the optic lobes. In the adult midgut, the overexpression of chinmo with RasACT in the stem cell and its immediate progeny, the enteroblast, promoted massive tumor overgrowth, resulting in esgGFP expressing cells completely filling the lumen of the gut, and eventual host lethality. The luminal filling of esgGFP cells is reminiscent of the effects of RasACT expression in larval adult midgut progenitor cells. Together with the data linking Chinmo function to stem or progenitor cells, these data reinforce the idea that epithelial tumorigenesis can be primed by signals, such as chinmo over-expression, that promote a stem or progenitor cell state (Doggett, 2015).

The function of some Drosophila BTB-ZF proteins including Chinmo and Ab, has also been linked to heterochronic roles involving the conserved let-7 miRNA pathway and hormone signals, to regulate the timing of differentiation. Indeed, Ab can directly bind to the steroid hormone receptor co-activator Taiman (Tai or AIB1/SRC3 in humans), to represses the transcriptional response to ecdysone signaling. Thus, the capacity of BTB-ZF proteins to influence the timing of developmental transitions, particularly if they impede developmental transitions within stem or progenitor cells, could help account for their potent oncogenic activity. Indeed, ecdysone-response genes were repressed by JNK in the tumorigenic state, consistent with the failure of the larvae to pupate and a delay in developmental timing. Whether repressing the ecdysone response cell autonomously might contribute to tumor overgrowth and/or invasion will be an interesting area of future investigation, given the complex role of hormone signaling in mammalian stem cell biology and cancers (Doggett, 2015).

Previous studies have suggested that JNK-dependent tumor cell invasion is developmentally similar to the JNK-induced EMT-like events occurring during imaginal disc eversion. Thus the capacity of JNK to also promote tumor overgrowth is reminiscent of how EMT inducers such as Twist (Twi) and Snail (Sna) are associated with the acquisition of cancer stem cell properties. In Drosophila, however, twi and snawere not induced by JNK in the tumors, although transcription factors involved in mesoderm specification, including the NF-kappaB homologue, dl (a member of the 103 JNK signature), and Mef2 (a member of the 399 JNK signature), were amongst the up-regulated JNK targets. Mesoderm specification is not necessarily associated with a mesenchymal-like cell morphology, however, dl is involved in the induction of EMT during embryonic development, and both dl and Mef2 act with Twi and Sna to coordinate mesoderm formation. Interestingly, recent studies have identified dl in an overexpression screen for genes capable of cooperating with scrib > in Drosophila tumorigenesis, and Mef2 has been identified as a cooperating oncogene in Drosophila, and possibly also in humans, where a correlation exists between the expression of Notch and Mef2 paralogues in human breast tumor samples. It is therefore possible that dl and Mef2 either act in combination with Twi or Sna, or independently of them but in a similar oncogenic capacity, to promote a mesodermal cell fate in scrib > RasACT/NACT tumors. The potential relevance of this to the mesenchymal cell morphology associated with tumor cell invasion, as well as the acquisition of progenitor states is worthy of further investigation (Doggett, 2015).

In mef2-driven tumors both overgrowth and invasion depend upon activation of JNK signaling, suggesting that Mef2 is not capable of promoting invasive capabilities independent of JNK. In contrast, chinmo+RasACT/NACT tumors appeared non-invasive and retained epithelial morphology despite the massive overgrowth, although closer examination of cell polarity markers will be required to confirm this. Furthermore, the overgrowth of chinmo+RasACT/NACT tumors was not dependent upon JNK signaling, suggesting that the maintenance of a progenitor-like state could be uncoupled from JNK-induced EMT-effectors associated with invasion. Whether clear divisions between mesenchymal behaviour and progenitor states in tumors can be clearly separated in this manner is not yet clear, however, overall, it is likely that multiple JNK-regulated genes will participate in both promoting tumor overgrowth as well as migration/invasion. Although this study used the 103 JNK signature as a means to focus upon potential key candidates, an analysis of the 399 JNK-regulated probe sets common to both Ras and Notch-driven tumours has the potential to provide deeper insights into the multiple effectors of JNK signaling during tumorigenesis. Whilst the individual role of these genes can be probed with knockdowns, the complexity of the response, potentially with multiple redundancies and cross-talk, will ultimately need a network level of understanding to more fully expose key nodes participating in overgrowth and invasion. This approach has considerable potential to further expose core principles and mechanisms that drive human tumorigenesis, since it is clear that many fundamental commonalities underlie the development of tumors in Drosophila and mammals (Doggett, 2015).

The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche

Local signals maintain adult stem cells in many tissues. Whether the sexual identity of adult stem cells must also be maintained was not known. In the adult Drosophila testis niche, local Jak-STAT signaling promotes somatic cyst stem cell (CySC) renewal through several effectors, including the putative transcription factor Chronologically inappropriate morphogenesis (Chinmo). This study found that Chinmo also prevents feminization of CySCs. Chinmo promotes expression of the canonical male sex determination factor Doublesex^M (Dsx^M) within CySCs and their progeny, and ectopic expression of Dsx^M in the CySC lineage partially rescues the chinmo sex transformation phenotype, placing Chinmo upstream of Dsx^M. The Dsx homolog DMRT1 prevents the male-to-female conversion of differentiated somatic cells in the adult mammalian testis, but its regulation is not well understood. This work indicates that sex maintenance occurs in adult somatic stem cells and that this highly conserved process is governed by effectors of niche signals (Ma, 2014).

CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster

Stem cells and their daughters are often associated with and depend on cues from their cellular microenvironment. In Drosophila testes, each Germline Stem Cell (GSC) contacts apical hub cells and is enclosed by cytoplasmic extensions from two Cyst Stem Cells (CySCs). Each GSC daughter becomes enclosed by cytoplasmic extensions from two CySC daughters, called cyst cells. CySC fate depends on an Unpaired (Upd) signal from the hub cells, which activates the Janus Kinase and Signal Transducer and Activator of Transcription (Jak/STAT) pathway in the stem cells. Germline enclosure depends on Epidermal Growth Factor (EGF) signals from the germline to the somatic support cells. Expression of RNA-hairpins against subunits of the COnstitutively Photomorphogenic-9- (COP9-) signalosome (CSN; see CSN5) in somatic support cells disrupted germline enclosure. Furthermore, CSN-depleted somatic support cells in the CySC position next to the hub had reduced levels of the Jak/STAT effectors Zinc finger homeotic-1 (Zfh-1) and Chronologically inappropriate morphogenesis (Chinmo). Knockdown of CSN in the somatic support cells does not disrupt EGF and Upd signal transduction as downstream signal transducers, phosphorylated STAT (pSTAT) and phosphorylated Mitogen Activated Protein Kinase (pMAPK), were still localized to the somatic support cell nuclei. The CSN modifies fully formed Cullin RING ubiquitin ligase (CRL) complexes to regulate selective proteolysis. Reducing cullin2 (cul2) from the somatic support cells disrupted germline enclosure, while reducing cullin1 (cul1) from the somatic support cells led to a low level of Chinmo. It is proposed that different CRLs enable the responses of somatic support cells to Upd and EGF (Qian, 2014).

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. This study shows that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors (Syed, 2017).

This study shows that the steroid hormone ecdysone is required to trigger a major gene expression transition at mid-larval stages: central brain neuroblasts transition from Chinmo/Imp to Broad/Syncrip/E93. Furthermore, it was shown that Svp activates expression of EcR-B1 in larval neuroblasts, which gives them competence to respond to ecdysone signaling, thereby triggering this gene expression transition. Although a global reduction of ecdysone levels is likely to have pleiotropic effects on larval development, multiple experiments were performed to show that the absence or delay in late temporal factor expression following reduced ecdysone signaling is not due to general developmental delay. First, the EcR gene itself is expressed at the normal time (~56 hr) in the whole organism ecdysoneless¹ mutant, arguing strongly against a general developmental delay. Second, a type II neuroblast seven-up mutant clone shows a complete failure to express EcR and other late factors, in the background of an entirely wild type larvae; this is perhaps the strongest evidence that the phenotypes that are described are not due to a general developmental delay. Third, lineage-specific expression of EcR dominant negative leads to loss of Syncrip and E93 expression without affecting Broad expression; the normal Broad expression argues against a general developmental delay. Fourth, live imaging was used to directly measure cell cycle times, and it was found that lack of ecdysone did not slow neuroblast cell cycle times. Taken together, these data support the conclusion that ecdysone signaling acts directly on larval neuroblasts to promote an early-to-late gene expression transition (Syed, 2017).

The role of ecdysone in regulating developmental transitions during larval stages has been well studied; it can induce activation or repression of suites of genes in a concentration dependent manner. Ecdysone induces these changes through a heteromeric complex of EcR and the retinoid X receptor homolog Ultraspiracle. Ecdysone is required for termination of neuroblast proliferation at the larval/pupal transition, and is known to play a significant role in remodeling of mushroom body neurons and at neuromuscular junctions. This study adds to this list another function: to trigger a major gene expression transition in mid-larval brain neuroblasts (Syed, 2017).

Does ecdysone signaling provide an extrinsic cue that synchronizes larval neuroblast gene expression? Good coordination of late gene expression is not seen, arguing against synchronization. For example, Syncrip can be detected in many neuroblasts by 60 hr, whereas Broad appears slightly later at ~72 hr, and E93 is only detected much later at ~96 hr, by which time Broad is low. This staggered expression of ecdysone target genes is reminiscent of early and late ecdysone-inducible genes in other tissues. In addition, for any particular temporal factor there are always some neuroblasts expressing it prior to others, but not in an obvious pattern. It seems the exact time of expression can vary between neuroblasts. Whether the pattern of response is due to different neuroblast identities, or a stochastic process, remains to be determined (Syed, 2017).

It has been shown preiously that the Hunchback-Krüppel-Pdm-Castor temporal gene transitions within embryonic neuroblasts are regulated by neuroblast-intrinsic mechanisms: they can occur normally in neuroblasts isolated in culture, and the last three factors are sequentially expressed in G2-arrested neuroblasts. Similarly, optic lobe neuroblasts are likely to undergo neuroblast-intrinsic temporal transcription factor transitions, based on the observation that these neuroblasts form over many hours of development and undergo their temporal transitions asynchronously. In contrast, this study shows that ecdysone signaling triggers a mid-larval transition in gene expression in all central brain neuroblasts (both type I and type II). Although ecdysone is present at all larval stages, it triggers central brain gene expression changes only following Svp-dependent expression of EcR-B1 in neuroblasts. Interestingly, precocious expression of EcR-B1 (worniu-gal4 UAS-EcR-B1) did not result in premature activation of the late factor Broad, despite the forced expression of high EcR-B1 levels in young neuroblasts. Perhaps there is another required factor that is also temporally expressed at 56 hr. It is also noted that reduced ecdysone signaling in ecd^ts mutants or following EcRDN expression does not permanently block the Chinmo/Imp to Broad/Syncrip/E93 transition; it occurs with variable expressivity at 120-160 hr animals (pupariation is significantly delayed in these ecd^ts mutants), either due to a failure to completely eliminate ecdysone signaling or the presence of an ecdysone-independent mechanism (Syed, 2017).

A small but reproducible difference was found in the effect of reducing ecdysone levels using the biosynthetic pathway mutant ecd^ts versus expressing a dominant negative EcR in type II neuroblasts. The former genotype shows a highly penetrant failure to activate Broad in old neuroblasts, whereas the latter genotype has normal expression of Broad (despite failure to down-regulate Chinmo/Imp or activate E93). This may be due to failure of the dominant negative protein to properly repress the Broad gene. Differences between EcRDN and other methods of reducing ecdysone signaling have been noted before (Syed, 2017).

Drosophila Svp is an orphan nuclear hormone receptor with an evolutionarily conserved role in promoting a switch between temporal identity factors. In Drosophila, Svp it is required to switch off hunchback expression in embryonic neuroblasts, and in mammals the related COUP-TF1/2 factors are required to terminate early-born cortical neuron production, as well as for the neurogenic to gliogenic switch. This study showed that Svp is required for activating expression of EcR, which drives the mid-larval switch in gene expression from Chinmo/Imp to Syncrip/Broad/E93 in central brain neuroblasts. The results are supported by independent findings that svp mutant clones lack expression of Syncrip and Broad in old type II neuroblasts (Tsumin Lee, personal communication to Chris Doe). Interestingly, Svp is required for neuroblast cell cycle exit at pupal stages, but how the early larval expression of Svp leads to pupal cell cycle exit was a mystery. The current results provide a satisfying link between these findings: Svp was shown to activate expression of EcR-B1, which is required for the expression of multiple late temporal factors in larval neuroblasts. Any one of these factors could terminate neuroblast proliferation at pupal stages, thereby explaining how an early larval factor (Svp) can induce cell cycle exit five days later in pupae. It is interesting that one orphan nuclear hormone receptor (Svp) activates expression of a second nuclear hormone receptor (EcR) in neuroblasts. This motif of nuclear hormone receptors regulating each other is widely used in Drosophila, C. elegans, and vertebrates (Syed, 2017).

The position of the Svp+ neuroblasts varied among the type II neuroblast population from brain-to-brain, suggesting that Svp may be expressed in all type II neuroblasts but in a transient, asynchronous manner. This conclusion is supported by two findings: the svp-lacZ transgene, which encodes a long-lived β-galactosidase protein, can be detected in nearly all type II neuroblasts; and the finding that Svp is required for EcR expression in all type II neuroblasts, consistent with transient Svp expression in all type II neuroblasts. It is unknown what activates Svp in type II neuroblasts; its asynchronous expression is more consistent with a neuroblast-intrinsic cue, perhaps linked to the time of quiescent neuroblast re-activation, than with a lineage-extrinsic cue. It would be interesting to test whether Svp expression in type II neuroblasts can occur normally in isolated neuroblasts cultured in vitro, similar to the embryonic temporal transcription factor cascade (Syed, 2017).

Castor and its vertebrate homolog Cas-Z1 specify temporal identity in Drosophila embryonic neuroblast lineages and vertebrate retinal progenitor lineages, respectively (Mattar, 2015). Although this study shows that Cas is not required for the Chinmo/Imp to Syncrip/Broad/E93 transition, it has other functions. Cas expression in larval neuroblasts is required to establish a temporal Hedgehog gradient that ultimately triggers neuroblast cell cycle exit at pupal stages (Syed, 2017).

Drosophila embryonic neuroblasts change gene expression rapidly, often producing just one progeny in each temporal transcription factor window. In contrast, larval neuroblasts divide ~50 times over their 120 hr lineage. Mushroom body neuroblasts make just four different neuronal classes over time, whereas the AD (ALad1) neuroblast makes ~40 distinct projection neuron subtypes. These neuroblasts probably represent the extremes (one low diversity, suitable for producing Kenyon cells; one high diversity, suitable for generating distinct olfactory projection neurons). This study found that larval type II neuroblasts undergo at least seven molecularly distinct temporal windows. If it is assumed that the graded expression of Imp (high early) and Syncrip (high late) can specify fates in a concentration-dependent manner, many more temporal windows could exist (Syed, 2017).

This study illuminates how the major mid-larval gene expression transition from Chinmo/Imp to Broad/Syncrip/E93 is regulated; yet many new questions have been generated. What activates Svp expression in early larval neuroblasts - intrinsic or extrinsic factors? How do type II neuroblast temporal factors act together with Dichaete, Grainy head, and Eyeless INP temporal factors to specify neuronal identity? Do neuroblast or INP temporal factors activate the expression of a tier of 'morphogenesis transcription factors' similar to leg motor neuron lineages? What are the targets of each temporal factor described here? What types of neurons (or glia) are made during each of the seven distinct temporal factor windows, and are these neurons specified by the factors present at their birth? The identification of new candidate temporal factors in central brain neuroblasts opens up the door for addressing these and other open questions (Syed, 2017).

Liu, L. Y., Long, X., Yang, C. P., Miyares, R. L., Sugino, K., Singer, R. H. and Lee, T. (2019). Mamo decodes hierarchical temporal gradients into terminal neuronal fate. Elife 8. PubMed ID: 31545163

Mamo decodes hierarchical temporal gradients into terminal neuronal fate

Temporal patterning is a seminal method of expanding neuronal diversity. This study has unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, Drosophila neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit chinmo translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of alpha'/beta' mushroom body neurons and identity maintenance. This study describes a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development (Liu, 2019).

The brain is a complicated organ which not only requires specific connections between neurons to form circuits, but also many neuronal types with variations in morphology, neurotransmitters and receptors. While mechanisms controlling neuronal diversity have not been globally examined, studying neural stem cells in the mouse and fruit fly have given insight into key aspects of neuronal specification. For example, in the mouse neocortex, radial glial progenitors (RGP) are multipotent-they produce a variety of neuron types organized sequentially into six layers, and then produce glia. In vivo lineage analysis demonstrated that after a stage of symmetric cell division, an individual neurogenic RGP produces an average of 8-9 progeny (range of 3-16) that can span all cortical layers. In Drosophila, clonal analysis has demonstrated a vast range of stem cell-specific lineage programs. On one extreme, lineage tracing of a single antennal lobe (AL) stem cell revealed a remarkable series of 40 morphologically distinct neuronal types generated sequentially. In light of these observations, a fundamental goal is to understand how distinct neuronal types correctly differentiate from a single progenitor. Despite a fundamental role for temporal patterning to create diverse neuronal lineages, understanding of neuronal temporal patterning is still limited. While scientists have discovered key temporal factors expressed in neural progenitors, much less is understood about how these signals are interpreted, that is what factors lie downstream of the specification signals to determine distinct neuronal temporal fates (Liu, 2019).

Despite its relatively small brain, Drosophila is leading the charge on studies of neuronal temporal fate specification. Many temporal transcription factors originally discovered in the fly have since been confirmed to have conserved roles in mouse retinal and cortical development. Moreover, temporal expression of an RNA binding protein, IGF-II mRNA-binding protein (Imp), that guides temporal patterning in the postembryonic fly brain is also implicated in mouse brain development. Drosophila brain development is an excellent model for studying neurogenesis; the neural stem cells, called neuroblasts (NB), are fixed in number, their modes of division are well characterized, and each NB produces a distinctive series of neurons which change fate based on birth order. Finally, the fruit fly is a genetically tractable system making it ideal for studying gene networks involved in cell fate decisions (Liu, 2019).

In Drosophila, cycling NBs express age-dependent genes that provide the serially derived newborn neurons with different temporal factors. In the embryonic ventral nerve cord and the optic lobe, the NBs express a rapidly changing series of four to six temporal transcription factors (tTF), some of which are directly inherited by the daughter neurons. Each tTF directly acts to specify a small number (two to four) of neuronal progeny. The neuronal progeny produced from one tTF window to the next can be quite different. The tTF series are intrinsically controlled, which ensures reliable production of all neuron types, but lacks the ability to adapt to complicated or changing conditions (Liu, 2019).

A separate mechanism is therefore required for adult brain development-both to produce very long series of related neuronal types and to coordinate with organism development. This can be accomplished utilizing protein gradients and hierarchical gene regulation, such as the mechanism used to pattern the fly's anterior/posterior (A/P) axis. In Drosophila A/P patterning, the embryo is progressively partitioned into smaller and smaller domains through layered gene regulation. This is initiated by asymmetric localization maternal mRNAs, bicoid (anterior) and nanos (posterior). The resulting opposing proteins gradients then act on maternal mRNA translation, and in the case of Bicoid, zygotic transcription. The embryo then progresses through expression of maternal morphogen gradients, then zygotic expression of gap genes to determine broad embryo regions, followed by progressive segmentation by the pair-rule and segment polarity genes, and finally specification by the homeotic selector genes (Liu, 2019).

Notably, in postembryonic brain development, two proteins in opposing temporal gradients expressed in NBs have been discovered. These proteins are Imp and Syncrip (Syp) RNA-binding proteins. Imp and Syp control neuronal temporal fate specification as well as the timing of NB termination (decommissioning). Imp promotes and Syp inhibits translation of the BTB-zinc finger nuclear protein, chinmo (chronologically inappropriate morphogenesis), so that protein levels in newborn neurons descend over time. The level of Chinmo correlates with the specification of multiple neuronal temporal fates. Discovering downstream layers in the Imp/Syp/Chinmo hierarchy is essential to fully comprehend the intricacies of temporal patterning in brain development (Liu, 2019).

Temporal regulation in the fly brain is easily studied in the relatively simple mushroom body (MB) neuronal lineages which are comprised of only three major cell types. These neuronal types are born in sequential order: beginning with γ neurons, followed by α'/β' neurons, and finally α/β neurons. Imp and Syp are expressed in relatively shallow, opposing temporal gradients in the MB NBs. Modulation of Imp or Syp expression results in shifts in the neuronal temporal fate. Imp and Syp post-transcriptionally control Chinmo so that it is expressed in a gradient in the first two temporal windows. γ neurons are produced in a high Chinmo window, α'/β' neurons are produced in a low Chinmo window, and α/β neurons are produced in a window absent of Chinmo expression. Moreover, altering Chinmo levels can shift the temporal fate of MB neurons accordingly, strongly implicating dose-dependent actions, similar to that of a morphogen. Despite its importance in temporal patterning, the mechanisms underlying the dosage-dependent effects of Chinmo on neuronal temporal identity is unknown (Liu, 2019).

This study describes Mamo (maternal gene required for meiosis), a BTB zinc finger transcription factor critical for temporal specification of α'/β' neurons. Mamo is expressed in a low Chinmo temporal window and Mamo expression can be inhibited both by high Chinmo levels and loss of chinmo. Additionally, Mamo is post-transcriptionally regulated by the Syp RNA binding protein. This layered regulation, which is utilized in both MB and AL lineages results in a discrete window of Mamo expression in young, post-mitotic neurons. In the MB lineages, this window corresponds to the middle window of neurogenesis and it was establish that Mamo codes for middle temporal fate(s); α'/β' neuronal characteristics are lost when Mamo levels are reduced and ectopic Mamo drives an increase in α'/β' neuron production. The temporal fate determination paradigm described in this study utilizes multiple levels of gene regulation. Temporal fate specification begins in the stem cell and proceeds in a hierarchical manner in successive stages where top and second-tier factors work together to specify neuronal temporal fate. These data suggest that Mamo deciphers the upstream temporal specification code and acts as a terminal selector to determine neuronal fate (Liu, 2019).

Chinmo levels in newborn neurons correlate with adult neuron identity. Based on smFISH, mamo transcription is initiated in newborn MB neurons around 72 hr ALH, which corresponds to weak Chinmo expression. Moreover, Mamo is only expressed when Chinmo levels are low, as Mamo is not expressed after either eliminating or overexpressing Chinmo. Together these data indicate that low Chinmo levels activate mamo transcription in young/maturing neurons (Liu, 2019).

Transcription initiation is not the only requirement for Mamo protein expression; Syp is also required as discussed below. This could explain why no Mamo expression turn on is seen in γ neurons, even as they age and Chinmo levels decrease, becoming quite low around wandering larval stage. γ neurons begin to express Mamo later, around pupation, despite lacking Syp. It has not yet been tested whether weak Chinmo levels are required for later Mamo expression in γ neurons. It is therefore possible that Mamo expression is controlled at this stage by an additional factor(s) (Liu, 2019).

ChIP-chip performed in embryos found five Chinmo binding sites within the mamo gene, consistent with direct activation of mamo transcription. However, the nature of Chinmo's concentration dependent actions is still unclear. Some morphogens such as Bicoid bind different targets at increasing concentrations based on the affinity of binding to different sites as well as the chromatin accessibility of the binding sites. This may also be the case with Chinmo, but would not easily explain why Mamo expression is inhibited at higher Chinmo concentrations. The gap gene Krüppel, on the other hand, has concentration dependent activities at the same binding site. Krüppel acts as an activator at lower concentrations and as a repressor at high concentrations. Krüppels C-terminus has the ability to activate genes and is also the location for dimerization. Upon dimerization, the C-terminus can no longer activate genes and Krüppel transforms from an activator to a repressor. The current data suggests that low concentrations of Chinmo activate mamo. However, in the testis, Chinmo is suspected to function as a transcriptional repressor. It is feasible that Chinmo, like Krüppel, could switch from an activator to a repressor. The protein concentration would affect whether Chinmo is a monomer (in the presence of other BTB proteins, a heterodimer) or a homodimer, and thus potentially which cofactors are recruited (Liu, 2019).

The ascending Syp RNA binding protein temporal gradient regulates Mamo expression both indirectly via its inhibition of Chinmo and also presumably directly, interacting with the mamo transcript and promoting its expression. The bi-modal, transcriptional (Chinmo) and post-transcriptional (Syp), regulation of the Mamo terminal selector is extremely advantageous. Given the finding that Mamo expression is positively autoregulated and that Mamo continues to be expressed into adult neurons, it is particularly important to control the timing of Mamo's onset. The additional layer of post-transcriptional regulation adds an extra safeguard, helping to guarantee that neuronal temporal patterning is a robust system. Indeed, as brain development needs to adapt to environmental conditions such as nutrient deprivation, it is crucial to ensure that there is no loss of neuronal diversity (Liu, 2019).

Syp is a homolog of mammalian SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein) also known as hnRNP-Q. SYNCRIP is involved with multiple facets of mRNA regulation including mRNA splicing and maturation, mRNA localization and stabilization as well as inhibiting mRNA translation and miRNA-mediated repression via competition with Poly(A) binding proteins. The Drosophila ortholog seems to have corresponding functions. Drosophila Syp was isolated from the spliceosome B complex, indicating a conserved role in mRNA splicing. Syp has likewise been found to operate in mRNA localization and stabilization. Furthermore, it has clear roles in altering protein expression of its mRNA targets, both positively and negatively. The bidirectional influence on protein expression likely reflects different Syp modalities (Liu, 2019).

This study shows that Syp is required for Mamo protein expression in the MB and AL neuronal lineages. To determine the nature of this regulation, single molecule fluorescence in situ hybridization (smFISH) was performed. In the absence of Syp, mamo transcription was initiated prematurely in response to weak Chinmo levels, yet mature transcripts failed to accumulate. This leads to the belief that Syp directly binds mamo mRNA and aids in its splicing, maturation and/or stabilization. This is consistent with the finding that overexpressing a mamo cDNA (lacking 5' UTR, 3' UTR and introns) was able to promote cell fate changes despite repression of Syp (Liu, 2019).

Mamo is required to produce the α'/β' neurons in the middle temporal window of the MB lineages. Trio positive α'/β' neurons are clearly absent after RNAi depletion of Mamo during development. Cell production does not appear to be altered, as mamo-RNAi expressing MBs are a normal size. This begs the question of which, if any terminal fate the middle-born neurons adopt in the absence of Mamo. The limited markers for each MB cell type makes it difficult to determine whether the middle-born neurons undergo fate transformation or simply lack terminal fate. The presence of a Fas-II negative lobe hints that some middle-born neurons may not carry temporal fate information, but phenotypic analysis is complicated by defects in γ neuron maturation/remodeling. Removing the γ neurons with chinmo-RNAi eliminates this complication, but it is still unclear whether, without Mamo, the neurons are transformed to the α/β fate. The Fas-II positive, α/β lobe appears enlarged, but it is difficult to tell whether all axons are Fas-II positive or whether Fas-II negative axons are comingled with α/β axons. Without a cell type-specific, cell body marker for α/β neurons, it is ambiguous whether the middle-born cells are transformed to α/β or whether they simply lack α'/β' temporal fate. A transformation to α/β fate would suggest that either α/β is the default fate of MB neurons (requiring no additional terminal selector) or that Mamo expression inhibits α/β specific factors (Liu, 2019).

Mamo's role in promoting α'/β' fate is further supported by Mamo overexpression phenotypes. Overexpression of Mamo in the MB is able to transform α/β and γ neurons to α'/β' neurons. In an otherwise wildtype scenario, overexpression of mamo did not transform every cell to α'/β' fate. Instead the α'/β' lobe was expanded and the other lobe seemed to be an amalgam of α and γ like lobes. This could be due to incomplete penetrance/low expression levels of the mamo transgene or it is possible that the α/β and γ cells retain their own terminal selector driven, cell-type specific gene expression, thus complicating the fate of the differentiated neuron. Mamo overexpression does not alter the specification factors Imp, Syp or Chinmo and presumably there are terminal selector genes expressed downstream of high Chinmo and possibly in Chinmo-absent cells. This seems a likely possibility when overexpressing Mamo in γ neurons. With Syp-RNAi, NBs are 'forever young' and divide into adulthood, persistently producing 'early-born' γ neurons. Interestingly when combining Syp-RNAi with the Mamo transgene, the newborn cells begin to take on a γ-like fate (expressing Abrupt) before a majority transform into an α'/β'-specific, strong Trio expression pattern and adopt α'/β'-like axon morphology. This suggests that Mamo functions downstream of the temporal fate specification genes, but is capable of overriding downstream signals in α/β and γ neurons to promote α'/β' terminal fate (Liu, 2019).

What this study describes about the BTB-ZF transcription factor, Mamo's role in α'/β' cell fate easily fits into the definition of a terminal selector gene, coined by Oliver Hobert. Terminal selector genes are a category of 'master regulatory' transcription factors that control the specific terminal identity features of individual neuronal types. Key aspects of terminal selector genes are that they are expressed post-mitotically in neurons as they mature and they are continuously expressed (often via autoregulatory mechanisms) to maintain the terminal differentiated state of the neuron. Correspondingly, mamo transcription is initiated in newborn, post-mitotic neurons and Mamo protein expression is visible beginning in young/maturing neurons. After transcription initiation, Mamo positively regulates its own expression and continues to be expressed in α'/β' neurons into adulthood. The other quintessential feature of terminal selector genes is that they regulate a battery of terminal differentiation genes, so that removing a terminal selector gene results in a loss of the specific identity features of a neuron type and misexpression can drive those features in other neurons. Indeed, removing Mamo with RNAi results in the loss of α'/β' identity, both developmentally and into adulthood. Further, overexpressing Mamo in either α/β or γ MB neurons results in shift to α'/β' fate. Individual terminal selectors do not often function alone, but in combination with other terminal selectors. Therefore, there are likely terminal selectors downstream of the MB NB-specific genes that contribute to each of the MB neuron types. In this way, the lineage-specific and temporal patterning programs can combine to define individual neuron types. This feature enables the reutilization of terminal selector genes to create disparate neuron types when used in distinct combinations (Hobert, 2016). This further suggests that temporally expressed Mamo serves as a temporally defined terminal selector gene in other lineages, such as the AL lineages that are describe in this study (Liu, 2019).

Altering Chinmo levels via upstream RNA-binding proteins or miRNAs, or by reducing Chinmo with RNAi all result in shifts in the ratio of neurons with different neuronal temporal fates. This evidence suggests a mechanism where Chinmo acts in newborn neurons to promote temporal fate specification. A recent publication suggested that Chinmo affects temporal fate via a neuronal remodeling mechanism by controlling Ecdysone signaling . Marchetti demonstrates that Chinmo is required for EcR-B1 expression; however it remains unclear whether Chinmo directly affects EcR-B1 expression or if the Chinmo-dependent EcR-B1 expression is the sole mechanism for γ neuron temporal fate specification. Moreover, neuronal temporal fate is not accurately determined by neuronal morphology alone, particularly when ecdysone signaling has known effects on MB cell morphology and fate. Ecdysone receptor signaling is highly pleiotropic, including ligand-independent functions making dominant-negative and overexpression studies difficult to interpret. Therefore, further investigation is needed to clarify the roles of Ecdysone receptor signaling in MB neuronal temporal fate and remodeling. This will be addressed in a follow-up paper. This current manuscript strongly promotes the idea that Chinmo functions in newborn neurons to promote temporal fate as weak Chinmo expression directly precedes Mamo transcription and Mamo is essential for specification and maintenance of α'/β' fate (Liu, 2019).

This study describes a multilayered hierarchical system to define distinct neuronal temporal fate that culminates in the expression of a terminal selector gene. Analogous mechanisms likely underlie temporal patterning in mammalian brains. However, whether orthologous genes play equivalent roles in mammalian temporal patterning has not been fully investigated. The Imp and Syp RNA-binding proteins are evolutionarily conserved. Both homologs are highly expressed in the developing mouse brain and play vital roles in neural development and/or neuronal morphology. The opposing functions of Imp and Syp also appear to be conserved, as the murine orthologs IMP1 and SYNCRIP bind the identical RNA to either promote or repress axon growth, respectively. Moreover, IMP1 expression in fetal mouse neural stem cells plays important roles in stem cell maintenance and proper temporal progression of neurogenesis. It would likewise be very interesting to explore SYNCRIP in the context of temporal patterning (Liu, 2019).

While Chinmo and Mamo have no clear mammalian orthologs, they are both BTB-ZF (broad-complex, tram-track and bric-à-brac - zinc finger) transcription factors. The BTB domain is a protein interaction domain that can form homo or heterodimers and also binds transcriptional regulators such as repressors, activators and chromatin remodelers. The C2H2 (Krüppel-like) zinc fingers bind DNA-providing target specificity. BTB-ZF proteins have been found to be critical regulators of developmental processes, including neural development. Indeed, the BTB-zinc finger protein, Zbtb20, appears to be essential for early-to-late neuronal identity in the mouse cortex. Zbtb20 is temporally expressed in cortical progenitors and knockout results in cortical layering defects, as the inside-out layering of the cortex follows neuronal birth order. While mutations of other brain-expressed BTB-ZF proteins also show cortical layering phenotypes, potential roles in temporal patterning have not been explored (Liu, 2019).

This study illustrates a fate specification process in which a layered series of temporal protein gradients guide the expression of terminal selector genes. The first-tier temporal gradients are expressed in neural stem cells, followed by a restricted expression window in newborn neurons to finally induce a terminal selector gene in a subset of neurons as they mature. This time-based subdivision of neuronal fate can likely be further partitioned, finally resulting in sequentially born neurons with distinct cell fates. This study demonstrates that Mamo, a BTB-ZF transcription factor, delineates α'/β' neurons, the middle temporal window of the MB lineages. Corresponding data in the AL lineages suggest that Mamo may serve as a temporally defined, terminal selector gene in a variety of lineages in the Drosophila brain. Mamo expression is regulated transcriptionally by the descending Chinmo BTB-ZF transcription factor gradient and post-transcriptionally by the Syp RNA binding protein. This multi-tiered, bimodal regulation ensures that only the progeny in a precise temporal window (those with both weak Chinmo and significant Syp levels) can effectively activate the terminal selector gene, mamo. This discovery attests to the power of gradients in creating diverse cells from a single progenitor. Utilizing layers of temporal gradients to define discrete temporal windows mirrors how in early embryos the spatial gradients of RNA-binding proteins and transcription factors specify the fly's A/P axis. This paradigm provides considerable complexity of gene network regulation, leading to abundant neural cell diversity (Liu, 2019).

Chinmo prevents transformer alternative splicing to maintain male sex identity

Reproduction in sexually dimorphic animals relies on successful gamete production, executed by the germline and aided by somatic support cells. Somatic sex identity in Drosophila is instructed by sex-specific isoforms of the DMRT1 ortholog Doublesex (Dsx). Female-specific expression of Sex-lethal (Sxl) causes alternative splicing of transformer (tra) to the female isoform tra^F. In turn, Tra^F alternatively splices dsx to the female isoform dsx^F. Loss of the transcriptional repressor Chinmo in male somatic stem cells (CySCs; cyst stem cells) of the testis causes them to "feminize", resembling female somatic stem cells in the ovary. This somatic sex transformation causes a collapse of germline differentiation and male infertility. This feminization occurs by transcriptional and post-transcriptional regulation of tra^F. chinmo-deficient CySCs upregulate tra mRNA as well as transcripts encoding tra-splice factors Virilizer (Vir) and Female lethal (2)d (Fl(2)d). tra^F splicing in chinmo-deficient CySCs leads to the production of Dsx^F at the expense of the male isoform DsxM, and both Tra^F and Dsx^F are required for CySC sex transformation. Surprisingly, CySC feminization upon loss of chinmo does not require Sxl but does require Vir and Fl(2)d. Consistent with this, this study shows that both Vir and Fl(2)d are required for tra alternative splicing in the female somatic gonad. This work reveals the need for transcriptional regulation of tra in adult male stem cells and highlights a previously unobserved Sxl-independent mechanism of tra^F production in vivo. In sum, transcriptional control of the sex determination hierarchy by Chinmo is critical for sex maintenance in sexually dimorphic tissues and is vital in the preservation of fertility (Brmai, 2018).

This study shows that that one single factor, Chinmo, preserves the male identity of adult CySCs in the Drosophila testis by regulating the levels of canonical sex determinants. CySCs lacking chinmo lose DsxM expression not by transcriptional loss but rather by alternative splicing of dsx pre-mRNA into dsx^F. These chinmo-mutant CySCs ectopically express Tra^F and Dsx^F, and both factors are required for their feminization. Furthermore, the results demonstrate that tra alternative splicing in cyst cells lacking chinmo is achieved independently of Sxl. Instead, this work strongly suggests that tra^F production in the absence of chinmo is mediated by splicing factors Vir and Fl(2)d. It is proposed that male sex identity in CySCs is maintained by a two-step mechanism whereby tra^F is negatively regulated at both transcriptional and post-transcriptional levels by Chinmo (see Model for adult somatic sex maintenance in the Drosophila somatic gonad). In this model, loss of chinmo from male somatic stem cells first leads to transcriptional upregulation of tra pre-mRNA as well as of vir and fl(2)d. Then the tra pre-mRNA in these cells is spliced into tra^F by the ectopic Vir and Fl(2)d proteins. The ectopic Tra^F in chinmo-deficient CySCs then splices the dsx pre-mRNA into dsx^F, resulting in loss of Dsx^M and gain of Dsx^F, and finally induction of target genes usually restricted to follicle cells in the ovary (Brmai, 2018).

Chinmo has motifs associated with transcriptional repression and its loss clonally is associated with ectopic transcription. One interpretation of the data is that Chinmo directly represses tra, vir, and fl(2)d in male somatic gonadal cells. As the binding site and potential co-factors of Chinmo are not known, future work will be needed to determine whether Chinmo directly regulates expression of these genes. It is also noted that ~50% of chinmo-mutant testes still feminize in the genetic absence of tra or dsx^F. These latter data indicate that Chinmo regulates male sex identity through another, presumably parallel, mechanism that does not involve canonical sex determinants. However, this tra/dsx-independent mode of sex maintenance downstream of Chinmo is not characterized and will require the identification of direct Chinmo target genes (Brmai, 2018).

Previous work has shown that JAK/STAT signaling promotes chinmo in several cell types, including CySCs (Flaherty, 2010). Since JAK/STAT signaling is itself sex-biased and restricted to the embryonic male gonad, it is presumed that activated Stat92E establishes chinmo in male somatic gonadal precursors, perhaps as early as they are specified in the embryo. Because loss of Stat92E from CySCs does not result in an apparent sex transformation phenotype, the interpretation is favored that Stat92E induces expression of chinmo in CySCs but that other sexually biased factors maintain it. One potential candidate is Dsx^M, which is expressed specifically in early somatic gonads and at the same time when Stat92E activation is occurring in these cells. In fact, multiple Dsx^M ChIP-seq peaks were identified in the chinmo locus, suggesting potential regulation of chinmo by Dsx^M. This suggests a potential autoregulatory feedback loop whereby Dsx^M preserves its own expression in adult CySCs by maintaining Chinmo expression, which in turn prevents tra^F and dsx^F production (Brmai, 2018).

Recent studies on tissue-specific sex maintenance demonstrate that while the Sxl/Tra/Dsx hierarchy is an obligate and linear circuit during embryonic development, at later stages it is more modular than previously appreciated. For example, Sxl can regulate female-biased genes in a tra-independent manner. Additionally, Sxl and Tra^F regulate body size and gut plasticity independently of the only known Tra^F targets, ^dsx and fru. Negative regulation of the Tra^F-Dsx^F arm of this cascade is required to preserve male sexual identity in CySCs but unexpectedly is independent of Sxl. Because depletion of Vir or Fl(2)d significantly blocks sex transformation and both are required for tra alternative splicing in the ovary, this work reveals they can alternatively splice tra pre-mRNA even in the absence of Sxl. This is the first demonstration of Sxl-independent, Tra-dependent feminization. These results raise the broader question of whether other male somatic cells have to safeguard against this novel mechanism. Because recent work has determined that sex maintenance is important in systemic functions regulated by adipose tissue and intestinal stem cells, it will be important to determine whether Chinmo represses tra^F in these settings. Finally, since the transcriptional output of the sex determination pathway is conserved from Drosophila (Dsx) to mammals (DMRT1), it is possible that transcriptional regulation of sex determinants plays a similar role in adult tissue homeostasis and fertility in higher organisms (Brmai, 2018).

An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling

Polycomb group proteins are epigenetic regulators maintaining transcriptional memory during cellular proliferation. In Drosophila larvae, malfunction of Polyhomeotic (Ph), a member of the PRC1 silencing complex, results in neoplastic growth. This study reports an intrinsic tumour suppression mechanism mediated by the steroid hormone ecdysone during metamorphosis. Ecdysone alters neoplastic growth into a nontumorigenic state of the mutant ph cells which then become eliminated during adult stage. This study demonstrates that ecdysone exerts this function by inducing a heterochronic network encompassing the activation of the microRNA lethal-7, which suppresses its target gene chronologically inappropriate morphogenesis (chinmo). This pathway can also promote remission of brain tumours formed in brain tumour mutants, revealing a restraining of neoplastic growth in different tumour types. Given the conserved role of let-7, the identification and molecular characterization of this innate tumour eviction mechanism in flies might provide important clues towards the exploitation of related pathways for human tumour therapy (Jiang, 2018).

Deregulation of PcG gene expression has been associated with various types of human cancer. For example, loss of expression of the human ph homologue has been linked to the formation of osteosarcomas. As the molecular mechanisms of PcG proteins in human cancers are largely unknown, understanding the tumour-suppressor function of PcG genes in Drosophila therefore could provide insights in human cancer biology. During the past decades imaginal discs have been used as a powerful paradigm to investigate mechanisms underlying the formation and progression of several types of tumour, including Ras-, PcG-, or Hippo pathway-induced tumours. In addition, it is worth noting that the let-7 consensus sequence is identical from Caenorhabditis elegans to humans, suggesting that let-7 may control functionally conserved targets in regulating proliferation and differentiation during development. In various types of human cancer, downregulation of one or more let-7 members has been observed. Moreover, induced expression of let-7 in cancer cell lines can suppress cell proliferation and tumour growth. In the human genome, the let-7 family consists of more than ten members. However, the transcriptional regulation, spatial and temporal expression, and their tissue-specific and/or redundant functions of the let-7 family in human are far more complicated and still remain elusive (Jiang, 2018).

This study identified an intrinsic mechanism reprogramming tumorigenic to nontumorigenic cells of at least two different tumour types, by marking the cells for destruction in adult Drosophila. The steroid hormone-induced miRNA let-7 is a key mediator of this mechanism. Interestingly, let-7 and its target genes, including chinmo have been shown to act as heterochronic genes that regulate developmental transitions. Other findings from this lab indicate that ph⁵⁰⁵ tumour cells are reprogrammed from the original larval imaginal disc identity to an early embryonic state. By artificially overexpressing a differentiation factor, these cancer cells can be induced to lose their neoplastic state, however. It appears as if these tumour cells are trapped in an immature condition, unable to differentiate. Pulses of ecdysone are a major timer of developmental transitions in flies and their target, let-7, is for example required for enforcing the terminal cell cycle arrest in pupal stage wing discs. These findings suggest that flies have evolved tumour suppressive mechanisms by inducing let-7-controlled heterochronic gene networks to enforce cellular differentiation in epigenetically derailed tumours. Indeed, differentiation therapy is considered a promising approach for curing human cancers. However, the strategy has been applied only in limited cases. As such, identification of an innate tumour eviction mechanism in flies based on these principles may provide new ideas how such cancer treatments could be further improved in human patients (Jiang, 2018).

Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors

It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. Previous work has shown that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation. Using single-cell transcriptomics, clonal analysis and numerical modeling, this study now identifies a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin (Genovese, 2019).

Central nervous system (CNS) tumors are rare and constitute less than 2% of all cancers in adults. In contrast, they represent more than 25% of cancer cases in children (including medulloblastoma, retinoblastoma, rhabdoid tumors (AT/RT), gliomas etc), suggesting that the developing CNS is particularly sensitive to malignant transformation. Moreover, unlike most adult tumors, pediatric tumors are often genetically stable and their initiation and progression do not necessarily require the accumulation of mutations in multiple genes. For example, the biallelic inactivation of a single gene is sometimes sufficient to trigger malignant growth as illustrated by mutations in the RB1 and SMARCB1 genes in retinoblastoma and rhabdoid tumors respectively. Recent studies suggest that CNS pediatric tumors such as medulloblastomas recapitulate the fetal transcription program that was active in the cell of origin. However, it remains unclear how the invalidation of single genes during fetal stages can disrupt on-going developmental programs to trigger malignant growth, and whether these fetal/developmental programs influence the heterogeneity, composition, and proliferative properties of cells composing CNS tumors (Genovese, 2019).

Faced with the complexity of brain development and neural tumors in mammals, simple animal models can represent a powerful alternative to investigate basic and evolutionary conserved principles. The development of the CNS is undoubtedly best understood in Drosophila. The Drosophila CNS arises from a small pool of asymmetrically-dividing neural stem cells (NSCs), called neuroblasts (NBs). NBs possess a limited self-renewing potential. They divide all along development (embryonic and larval stages) to self-renew while generating daughter cells named Ganglion Mother Cells (GMCs). GMCs then usually divide once to produce two post-mitotic neurons or glia. NBs are the fastest cycling cells during development, able to divide every hour during larval stages when most of the neurons are produced. However, all NBs terminate during metamorphosis and are absent in adults. Two antagonistic RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp) are essential to first promote and then conclude this formidable period of activity. During early larval development (L1/L2), NBs express Imp that promotes NB self-renewal. Around late L2/early L3, NBs silence Imp to express Syp that remains expressed until NB decommissioning during metamorphosis. This Imp-to-Syp transition is essential to render NBs competent to respond to subsequent pupal pulses of the steroid hormone ecdysone and initiate a last differentiative division. Failure to trigger the transition results in NBs permanently dividing in adults. The Imp-to-Syp transition appears to be mainly regulated by a NB intrinsic timing mechanism driven by the sequential expression of transcription factors. This series of factors, also known as temporal transcription factors, has been first identified for its ability to specify different neuronal fates produced by NBs as they divide. In addition, temporal transcription factors also schedule the Imp-to-Syp transition to ensure that NBs will not continue cycling in adults. Recent transcriptomic analyses indicate that other genes are dynamically transcribed in NBs throughout larval stages, although their function and epistatic relationship with temporal transcription factors and the Imp/Syp module are unclear. All together, these studies highlight a complex, but still relatively unexplored, temporal patterning system in larval NBs (Genovese, 2019).

Perturbation of the asymmetric division process during early development can lead to NB exponential amplification. In such conditions, the NB-intrinsic temporal program limiting self-renewal appears to become inoperant, and uncontrolled NB amplification is observed. Serial transplantations of asymmetric division-defective NBs have revealed an ability to proliferate for months, if not years, demonstrating tumorigenic characteristics. Perturbation of asymmetric divisions can be induced by the inactivation of the transcription factor Prospero (Pros) in type I NB lineages (most lineages in the ventral nerve cord (VNC) and central brain (CB)). During development, Pros is strongly expressed in GMCs where it accumulates to induce cell cycle-exit and neuronal or glial differentiation. GMCs that lack pros fail to differentiate and revert to a NB-like state. This triggers rapid NB amplification at the expense of neuron production. Previous work has shown that inactivation of pros in NBs, and their subsequent GMCs, before mid-L3 (L3 being the last larval stage) leads to aggressive NB tumors that persist growing in adults. In contrast, inactivation of pros after mid-L3 leads to transient NB amplification and most supernumerary NB properly differentiate during metamorphosis, leading to an absence of growing tumors in adults. Interestingly, propagation of NB tumor growth beyond normal developmental stages is caused by the aberrant maintenance of Imp and the transcription factor Chinmo from early-born GMCs, the latter representing the cells of origin of such aggressive tumors (Narbonne-Reveau, 2016). Chinmo and Imp positively cross-regulate and inactivation of either in NB tumors stops tumorigenic growth. Because pros-/- NB tumors can only be induced during an early window of development, and are caused by the biallelic inactivation of a single gene, they represent an exciting and simple model to investigate the basic mechanisms driving the growth of tumors with an early developmental origin, such as in the case of pediatric CNS cancers (Genovese, 2019).

NB tumors can also be induced from type II NBs (a small subset of NBs in the central brain) or from neurons upon inactivation of the NHL-domain family protein Brat or Nerfin-1 respectively. In both cases, tumor growth appears to rely on the aberrant expression of the Chinmo/Imp module arguing for a general tumor-driving mechanism in the developing Drosophila CNS (Narbonne-Reveau, 2016). Interestingly, in the different types of NB tumors, Chinmo and Imp are only expressed in a subpopulation of cells, demonstrating heterogeneity in the population of tumor NBs (tNBs). However, the full repertoire of cells composing the tumor, the rules governing the cellular heterogeneity and the mechanisms determining the proliferative potential of each cell type remain to be investigated (Genovese, 2019).

This study used single-cell RNA-seq, clonal analysis and numerical modeling to investigate these questions. A subset of genes involved in the temporal patterning of larval NBs were identified that are redeployed in tumors to generate a differentiation trajectory responsible for creating tumor cell heterogeneity. This cellular heterogeneity results in NBs with different types of metabolism and different proliferative properties. This study also deciphered a robust hierarchical scheme that drives reproducible heterogeneity through the dysregulated but fine-tuned transition between the two RNA-binding proteins Imp and Syp. This work thus identifies a core larval NB temporal patterning program, the disruption of which not only causes unlimited growth but has an overarching role in governing the cellular hierarchy, heterogeneity and metabolism of NB tumors (Genovese, 2019).

This study demonstrates that temporal patterning, not only determines which cells are susceptible to cancer transformation during development (Narbonne-Reveau, 2016), but also has an overarching role in governing different aspects of CNS tumor organization such as hierarchy, heterogeneity and the proliferative properties of the different types of cells via the regulation of their metabolism (Genovese, 2019).

Given the recent discovery that temporal patterning is conserved in the developing mammalian brain (Telley, 2019), this study could shed light on an ancestral mechanism that governs the progression of CNS tumors with developmental origins (Genovese, 2019).

The rules governing the initiation and progression of CNS pediatric tumors that often exhibit stable genomes are still unclear. Previousl work has demonstrated that temporal patterning in Drosophila larval NBs delineates a window of time during which the Chinmo/Imp oncogenic module is expressed and makes early larval NBs prone to malignant transformation (Narbonne-Reveau, 2016). This study finds that after tumor initiation, temporal patterning is partly recapitulated in tNBs where it generates differentiation trajectories to constrain tumor composition and growth. This is illustrated by the presence of about 20 genes (Imp, chinmo, Lin-28, E23, Oatp74D, Gapdh2, Sip1/CG10939, plum/CG6490, SP1173, Chd64, CG10512, CG44325, CG5953, Syp, E93, lncRNA:noe, CG15646 and stg), previously identified to be temporally regulated in some larval NBs, that are differentially regulated along the pseudotime/differentiation trajectory reconstructed from single-cell RNA-seq analysis of tNBs, and/or differentially expressed in Imp+ vs Syp+ tNBs. Thus, this study identified what appears to be a subset of a core temporal patterning program encoded in central brain and ventral nerve cord NBs that becomes deregulated upon asymmetric-division defects during early development (Genovese, 2019).

Notably, the larval temporal transcription factor Cas and Svp, known to schedule the Imp-to-Syp transition during development are not enriched in Imp+ tNBs suggesting that they do not play a role in regulating the Imp-to-Syp transition along the trajectory in tumors. Interestingly, while Syp is transcriptionally regulated in larval NBs, it seems rather post-transcriptionally regulated in tNBs as Syp RNAs are present throughout all clusters. This suggests that different mechanisms may be operating in tumors than during development to regulate the Imp-to-Syp transition (Genovese, 2019).

This study observed that the proportions of Imp+ and Syp+ tNBs systematically reach an equilibrium over a few days with a 20/80 (+/-10) ratio in poxn > prosRNAi tumors. This suggests that the regulation of the Imp-to-Syp transition in tumors is not random and the predictability of the final proportions possibly implies robust underlying constraints. By investigating the population dynamics of Imp+ and Syp+ tNBs in prosRNAi tumors, this study has deciphered a finely tuned hierarchical division scheme that appears to constrain the growth and cellular heterogeneity of the tumor. Imp+ tNBs is shown in the tumorigenic context favor a symmetric self-renewing mode of divisions (in more than 60% of divisions) while unlikely to exit the cell-cycle. This allows the perpetuation of a small subset of Imp+ tNBs that are endowed with a seemingly unlimited self-renewing potential by the Imp/Chinmo module. Imp+ tNBs can also make symmetric and asymmetric divisions that generate Syp+ tNBs, leading to the production of a population of Syp+E93+ tNBs that accumulates through limited self-renewal, and have a high propensity for exiting the cell-cycle. Moreover, this study could shows that Syp+E93+ tNBs are unable to generate Imp+ tNBs, demonstrating a rigid cellular hierarchy reminiscent of development. In addition, in this context, Syp acts as a tumor suppressor by limiting tNB proliferation while Imp acts as an oncogene by promoting tNB proliferation and propagation of tumor growth. Together, these data argue for a scenario where cooption of the Imp-to-Syp transition is responsible for installing a hierarchical mode of tumor growth with Imp+ tNBs propagating unlimited growth in a CSC-like manner, while Syp+E93+ tNBs acts as transient amplifying progenitors with limited self-renewing abilities. Although the Imp/Syp RNA-binding proteins have an essential and antagonistic role in governing the proliferative properties of tumor cells, the function of the other redeployed temporal patterning genes is unknown (except for chinmo, downstream to Imp and Syp, that is essential for tumor growth). As many are linked with the Imp+ tNB state, it will be important in the future to decipher how they contribute to establish or maintain the CSC-like identity (Genovese, 2019).

The division parameters defined by clonal analysis and modeling approach could capture both the hierarchical aspect of tumor growth as well as the global population dynamics: from an initial homogenous pool of larval Imp+ tNBs to the stable heterogeneity observed during adulthood. It could also resolve the paradoxical observation that Chinmo+Imp+ tNBs end up in minority despite exhibiting a higher average mitotic rate. Although, like all models, it is not expected that this model would perfectly recapitulate all the parameters regulating tumor growth and heterogeneity (for example, this study has neglected apoptosis and neuronal differentiation that occur at low levels), it is thought that this model provides a reasonable and useful ground on which further studies can be performed for a more detailed understanding. On these lines, while the division pattern this study has described with a numerical model provides estimates of division probabilities in poxn > prosRNAi tumors, it says nothing as to how these probabilities are biologically set within the tumor. A possible scenario is that cell fate determination upon division relies on signals received by immediate neighboring tumor cells, resulting in effective probabilities at the scale of the whole tumor. Such a micro-environment dependent regulation of the Imp-to-Syp transition in tumors would strongly contrast with the cell-intrinsic regulation of the Imp-to-Syp transition that systematically occurs in NBs around early L3. Future studies will aim at deciphering the mechanisms that interfere with the developmental progression of the temporal patterning, upon asymmetric-division defects, to favor the self-renewing mode of divisions undergone by the Chinmo+Imp+ tNBs, allowing perpetuation of a population of CSC-like cells (Genovese, 2019).

Noteworthy, prosRNAi and snr1/dSmarcb1RNAi tumors exhibit different but reproducible ratios of Imp+ and Syp+ tNBs. This suggests the existence of tumor-specific mechanisms that fine-tune the Imp-to-Syp transition. Such mechanisms may be related to the tumor cell of origin, or to the genetic insult that initiated NB amplification. Further analysis will help identifying tumor-intrinsic signals regulating the balance between Chinmo+Imp+ tNBs and Syp+E93+ tNBs in various types of NB tumors (Genovese, 2019).

Until recently, the existence of temporal patterning in mammalian neural progenitors remained uncertain. Elegant single-cell transcriptomic studies of embryonic cortical and retinal progenitors in mice have now revealed that they transit through different transcriptional states that are transmitted to their progeny to generate neuronal diversity, similar to temporal patterning in Drosophila (Clark, 2019; Telley, 2019). However, it remains unknown whether temporal patterning determines the cell of origin and governs the growth of CNS tumors in children. Along these lines, the finding that the transcriptional programs operating in cerebellar progenitors during fetal development are recapitulated in medulloblastomas is promising (Vladoiu, 2019). By uncovering the overarching role of temporal patterning in governing tumor susceptibility during CNS development and in constraining tumor properties during cancer progression in Drosophila, this work thus possibly provides a new conceptual framework to better understand CNS tumors in children (Genovese, 2019).

Because of the difficulty to investigate metabolism at the single-cell level, it has been difficult to determine how heterogeneous is the metabolic activity of cells in tumors, and how it is controlled. Using a combination of single-cell and bulk RNA-seq approaches, this study has found that progression of temporal patterning provides a tumor-intrinsic mechanism that generates heterogeneity in the proliferative abilities of tumor cells through the progressive silencing of glucose and glutamine metabolism genes (Genovese, 2019).

Consequently, Chinmo+Imp+ tNBs, that lie at the top of the hierarchy, highly express glycolytic and respiratory/OXPHOS genes, as well as Gdh, that are down-regulated by the Imp-to-Syp transition. This default high expression of both glutamine and glucose metabolism genes in CSC-like Chinmo+Imp+ tNBs likely favors sustained self-renewal, but could also confer plasticity and a way to adapt cellular metabolism to different environmental conditions as frequently observed in CSCs (e.g., glutamine can compensate for glucose shortage) (Sancho, 2016) (Genovese, 2019).

This study showed that Syp+E93+ tNBs exhibit a reduced size, and that knock-down of glycolytic (Gapdh1 or Pglym78) or respiratory/OXPHOS genes (Cyt-c-p or Cyt-C1) prevented propagation of tumor growth in adults. Thus, reduction of biosynthesis and energy production through down-regulation of glucose and glutamine metabolism genes after the Imp-to-Syp transition could progressively exhaust Syp+E93+ tNB growth and self-renewing ability, ultimately leading to cell-cycle exit (Genovese, 2019).

With the demonstration that temporal patterning regulates glycolytic, TCA cycle and OXPHOS genes in NB tumors, this work provides a tumor-intrinsic mechanism that creates metabolic heterogeneity to control the proliferative potential of the various tumor cells. It was also observed that Syp+E93+ tNBs associated with lowest levels of metabolic and cell-cycle genes also upregulate genes of the E(spl) genes. Interestingly, expression of Hes genes (orthologs of Enhancer of split genes) in vertebrate neural stem cells is associated with the maintenance of a quiescent state in adults. Thus, E(spl) genes may promote the quiescent tNB state identified with the clonal and numerical analysis while preventing their differentiation in neurons (Genovese, 2019).

Down-regulation of the mRNA levels of metabolic genes after the Imp-to-Syp transition could be due to the silencing of a transcriptional activator or to an increased mRNA degradation. On one hand, Chinmo is a likely candidate for the first scenario, as its inactivation reduces growth in NBs (Narbonne-Reveau, 2016) and this study showed that it is a direct target of both Imp and Syp. On the other hand, the second scenario is consistent with Imp orthologs in human being able to promote OXPHOS and proliferation in glioma cells, through the post-transcriptional regulation of mitochondrial respiratory chain complex subunits (Genovese, 2019).

This study has also identified a small population of tNBs expressing various stress or growth arrest factors. One of these factors, Xrp1, is a transcriptional target of p53 in the response to irradiation. Xrp1 expression has also recently been linked to defects in translation rates, together with the expression of Irbp18 and GstE6. Thus, these factors may label a subset of tNBs undergoing DNA or translational stress. The reason and consequences of such cellular stresses in tumor progression need to be further investigated (Genovese, 2019).

Transcriptomic analyses have revealed strong similarities in the differentiation trajectories of tNBs in tumors and of NBs in larvae. Yet, it is surprising that the down-regulation of glutamine and glucose metabolism genes has not been detected in NBs during larval development, after the Imp-to-Syp transition (Ren, 2017). It is possible that the glial niche surrounding NBs, that is known to influence NB growth properties during larval stages, somehow sustains high levels of glucose metabolism genes in late Syp+E93+ NBs. Given that this glial niche is absent in tumors, Syp+E93+ tNBs may not be able to sustain the high expression of metabolic genes imposed by the Imp/Chinmo module, leading to progressive cell-cycle exit (Genovese, 2019).

Chinmo and Imp are reminiscent to oncofetal genes in mammals, in that their expression decrease rapidly as development progresses while they are mis-expressed in tumors. Along these lines, the three IMP orthologs in humans (also called IGF2BP1-3) are also known as oncofetal genes. They emerge as important regulators of cell proliferation and metabolism in many types of cancers including pediatric neural cancers. Along evolution, the ancestral Syncrip gene has been subjected to several rounds of duplication and has diverged into five paralogs in mammals, some of them emerging as tumor suppressors with an important role in tumor progression (Genovese, 2019).

Thus, the respective oncogenic and tumor suppressor roles of IMP and SYNCRIP gene families appear to have been conserved in humans and they may not be restricted to tumors of neural origin. This study therefore raises the exciting possibility that these two families of RNA-binding proteins form a master module at the top of the self-renewal/differentiation cascades, that regulates CSC populations and hierarchy in a spectrum of human cancers (Genovese, 2019).

chinmo^-mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche

Niches maintain a finite pool of stem cells via restricted space and short-range signals. Stem cells compete for limited niche resources, but the mechanisms regulating competition are poorly understood. Using the Drosophila testis model, this study showed that germline stem cells (GSCs) lacking the transcription factor Chinmo gain a competitive advantage for niche access. Surprisingly, chinmo(-/-) GSCs rely on a new mechanism of competition in which they secrete the extracellular matrix protein Perlecan to selectively evict non-mutant GSCs and then upregulate Perlecan-binding proteins to remain in the altered niche. Over time, the GSC pool can be entirely replaced with chinmo^-/- cells. As a consequence, the mutant chinmo allele acts as a gene drive element; the majority of offspring inherit the allele despite the heterozygous genotype of the parent. These results suggest that the influence of GSC competition may extend beyond individual stem cell niche dynamics to population-level allelic drift and evolution (Tseng, 2022).

This work reveals an unexpected model of GSC competition that results in biased inheritance. These results provide a mechanistic demonstration of the postulated 'mitotic drive' by which GSCs with a competition advantage transmit competitive alleles at a greater than the expected 50% Mendelian ratio. Studies in plants, yeast, flies, and mice have shown that selfish genetic elements can cause gene drive through various molecular mechanisms. These include 'meiotic drivers' that co-opt meiotic divisions or that kill viable gametes that do not inherit the selfish element. 'Mitotic drive' occurs earlier in the germline lineage, at the stem cell level, and is an understudied area. Prior work in the Drosophila ovary has shown that dedifferentiation-defective female GSCs 'win' by upregulating E-Cad at the GSC-niche interface and gradually pushing WT GSCs out of the niche. Since differentiation-defective GSCs do not differentiate into gametes, this study could not examine biased allele inheritance. Female GSCs with 4x gene dose of Myc were reported to be 'winners,' but biased inheritance was not assessed (Tseng, 2022).

Given the competitive advantage of GSCs lacking chinmo, why has evolution not selected for male GSCs with no chinmo expression? Since chinmo is an essential gene required for development (Zhu et al., 2006), loss of chinmo in GSCs might cause reduced chinmo expression in other tissues, likely reducing organismal fitness. Furthermore, chinmo^- dependent competition is a progressive phenotype requiring at least 2 weeks of adulthood. If males with mosaic chinmo^- clones in the germline were mated as young adults, both the chinmo⁺ and chinmo^- allele would be passed on to offspring, and this would be sufficient to maintain the chinmo⁺ allele in the population. Although the GSC pool in testes with chinmo^- cells is often monoclonal, why is the chinmo^- allele is not passed on to 100% of offspring? Males are maintained as virgins until their testes are dissected or until mating. This means that both chinmo^- GFP-negative and ubi-GFP-positive spermatids are stored in the seminal vesicle throughout the male's lifetime, and the single round of mating in these experiments allows for the transmission of both types of sperm (Tseng, 2022).

This work identified three phenotypes-competition, aging, and transdifferentiation-that are dependent on ectopic Pcan expression in chinmo^-deficient stem cells. This remarkable finding raises the important question of which factors regulate Chinmo expression in GSCs during adulthood and which genes are direct targets of Chinmo. This study identified chinmo as a JAK/STAT target gene, but STAT-deficient GSCs still express Chinmo protein, indicating that as-yet unidentified factors regulate Chinmo in GSCs and perhaps in other stem cells. Additionally, future molecular work will be needed to determine whether Pcan, Dg, and βPS are direct Chinmo target genes in germline and somatic stem cells (Tseng, 2022).

This work raises the possibility that other mutant stem cells can 'cheat' by resculpting their microenvironment and then ensuring their own retention in this remodeled milieu. Paternal age effect disorders (PAEs) encompass a broad spectrum of spontaneous congenital disorders and are thought to arise from rare selfish GSCs that are positively selected and clonally expanded. While the current model of PAE postulates increased proliferation of mutant GSCs as the competitive mechanism, other selfish cellular behaviors, such as the ones this study has discovered, could also be functioning in the mammalian testis. Cancer stem cells could utilize the mechanisms described in this study to colonize a tissue. Leukemic stem cells induce progressive remodeling of the bone marrow niche, and this altered niche favors the mutant stem cells while impairing normal hematopoietic stem cell residence and contributes to bone marrow fibrosis. In sum, this work raises the possibility that selfish stem cells across species cheat using the mechanisms this study has discovered in competitive GSCs in the Drosophila testis (Tseng, 2022).

Although this study found significant increases in Pcan, Dg, and βPS transcripts in GSCs deficient for Chinmo, HCR-FISH analyses of relative intensity are not strictly quantitative. As such, it cannot be ruled out that Chinmo affects other aspects of mRNA regulation, such as splicing. Because the transcriptome of chinmo^-/- GSCs have not been attained at sufficient resolution and this study has not been able to perform Chinmo ChIP-seq in GSCs, it is not possible to conclude that Chinmo directly represses these genes. It will be important in the future to determine Chinmo occupancy on chromatin in GSCs and in other stem cells. Surprisingly, the niche spaces vacated by non-mutant neighbor GSCs are occupied by cyst stem cells (CySCs) and not by chinmo^-/- GSCs. It is not understood why the CySCs predominate, and future studies employing ex vivo live-imaging will be important to gain insights into this process (Tseng, 2022).

Hierarchical deployment of factors regulating temporal fate in a diverse neuronal lineage of the Drosophila central brain

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).

Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates

Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons.Factors that could regulate neural temporal fate were sought by RNA-sequencing of lineage-specific neuroblasts at various developmental times. Two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), were found to display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage (Liu, 2015).

Diverse neural stem cells produce distinct sets of specialized neurons. The suite of daughter neurons generated by common neural stem cells can further change as development progresses, which suggests that the neural stem cells themselves change over time. In Drosophila, the neuroblast, a type of neural stem cell, can bud off about 100 ganglion mother cells that each divide once to produce two, often different, daughter neurons. Mapping the serially derived neurons based on birth order/time has revealed that each individual neuroblast makes an invariant series of morphologically distinct neuronal types (Liu, 2015).

Drosophila central brain neuroblasts differ greatly in the number of neuronal types produced and the tempo at which changes occur. The four mushroom body neuroblasts divide continuously throughout larval and pupal development, but each produces only three classes of neurons. By contrast, the antennal lobe anterodorsal 1 (ALad1) neuroblast generates 22 neuronal types during larval development. Although sequential neuroblast expression of temporal transcription factors specifies neuronal cell fates in lineages of the Drosophila ventral nerve cord and optic lobes, this mechanism is not easily applied to central brain. This study analyzed fate determinants that direct neuronal diversification based on the age of the neuroblast (Liu, 2015).

Genes with age-dependent changes in expression levels throughout the life of a neuroblast could confer different temporal fates upon the neuronal daughter cells born at different times. Thus, this study aimed to find genes dynamically expressed in mushroom body and antennal lobe neuroblasts by sequencing transcriptomes over the course of larval and pupal neurogenesis. Mushroom body or antennal lobe neuroblasts were marked persistently and exclusively with green fluorescent protein (GFP) by genetic intersection and immortalization tactics. Approximately 100 GFP+ neuroblasts were isolated for each RNA/cDNA preparation. Quantitative polymerase chain reaction (qPCR) showed that known neuroblast genes, including deadpan (dpn) and asense, are enriched in mushroom body and antennal lobe neuroblasts compared with total larval brains. Samples passing this qPCR quality check were sequenced. The transcriptomes of mushroom body neuroblasts were obtained at 24, 50, and 84 hours after larval hatching (ALH), as well as 36 hours after puparium formation (APF), and the transcriptomes of antennal lobe neuroblasts at 24, 36, 50, and 84 hours ALH. The antennal lobe neuroblasts were not sequenced at 36 hours APF because they stop producing neurons around puparium formation. Also, mushroom body neuroblasts were not analyzed at 36 hours ALH because the mushroom body lineages do not undergo detectable fate or molecular changes between 24 and 50 hours ALH (Liu, 2015).

Strongly dynamic genes were defined as those with a greater than fivefold change in expression level across different time points and a maximum average abundance higher than 50 transcripts per million. Eighty-three strongly dynamic genes in the mushroom body and 63 in the antennal lobe. The two sets shared 16 genes in common. Among these 16 common genes, pncr002:3R, a putative noncoding RNA, ranks highest in absolute abundance, the importance of which remains unclear (Liu, 2015).

IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), which code for two evolutionally conserved RNA-binding proteins, rank second and third in absolute abundance (McDermott, 2012; McDermott, 2014; Munro, 2006). Imp is expressed abundantly at 24 hours ALH and declines to a minimum at 84 hours ALH in antennal lobe and 36 hours APF in mushroom body neuroblasts, whereas Syp increases from minimal expression at 24 hours ALH to become one of the most abundant genes at late larval stages. Imp/Syp gradients with larger amplitudes and steeper slopes characterize antennal lobe neuroblasts, which yield more diverse neuron types at faster tempos than the mushroom body neuroblasts. Antibodies to Imp and Syp showed similar patterns in shifts of protein abundance in both mushroom body neuroblasts and neuronal daughter cells. The roles of Imp and Syp in neuronal temporal fate specification were investigated in both mushroom body and antennal lobe lineages (Liu, 2015).

The post-embryonic mushroom body neuroblasts sequentially produce γ, α'/β', and α/β neurons, which can be distinguished by a variety of markers. Fasciclin II (FasII) is expressed in the perimeter of the α/β lobes, is weakly expressed in the γ lobe, and is not expressed in the α'/β' lobes. Trio, a Dbl family protein, is expressed in the γ and α'/β' lobes. The γ neurons can also be identified in wandering larvae by expression of ecdysone receptor B1 isoform (EcR-B1). Moreover, one can predict the fate of newborn mushroom body neurons based on the protein levels of Chinmo, a known temporal transcription factor, as abundant Chinmo specifies the γ fate, weak Chinmo expression confers the α'/β' fate, and absence of Chinmo permits the α/β fates (Liu, 2015).

RNA interference (RNAi) aimed to reduce Imp expression resulted in up-regulated Syp, whereas knocking down Syp expression caused increased Imp expression. This reciprocal derepression was evident in protein and transcript content as well as phenotype. Silencing Imp triggered precocious production of α/β neurons throughout larval development; these neurons lacked EcR-B1 at the wandering larval stage and showed no Chinmo at 24 hours ALH. Imp-depleted neuroblasts ended neurogenesis prematurely: By 28 hours APF, no neuroblasts remained in the mushroom body. This resulted in small adult mushroom bodies with only α/β lobes. By contrast, silencing Syp extended the production of Chinmo-positive γ neurons through pupal development. The Syp-depleted adult mushroom bodies consisted of a single prominent γ lobe. The reciprocal temporal fate transformations were also seen in the mushroom body neuroblast clones homozygous for various Imp or Syp loss-of-function mutations. Thus, Imp specifies early γ neurons and Syp specifies late α/β neurons (Liu, 2015).

It was next asked whether prolonged coexpression of Imp and Syp can increase the number of α'/β' neurons, which are typically born at a time when Imp and Syp expression levels are similar. Ectopic induction of Imp or Syp transgenes enhanced Imp or Syp protein levels in mushroom body newborn neuronal daughter cells but not in the neuroblasts. In the case of Syp overexpression, the early larval neuroblasts still contained abundant Imp, as did their newborn neurons expressing ectopic Syp. Analogously, overexpressing Imp rendered the pupal-born neurons strongly positive for Syp as well as ectopic Imp. Therefore, after Imp or Syp overexpression, many more newborn neurons simultaneously expressed Imp and Syp, and the normally modest α'/β' neuronal lobes were enlarged in adult brains. Cell death was unlikely to distort the developmental outcomes, as rare sporadic cell death was only detected in mushroom body neurons expressing ectopic Syp at 50 hours ALH. Taken together, these data demonstrate that relative levels of Imp and Syp dictate mushroom body neuronal temporal fates (Liu, 2015).

The altered Chinmo protein levels upon Imp or Syp depletion prompted a look at whether Imp and Syp regulate chinmo expression. The abundance of chinmo transcripts normally decreases as neuroblasts age. This pattern was unperturbed in Imp or Syp-depleted neuroblasts. Together, these data suggest that Imp and Syp regulate chinmo expression at a posttranscriptional level (Liu, 2015).

Epistasis was used to explore whether Imp and Syp act to regulate mushroom body neuronal temporal fates through Chinmo. Overexpressing a chinmo transgene partially restored the production of γ neurons by the short-lived, Imp-depleted neuroblasts. Moreover, silencing chinmo transformed the supernumerary γ neurons made by the Syp-depleted neuroblasts into α/β neurons. Together, these observations place Chinmo downstream of Imp and/or Syp in the temporal fate specification of mushroom body (Liu, 2015).

To ascertain whether Imp/Syp gradients serve as a general temporal-fating mechanism, the roles of Imp and Syp were examined in the rapidly changing antennal lobe anterodorsal 1 (ALad1) lineage that yields ~60 larval-born neurons of 22 types. Although all 22 types express acj6-GAL4, only the first 12 types generated express GAL4-GH146. Imp depletion reduced the ALad1 daughter cell number (acj6+) but increased the ratio of late-type to early-type neurons. By contrast, Syp depletion increased total ALad1 daughter cells and increased the percentage of the early-type (GH146+) neurons. Despite precocious production of late-type neurons or prolonged generation of early-type neurons, 21 of the 22 neuron types were preserved. In summary, the opposing Imp/Syp gradients govern temporal fates in at least two different neuroblast lineages that produce mushroom body and antennal lobe neurons functioning in memory and olfaction, respectively (Liu, 2015).

The opposite temporal gradients of Imp and Syp in neuroblasts confer the serially derived daughter cells with graded levels of Imp/Syp. The acquisition of distinct daughter cell fates based on the Imp/Syp morphogens is reminiscent of early embryonic patterning by the opposite spatial gradients of maternally inherited Bicoid and Nanos. Different levels of Bicoid and Nanos are incorporated into cells along the anterior-posterior axis after cellularization of the blastoderm. Bicoid and Nanos function as RNA-binding proteins to initiate anterior-posterior spatial patterning via translational control of maternal transcripts that encode transcription factors. Analogously, temporal fate patterning of newborn neurons is orchestrated by post-transcriptional control of chinmo and potentially other genes by Imp and Syp. Because Imp and Syp may share common targets but show affinity for different RNA motifs, it is possible that Imp and Syp can both bind chinmo transcripts, but they may differentially target chinmo transcripts for translation versus sequestration. Descending Imp temporal gradients governs aging of Drosophila testis stem cell niche. Imp-1, the mammalian ortholog of Imp, is also needed to maintain mouse neural stem cells. It is proposed that graded Imp/Syp expression constitutes an evolutionally conserved mechanism for governing time-dependent stem cell fates, including temporal fate progression in neural stem cells and their derived neuronal lineages (Liu, 2015).

Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche

In the Drosophila testis, germline stem cells (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells, termed the hub, which produce a variety of growth factors contributing to the niche microenvironment that regulates both stem cell pools. This study shows that CySC but not GSC maintenance requires Hedgehog (Hh) signalling in addition to Jak/Stat pathway activation. CySC clones unable to transduce the Hh signal are lost by differentiation, whereas pathway overactivation leads to an increase in proliferation. However, unlike cells ectopically overexpressing Jak/Stat targets, the additional cells generated by excessive Hh signalling remain confined to the testis tip and retain the ability to differentiate. Interestingly, Hh signalling also controls somatic cell populations in the fly ovary and the mammalian testis. These observations might therefore point towards a higher degree of organisational homology between the somatic components of gonads across the sexes and phyla than previously appreciated (Michel, 2012).

Hh thus provides a niche signal for the maintenance and proliferation of the somatic stem cells of the testis. CySCs that are unable to transduce the Hh signal are lost through differentiation, whereas pathway overactivation causes overproliferation. Hh signalling thereby resembles Jak/Stat signalling via Upd. Partial redundancy between these pathways might explain why neither depletion of Stat activity nor loss of Hh signalling causes complete CySC loss (Michel, 2012).

This study has shown that loss of Hh signalling in smo mutant cells blocks expression of the Jak/Stat target Zfh1, whereas mutation of ptc expands the Zfh1-positive pool. Overexpression of Zfh1 or another Jak/Stat target, Chinmo, is sufficient to induce CySC-like behaviour in somatic cells irrespective of their distance. By contrast, Hh overexpression in the hub using the hh::Gal4 driver only caused a moderate increase in the number of Zfh1-positive cells relative to a GFP control. Ectopic Hh overexpression in somatic cells under c587::Gal4 control increased this number further. However, unlike in somatic cells with constitutively active Jak/Stat signalling, the additional Zfh1-positive cells remained largely confined to the testis tip, although their average range was increased threefold. Thus, Hh appears to promote stem cell proliferation, in part, also independently of competition (Michel, 2012).

It is tempting to speculate that further stem cell expansion is limited by Upd range. Consistently, cells with an ectopically activated Jak/Stat pathway remain undifferentiated, whereas ptc cells can still differentiate. Future experiments will need to formally address the epistasis between these pathways. However, the observations already show that Hh signalling influences expression of the bona fide Upd target gene zfh1, and therefore presumably acts upstream, or in parallel to, Upd in maintaining CySC fate (Michel, 2012).

In addition, the reduction in GSC number following somatic stem cell loss implies cross-regulation between the different stem cell populations that presumably involves additional signalling cascades, such as the EGF pathway (Michel, 2012).

In recent years, research has focused on the differences between the male and female gonadal niches. This paper instead emphasizes the similarities: in both cases, Jak/Stat signalling is responsible for the maintenance and activity of cells that contribute to the GSC niche, and Hh signalling promotes the proliferation of stem cells that provide somatic cells ensheathing germline cysts. In the testis, both functions are fulfilled by the CySCs, whereas in the ovary the former task is fulfilled by the postmitotic escort stem cells/escort cells and the latter by the FSCs. Finally, male desert hedgehog (Dhh) knockout mice are sterile. Dhh is expressed in the Sertoli cells and is thought to primarily act on the somatic Leydig cells. However, the signalling microenvironment of the vertebrate spermatogonial niche is, as yet, not fully defined. Future experiments will need to clarify whether these similarities reflect convergence or an ancestral Hh function in the metazoan gonad (Michel, 2012).

Search PubMed for articles about Drosophila Chinmo

Almeida, M. S. and Bray, S. J. (2005). Regulation of post-embryonic neuroblasts by Drosophila Grainyhead. Mech. Dev. 122: 1282-1293. PubMed ID: 16275038

Bello, B., Reichert, H. and Hirth, F. (2006). The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila. Development 133: 2639-2648. PubMed ID: 16774999

Bray, S. J., Burke, B., Brown, N. H. and Hirsh, J. (1989). Embryonic expression pattern of a family of Drosophila proteins that interact with a central nervous system regulatory element. Genes Dev. 3: 1130-1145. PubMed ID: 2792757

Brody, T. and Odenwald, W. F. (2002). Cellular diversity in the developing nervous system: a temporal view from Drosophila. Development 129: 3763-3770. PubMed ID: 12135915

Brody, T., and Odenwald, W. F. (2000). Programmed transformations in neuroblast gene expression during Drosophila CNS lineage development. Dev. Biol. 226: 34-44. PubMed ID: 10993672

Cenci, C., and Gould, A. P. (2005). Drosophila Grainyhead specifies late programmes of neural proliferation by regulating the mitotic activity and Hox-dependent apoptosis of neuroblasts. Development 132: 3835-3845. PubMed ID: 16049114

Clark, B. S., Stein-O'Brien, G. L., Shiau, F., Cannon, G. H., Davis-Marcisak, E., Sherman, T., Santiago, C. P., Hoang, T. V., Rajaii, F., James-Esposito, R. E., Gronostajski, R. M., Fertig, E. J., Goff, L. A. and Blackshaw, S. (2019). Single-cell RNA-Seq analysis of retinal development identifies NFI factors as regulating mitotic exit and late-born cell specification. Neuron 102(6): 1111-1126 e1115. PubMed ID: 31128945

Doggett, K., Turkel, N., Willoughby, L. F., Ellul, J., Murray, M. J., Richardson, H. E. and Brumby, A. M. (2015). BTB-zinc finger oncogenes are required for Ras and Notch-driven tumorigenesis in Drosophila. PLoS One 10: e0132987. PubMed ID: 26207831

Dyer, M. A., Livesey, F. J., Cepko, C. L., and Oliver, G. (2003). Prox1 function controls progenitor cell proliferation and horizontal cell genesis in the mammalian retina. Nat. Genet. 34: 53-58. PubMed ID: 12692551

Flaherty, M. S., Zavadil, J., Ekas, L. A. and Bach, E. A. (2009). Genome-wide expression profiling in the Drosophila eye reveals unexpected repression of notch signaling by the JAK/STAT pathway. Dev. Dyn. 238: 2235-2253. PubMed ID: 19504457

Flaherty, M. S., et al. (2010). chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila. Dev. Cell 18: 556-568. PubMed ID: 20412771

Genovese, S., Clement, R., Gaultier, C., Besse, F., Narbonne-Reveau, K., Daian, F., Foppolo, S., Luis, N. M. and Maurange, C. (2019). Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors. Elife 8. PubMed ID: 31566561

Graham, V., Khudyakov, J., Ellis, P., and Pevny, L. (2003). SOX2 functions to maintain neural progenitor identity. Neuron 39: 749-765. PubMed ID: 12948443

Grmai, L., Hudry, B., Miguel-Aliaga, I. and Bach, E. A. (2018). Chinmo prevents transformer alternative splicing to maintain male sex identity. PLoS Genet 14(2): e1007203. PubMed ID: 29389999

Flaherty, M. S., Salis, P., Evans, C. J., Ekas, L. A., Marouf, A., Zavadil, J., Banerjee, U. and Bach, E. A. (2010). chinmo is a functional effector of the JAK/STAT pathway that regulates eye development, tumor formation, and stem cell self-renewal in Drosophila. Dev Cell 18(4): 556-568. PubMed ID: 20412771

Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q. (2001). Drosophila neuroblasts sequentially express transcription factors which specify the temporal identity of their neuronal progeny. Cell 106: 511-521. PubMed ID: 11525736

Jiang, Y., Seimiya, M., Schlumpf, T. B. and Paro, R. (2018). An intrinsic tumour eviction mechanism in Drosophila mediated by steroid hormone signalling. Nat Commun 9(1): 3293. PubMed ID: 30120247

Kao, C. F., Yu, H. H., He, Y., Kao, J. C. and Lee, T. (2012). Hierarchical deployment of factors regulating temporal fate in a diverse neuronal lineage of the Drosophila central brain. Neuron 73(4): 677-84. PubMed ID: 22365543

Leatherman, J. L. and Dinardo, S. (2008). Zfh-1 controls somatic stem cell self-renewal in the Drosophila testis and nonautonomously influences germline stem cell self-renewal. Cell Stem Cell 3(1): 44-54. PubMed ID: 18593558

Liu, Z., Yang, C. P., Sugino, K., Fu, C. C., Liu, L. Y., Yao, X., Lee, L. P. and Lee, T. (2015). Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates. Science 350: 317-320. PubMed ID: 26472907

Ma, Q., Wawersik, M. and Matunis, E. L. (2014). The Jak-STAT target Chinmo prevents sex transformation of adult stem cells in the Drosophila testis niche. Dev Cell 31: 474-486. PubMed ID: 25453558

Maurange, C., Cheng, L. and Gould, A. P. (2008). Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila. Cell 133(5): 891-902. PubMed ID: 18510932

McDermott, S. M., Meignin, C., Rappsilber, J. and Davis, I. (2012). Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification. Biol Open 1: 488-497. PubMed ID: 23213441

McDermott, S. M., Yang, L., Halstead, J. M., Hamilton, R. S., Meignin, C. and Davis, I. (2014). Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction. RNA 20: 1593-1606. PubMed ID: 25171822

Michel, M., Kupinski, A. P., Raabe, I. and Bõkel, C. (2012). Hh signalling is essential for somatic stem cell maintenance in the Drosophila testis niche. Development 139(15): 2663-9. PubMed ID: 22745310

Munro, T. P., Kwon, S., Schnapp, B. J. and St Johnston, D. (2006). A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP. J Cell Biol 172: 577-588. PubMed ID: 16476777

Narbonne-Reveau, K., Lanet, E., Dillard, C., Foppolo, S., Chen, C. H., Parrinello, H., Rialle, S., Sokol, N. S. and Maurange, C. (2016). Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila. Elife 5. PubMed ID: 27296804

Qian, Y., Ng, C. L. and Schulz, C. (2014). CSN maintains the germline cellular microenvironment and controls the level of stem cell genes via distinct CRLs in testes of Drosophila melanogaster. Dev Biol 398(1): 68-79. PubMed ID: 25459658

Ren, Q., Yang, C. P., Liu, Z., Sugino, K., Mok, K., He, Y., Ito, M., Nern, A., Otsuna, H. and Lee, T. (2017). Stem cell-intrinsic, Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors. Curr Biol [Epub ahead of print]. PubMed ID: 28434858

Shen, Q., Wang, Y., Dimos, J.T., Fasano, C. A., Phoenix, T. N., Lemischka, I. R., Ivanova, N. B., Stifani, S., Morrisey, E. E. and Temple, S. (2006). The timing of cortical neurogenesis is encoded within lineages of individual progenitor cells. Nat. Neurosci. 9: 743-751. PubMed ID: 16680166

Syed, M. H., Mark, B. and Doe, C. Q. (2017). Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity. Elife 6 [Epub ahead of print]. PubMed ID: 28394252

Telley, L., Agirman, G., Prados, J., Amberg, N., Fievre, S., Oberst, P., Bartolini, G., Vitali, I., Cadilhac, C., Hippenmeyer, S., Nguyen, L., Dayer, A. and Jabaudon, D. (2019). Temporal patterning of apical progenitors and their daughter neurons in the developing neocortex. Science 364(6440). PubMed ID: 31073041

Tseng, C. Y., Burel, M., Cammer, M., Harsh, S., Flaherty, M. S., Baumgartner, S. and Bach, E. A. (2022). chinmo^- mutant spermatogonial stem cells cause mitotic drive by evicting non-mutant neighbors from the niche. Dev Cell 57(1): 80-94.e87. PubMed ID: 34942115

Vladoiu, M. C., El-Hamamy, I., Donovan, L. K., Farooq, H., Holgado, B. L., Sundaravadanam, Y., Ramaswamy, V., Hendrikse, L. D., Kumar, S., Mack, S. C., Lee, J. J. Y., Fong, V., Juraschka, K., Przelicki, D., Michealraj, A., Skowron, P., Luu, B., Suzuki, H., Morrissy, A. S., Cavalli, F. M. G., Garzia, L., Daniels, C., Wu, X., Qazi, M. A., Singh, S. K., Chan, J. A., Marra, M. A., Malkin, D., Dirks, P., Heisler, L., Pugh, T., Ng, K., Notta, F., Thompson, E. M., Kleinman, C. L., Joyner, A. L., Jabado, N., Stein, L. and Taylor, M. D. (2019). Childhood cerebellar tumours mirror conserved fetal transcriptional programs. Nature 572(7767): 67-73. PubMed ID: 31043743

Zhu, S., Lin, S., Kao, C. F., Awasaki, T., Chiang, A. S. and Lee, T. (2006). Gradients of the Drosophila Chinmo BTB-zinc finger protein govern neuronal temporal identity. Cell 127: 409-422. PubMed ID: 17055440

date revised: 11 June 2022

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