seven up
A complex regulatory cascade is required for normal cardiac development, and many aspects of this network are conserved from Drosophila to mammals. In Drosophila, the seven-up (svp) gene, an ortholog of the vertebrate chick ovalbumin upstream promoter transcription factors (COUP-TFI and II), is initially activated in the cardiac mesoderm and is subsequently restricted to cells forming the cardiac inflow tracts. This study investigated svp regulation in the developing cardiac tube. Using bioinformatics, a 1007-bp enhancer of svp was identified which recapitulates its entire expression in the embryonic heart and other mesodermal derivatives; this enhancer is initially activated by the NK homeodomain factor Tinman (Tin) via two conserved Tin binding sites. Mutation of the Tin binding sites significantly reduces enhancer activity both during normal development and in response to ectopic Tin. This is the first identification of an enhancer for the complex svp gene, demonstrating the effectiveness of bioinformatics tools in assisting in unraveling transcriptional regulatory networks. These studies define a critical component of the svp regulatory cascade and place gene regulatory events in direct apposition to the formation of critical cardiac structures (Ryan, 2007).
In order to identify the svp cardiac enhancer (SCE), an initial goal was to identify a transcription factor whose function was required for svp expression in the dorsal vessel. Published data demonstrated that tinman (tin) and seven-up (svp) are co-expressed at stage 11, and become mutually exclusive shortly thereafter. Given the temporal and spatial coincidence of tin and svp expression during early cardiogenesis, and given the role that Tin plays in the expression of other important cardiac genes, it was hypothesized that tin function might be required for svp expression. Thus, Svp accumulation was evaluated in heterozygotes and homozygotes for the tin null allele tinEC40. svp expression was monitored in this experiment using the svp-lacZ enhancer trap line. The normal complement of seven bilateral pairs of Svp cells was visible in heterozygous embryos, whereas homozygous tinEC40 sibling embryos lacked cardiac svp expression, although Svp was still present in the ring gland. This result supported Tin as an upstream regulator of svp, however, whether Tin itself bound to the svp enhancer was to be determined. The possibility was considered that svp expression was not initiated in tin mutants simply because cardiac specification had failed to occur. However, since svp expression is initiated relatively early during cardiac specification, it was felt that Tin would still be a strong candidate activator (Ryan, 2007).
Given the dependence of svp expression on the presence of Tin, the svp gene was examined for consensus Tin binding sites. Seven sites, all located within the very large first intron of the svp-RC transcript variant, were identified. Since Tin targets have been shown to contain two closely apposed Tin binding sites, focus was placed upon the genomic regions where at least two sites lay within 300 bp of each other. Six of these putative Tin binding sites were present as three such pairs (Ryan, 2007).
It was then determined if any of the three genomic regions containing putative Tin binding sites were conserved in other Drosophila species. Only one region, encompassing the Tin sites at 8092711/8092853, showed strong sequence similarity between species. When compared with five additional Drosophila species, the putative Tin binding sites within this region were 100% conserved. To determine if Tin protein can bind to the putative Tin sites, an electrophoretic mobility shift assay, using Tin protein and radioactively labeled double-stranded oligonucleotides corresponding to each of the two putative Tin sites was performed. Tin protein, generated in vitro, bound to both of the radioactive DNA sequences, more strongly in the case of the Tin 1 site. Binding was effectively competed with identical respective unlabeled sequences, but not with respective mutated sequences. The higher affinity of the Tin1 binding was further reflected in the corresponding competition assay, in which a light band was still evident when competed with wild-type probe. These results further supported the notion that Tin might directly regulate svp gene expression via this genomic region (Ryan, 2007).
This study showd that Tin is an essential regulator of svp gene expression in the cardiac mesoderm, via activation of a an ~1 kb enhancer (termed the SCE) located in the first intron of the svp gene. Thus, it appears that Tin mediates this 'cardiac context' (Ryan, 2007).
The role of Tin in the initial activation of svp reflects the critical role tin plays in cardiac development in Drosophila. tin function is essential for cardiac specification, and a number of genes expressed in the dorsal vessel have been identified as direct transcriptional targets of Tin. It is anticipated that the SCE will ultimately provide greater insight into developmental patterning processes, since further analysis of the enhancer should identify how both Hox and Hh signals impact svp gene expression, as a model of how they impact cardiac patterning in general (Ryan, 2007).
Once svp expression is initiated, it soon becomes mutually exclusive with tin expression, and a svp-lacZ enhancer trap line is active in the Svp cells all through development to adulthood. In situ hybridization of SCE-lacZ embryos showed that the enhancer is active during embryogenesis through stage 14, although the activity had waned by stage 16. Thus, the SCE is responsible for the initial activation of cardiac svp gene expression at stages 12 to 14, yet other regulatory sequences mediate subsequent sustained svp expression. Since SCE activity is strong at stage 14, a time at which tin and svp expression do not overlap, it is reasonable to suggest that additional enhancer sequences must contribute to this period of expression. In contrast, mutation of the Tin sites also affects enhancer activity at stage 14, when Tin is absent from Svp cells. How can the integrity of Tin sites be required for enhancer activity at a stage when Tin is no longer present? One possibility is that initial binding of Tin to the enhancer might induce epigenetic changes to the genomic region, which can facilitate subsequent enhancer activity. In support of this notion, Tin has been shown to interact directly with the transcription cofactor P300 (Ryan, 2007).
Previous studies have demonstrated the importance of both the svp gene and its vertebrate ortholog, COUP-TFII, in cardiac development. Each factor is expressed in the cardiac inflow tracts: in the case of Drosophila, the inflow tracts are represented by the ostia, which form from the Svp cells and which require svp function for their formation; in vertebrates, COUP-TFII is expressed in and required for the formation of the atria. While neither upstream regulatory factors nor downstream targets of svp and COUP-TFII have been characterized to date, it is reasonable to speculate that such genes showing conserved functions might also share common upstream regulators. Thus, it is predicted that activation of COUP-TFII in vertebrate atrial cells might be mediated at least in part by NKX2.5, although no studies have directly assessed the expression of COUP-TFII in NKX2.5 mutants. Since this study used the dependence of svp expression upon tin function to predict the location of the SCE, such an approach might also be used to identify the COUP-TFII cardiac enhancer based upon the presence of conserved binding sites for NKX2.5. Given that COUP-TFII lies within a large, gene-poor genomic region in both mice and humans, this approach may facilitate the still significant task of illuminating the genetic regulation of COUP-TFII (Ryan, 2007).
Lozenge was initially identified by a mutation caused by a P-element in the X chromosome. Because the P-element contained two copies of the sevenless enhancer, DNA adjacent to the site of insertion was expressed in cells normally expressing sevenless (R7, the R3/R4 pair and cone cell precursors). The P-element caused a dominant mutant phenotype resembling loss-of-function mutations of seven-up. Consequently the dominant mutant was called Sprite. In Sprite/+ heterozygotes, 72% of the ommatidia show transformation of R4 into an R7 cell, and in 10% of ommatidia, both R3 and R4 become converted. The phenotype is more extreme in Sprite mutant homozygotes. One explanation for the mutant phenotype caused by the insertion was that the adjacent DNA coded for a protein which repressed seven-up. A null mutation was used to test whether lozenge regulates seven-up. Whereas svp is normally expressed in the R1/R6 pair and the R3/R4 pair, in flies that are lozenge mutants, svp is expressed in R7 and the four cone cell precursors as well. It is concluded that LZ negatively regulates svp in R7 cells, and in cone cell. In the absence of lozenge each of these cells develop a R7 fate. This transformation is shown to be partially dependent on the functioning of sevenless (Daga, 1996).
lozenge mutants do not express the two Bar genes, and the enhancer-trap O32 (associated with an unknown gene specific to cells R3/4 and R7) is expressed in too many cells. Thus the defective recruitment that occurs in lozenge mutants can be attributed to abnormalities in the expression of genes like Bar, the gene marked by O32, and seven-up, which are essential for establishing the correct cell fate for the final three photoreceptor cells, R1, R6 and R7. seven-up is derepressed in R7 cells in lozenge mutants. The derepression of seven-up is reminiscent of the derepression of svp in rough mutants. rough normally represses svp in R3/R4. Thus Lozenge both actively represses some genes and activates others (Crew, 1997).
The fat body precursors are present in serpent mutants. However, the cells do not proliferate and do not rearrange to form the continuous sheet of cells observed in wild-type embryos at late stage 11. Furthermore, the early events of fat body differentiation do not take place in srp mutants. Expression of seven-up, important in fat body development, is not initiated in the mesoderm of srp mutants (Rehorn, 1997).
Signaling from the EGF receptor can trigger the
differentiation of a wide variety of cell types in many
animal species. The mechanisms that
generate this diversity have been explored using the Drosophila peripheral
nervous system. In this context, Spitz ligand can
induce two alternative cell fates from the dorsolateral
ectoderm: chordotonal sensory organs and non-neural
oenocytes. The overall number of both cell
types that are induced is controlled by the degree of Egfr
signaling. In addition, the spalt gene is identified as a
critical component of the oenocyte/chordotonal fate switch.
Genetic and expression analyses indicate that the Sal zinc-finger
protein promotes oenocyte formation and suppresses
chordotonal organ induction by acting both downstream of
and in parallel to the Egfr. To explain these findings, a prime-and-respond model is proposed. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to
raise the apparent threshold for induction by Spi.
Subsequently, sal-dependent Sal upregulation is triggered
as part of the oenocyte-specific Egfr response. Thus, a
combination of Sal in the responding nucleus and
increased Spi ligand production sets the binary cell-fate
switch in favour of oenocytes. Together, these studies help
to explain how one generic signaling pathway can trigger
the differentiation of two distinct cell types (Elstob, 2001).
The larval oenocytes of Drosophila are conspicuous secretory
cells of ectodermal origin. They are arranged in clusters of, on average, 6 cells
per abdominal hemisegment, occupying a characteristic
lateral and subepidermal location. In contrast to the invariant
peripheral nervous system, the number of cells in each larval
oenocyte cluster can vary between 4 and 9. Using many
different molecular markers, the development
of larval oenocytes has been followed from the
third larval instar back to the extended germ band stage of
embryogenesis. Developing oenocytes express four genes
from very early stages, all of which encode DNA-binding
proteins. These are seven up, pointed, spalt
and ventral veins lacking which produce proteins of the nuclear receptor, ETS-domain, zinc-finger and POU-homeodomain class, respectively (Elstob, 2001).
In the late embryo, immunolabelling experiments were carried
out with two independent oenocyte markers: svp-lacZ, an
enhancer trap into the svp gene and BO-lacZ, a regulatory
construct containing an oenocyte-specific enhancer from the
sal complex. Using these markers,
in conjunction with the sensory neuronal marker anti-Futsch/
22C10, it can be seen that each cluster of oenocytes is closely associated
with an array of five lateral chordotonal organs, termed an Lch5. In each abdominal hemisegment, there are eight chordotonal organs that are partitioned into arrays
consisting of one dorsolateral (V'ch1), five lateral (Lch5) and
two ventral (VchAB) organs. The close apposition of mature oenocyte clusters and Lch5 arrays in late embryos suggests that their formation might be
linked in some way. In order to investigate this possibility, the spatial relationship between the precursors of both cell types were examined in early embryos. Each chordotonal organ is formed by a single chordotonal
organ precursor (COP) that divides asymmetrically to produce
four cells, a sensory neuron, scolopale, ligament and cap cell. The progeny of the most dorsal COP (C1) constitute the most anterior chordotonal organ of the lateral
cluster (Lch5a). A rho-lacZ insertion that is specific for Lch5a and its precursor COP was used, together with anti-Sal, to follow the development of C1 and oenocytes simultaneously. By stage 10, C1 has delaminated and does not
express Sal, despite lying directly underneath a dorsal domain
of Sal-positive ectoderm (termed the dorsal Sal domain). By stage 11, C1 has already divided and its progeny are surrounded by a whorl of
sickle-shaped nuclei expressing higher levels of Sal than
surrounding cells. The whorl structure always
appears in a dorsal and posterior segmental position, close to
the ventral limit of the Sal domain, and corresponds to oenocyte precursors in the process of delamination. In addition to high levels of Sal, the oenocyte precursor whorl also expresses svp-lacZ and vvl. Since only one oenocyte precursor whorl per hemisegment is seen, and this surrounds C1, it is concluded that more ventral COPs are not associated with the formation of oenocytes (Elstob, 2001).
A prime-and-respond model is presented to integrate the dual roles of sal downstream and also in parallel to the Egfr. In this model, sal functions in the parallel pathway as a competence switch. Thus, Sal prepatterns the dorsal
ectoderm so that, on receipt of the Egf signal, oenocytes rather than COPs are induced. One consequence of the Sal oenocyte prepattern is to increase the apparent induction threshold in responding cells. This makes the prediction that the signaling cell inducing oenocytes needs to express more ligand than those that recruit secondary COPs, and this is indeed the case. C1 is known to express high levels of rho for longer than any of the other primary COPs. Thus, the Egf pathway does contribute to the cell-type specificity of the induction event in the sense that more Spi ligand is required to induce oenocytes than to recruit
chordotonal organs (Elstob, 2001).
In the prime-and-respond model, it is implicit that the early
and late phases of sal expression produce distinct effects on
the responding cell. As the levels of Sal are different in the
two phases, it may be that there are at least two different
concentration-dependent effects for this transcription factor. In
agreement with this, it has been shown that strong expression of
the sal target gene, svp, correlates with the domain of sal
upregulation and not with the lower-level prepattern. In another
system, wing vein development, there is a very extreme
example of a concentration difference, with low and high levels
of Sal producing completely opposite transcriptional effects
on the knirps target gene (Elstob, 2001 and references therein).
According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial
cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited
ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three
developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions
where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker
genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus,
indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).
In contrast to ato broad expression just anterior to the
furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes
evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential
for uncommitted cells to acquire ommatidial cell fate or the
ability to express ommatidial marker genes. Consistent with
this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least
for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).
In the eye developmental field posterior to the morphogenetic
furrow, three ommatidial cell marker genes, ro, svp,
and pros, were found to be misexpressed zonally in many
uncommitted cells along the A/P axis when Ras1 was transiently
but ubiquitously activated. The Ras1-dependent misexpression regions appear to correspond to those regions where expression of these genes is normally
initiated, indicating that Ras1-dependent zonal marker gene
misexpression in presumptive uncommitted cells may
reflect directly their developmental potentials.
Subsequent to Ras1 activation, posterior ro misexpression
is restricted only to the vicinity of the furrow. Wild type cells just posterior to the morphogenetic furrow may thus possess developmental potential for
ro expression. Misexpression of svp and pros, subsequent to
Ras1 activation, is strongly present from row 2 to row 6
and from row 4 to row 9, respectively, causing the region strongly misexpressing svp partially to overlap that of pros. The posterior half of the svp expression region overlaps the anterior half of the pros
expression region, but this does not necessarily
mean that cells in the overlapped region (cells in or near
rows 4-6) possess two different developmental potentials at
the same time for svp and pros expression. Indeed, optical
section analysis has indicated that svp and pros expression
occurs mutually exclusively (Hayashi, 2001).
In contrast to Ras1 activation in the present study, differentiating
wild type cells may receive Ras1 activation signals
reiteratively or for a relatively long period, this possibly
being essential in order to exclude any ambiguities in developmental
potential. Preliminary inspection indicates that in
the overlapping region, svp- but not pros-misexpressing cells
strongly tend to be localized near differentiating ommatidia,
which may secrete short-ranged Spitz or Ras1 activation
signals. This may suggest that the threshold of Ras1 signal
for svp expression is higher than that for pros expression. In
addition to the Ras1 signal, the involvement of the Notch (N) signal also has to be considered. N has been shown to play important roles in ommatidial cell fate determination. Therefore, although developmental potential appears to be essential for cell fate specification, it is not the only mechanism used and the strength or
duration of Ras1 signal and the pattern of N signal activation
may also be important for making a perfect ommatidium (Hayashi, 2001).
This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow.
Uncommitted cells just posterior to the morphogenetic
furrow are presumed to acquire ro expression potential at the earliest
stage of the model (stage 1). In stage 2, R3/R4 precursors expressing ro acquire svp
expression potential. svp expression in wild type
R3/R4 precursors along with Ras1 activation-dependent svp
misexpression in uncommitted cells is assumed to be not
only positively regulated by the concerted action of Ras1
signaling and Dac and Eya but also negatively regulated
by the protein product of the prepattern gene, lz.
R1/R6 photoreceptors are recruited into ommatidia between
stages 2 and 3. R1/R6 fate is previously shown specified by dual Bar homeobox genes, BarH1 and BarH2, whose expression is positively regulated by the cell-autonomous function of lz and svp. Consistent with this, in the putative R1/R6 arising area (around row 6), considerable svp expression occurs even in the presence of Lz. svp expression
is regulated by Dac and Eya, so that normal Bar expression
or R1/R6 fate eventually comes under the control of
putative eye prepattern genes Lz, Dac, and Eya (Hayashi, 2001).
In stage 3, which may correspond to R7 and cone cell
formation stages, pros is positively regulated through the
concerted action of Ras1 signaling and prepattern gene lz (Hayashi, 2001).
dac and eya are expressed and may serve as eye prepattern
genes in the region anterior to the morphogenetic
furrow. This is supported by the finding that ommatidial
marker gene misexpression subsequent to Ras1 activation
occurs only within the Dac/Eya expression domain. pros expression is considerably restricted but ro and svp misexpression is evident throughout the entire Dac/Eya expression domain. Considerably strong misexpression
of ELAV, a neuron-specific antigen, is also apparent anterior to the furrow. Thus, as with posterior cells, those anterior to the morphogenetic
furrow are capable of developing into photoreceptors or
ommatidial cells with receipt of Ras1 signals. However,
no apparent regularity in ommatidial marker gene expression
can be detected in the region anterior to the furrow, indicating that developmental potential of anterior cells is necessarily reset to some extent before the onset of normal eye development at the morphogenetic
furrow or before first receiving Spitz signals from nascent R8 (Hayashi, 2001).
ro expressed along the morphogenetic furrow may be
involved in this process, in that svp expression near the morphogenetic furrow is significantly repressed by ro expression along the morphogenetic
furrow. Furthermore, ro has also been shown to repress ato
expression near the furrow (Hayashi, 2001).
In the developing Drosophila eye, differentiation of undetermined
cells is triggered by Ras1 activation but their ultimate
fate is determined by individual developmental
potential. Presently available data suggest that developmental
potential is important in the neurogenesis of vertebrates
and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along
the dorso-ventral axis in response to graded Sonic Hedgehog
signals and this presages their future fates. Subdivision of originally
equivalent neural progenitors through the action of
prepattern genes may accordingly be a general strategy by
which diversified cell types are produced through neurogenesis (Hayashi, 2001).
Cell proliferation in the developing renal tubules of Drosophila is strikingly patterned, occurring in two phases to
generate a consistent number of tubule cells. The later phase of cell division is promoted by EGF receptor signaling
from a specialized subset of tubule cells, the tip cells, which express the protease Rhomboid and are thus able to
secrete the EGF ligand, Spitz. The response to EGF signaling, and in consequence cell division, is patterned by the specification of a second cell type in the tubules. These cells are primed to respond to EGF signaling
by the transcription of two pathway effectors, PointedP2, which is phosphorylated on pathway activation, and Seven up. While expression of
pointedP2 is induced by Wingless signaling, seven up is initiated in a subset of the PointedP2 cells through the activity of the proneural genes. Both signaling and responsive cells are set aside in each tubule primordium from a proneural gene-expressing cluster of cells, in a
two-step process: (1) a proneural cluster develops within the domain of Wingless-activated, pointedP2-expressing cells to initiate the co-expression
of seven up; (2) lateral inhibition, mediated by the neurogenic genes, acts within this cluster of cells to segregate the tip cell precursor, in which
proneural gene expression strengthens to initiate rhomboid expression. As a consequence, when the precursor cell divides, both daughters secrete
Spitz and become signaling cells. Establishing domains of cells competent to transduce the EGF signal and divide ensures a rapid and reliable
response to mitogenic signaling in the tubules and also imposes a limit on the extent of cell division, thus preventing tubule hyperplasia (Sudarsan, 2002).
To understand how the proneural and neurogenic genes pattern the response to EGFR activation, the expression of genes involved in transduction of the pathway was analyzed. The orphan nuclear-receptor svp functions downstream of the EGF receptor to promote cell divisions in the tubules. In the absence of Svp function, cycE and stg transcription is abolished, with a consequent reduction in EGFR-driven cell divisions. These late divisions in the tubules of stage 12 wild-type embryos were followed and it was found that BrdU incorporation (and hence, cell division) is confined within the svp-lacZ domain. These results define the svp domain of expression as including those cells which will divide in response to Egfr activation. However, the expression of svp-lacZ is initiated in a group of cells surrounding the tip mother cell, before the birth of the TC. This early onset of svp expression occurs before the late divisions start (cycle 17 onwards), when neither Svp function nor Egfr activation is required for cell proliferation. The pattern of gene expression observed suggests that the Svp-positive cells surrounding the tip mother cell derive from the proneural cluster (Sudarsan, 2002).
To test this hypothesis, the expression of svp-lacZ in embryos lacking proneural gene function was examined. Indeed, in AS-C-/- embryos, the expression of svp is not initiated in the tubules. Conversely, in N mutants, where all cells in the cluster adopt the primary tip cell (TC) fate, the expression of svp is confined to the transformed cells. After the initiation of Spi signaling from the TC/SC, svp expression depends on Egf receptor activation. However that the early expression of svp is not dependent on Egfr function is shown in topCO mutants, where svp-lacZ expression is still initiated normally, but is not maintained. In AS-C–/– embryos expressing lambdatop in the tubules, svp expression is not detected. Together these data suggest that the initiation of svp depends on the proneural genes but is independent of Egf receptor signaling, which acts only from cycle 17 to maintain svp expression (Sudarsan, 2002).
These results show that the expression of proneural genes in the tubules not only confers tip cell potential but also initiates the expression of an effector of the Egf pathway, svp. It is suggested that this primes cells to divide in response to EGF receptor activation. Proneural genes are therefore required to specify two cell fates in the tubule proneural clusters (PNCs); the tip mother cell and cells competent to respond to Egfr activation (Sudarsan, 2002).
These results suggest that, while the segregation of single cells from an equivalence domain is a unifying theme in the generation of tissues from a wide range of organisms, PNCs in specific tissues have developed an additional function: to establish a second cell fate that cooperates with the first to implement the subsequent program of tissue differentiation (Sudarsan, 2002).
The Drosophila larval cardiac tube is composed of 104 cardiomyocytes that exhibit genetic and functional diversity. The tube is divided into the aorta and the heart proper that encompass the anterior and posterior parts of the tube, respectively. Differentiation into aorta and heart cardiomyocytes takes place during embryogenesis. Living embryos have been observed to correlate
morphological changes occurring during the late phases of cardiogenesis with the acquisition of organ function, including functional inlets, or ostiae. Cardiac cell diversity originates in response to two types of spatial information such that cells differentiate according to their position, both within a segment and along the anteroposterior axis. Axial patterning is controlled by homeotic genes of the Bithorax Complex (BXC) that are regionally expressed within the cardiac tube in non-overlapping domains. The segmentally repeated expression of svp is regulated by a positive inductive effect of Hh secreted by cells from the overlying ectoderm. A role for hh signaling in Drosophila cardiogenesis has not previously been acknowledged. It has been observed that in hh mutant embryos heart progenitors are lacking, however this has been interpreted to be an indirect influence of hh upon wg signaling. The results reported here strongly favor the idea that hh has a direct and positive effect on the determination and specification of the sub-population of cardioblasts that expresses svp. It has been proposed that each segment of the trunk is sub-divided into two domains, A and P. The cells from the anterior domain (A domain) of the dorsal mesoderm would be directed towards a cardiogenic fate while cells from the posterior domain (P domain) would adopt a visceral mesoderm fate. The wg and hh signals released, respectively, from the anterior and posterior compartments of the ectodermal parasegments have been proposed to be the determinants in specification of the two domains. The observations in this study, however, provide strong evidence that a subtype of cardiac cells can originate from the mesodermal P domain. The P domain origin of some cardioblast progenitors has been suggested by the presence at stage 11-12, within the P domains, of bkh-expressing cells, which contribute to the cardiac epithelium later in development. It seems, therefore, that there is not a perfect superimposition between A domains within mesodermal segments and the capacity of the cardiac cells to be integrated into the cardiac tube (Ponzielli, 2002).
The hh signal secreted by cells belonging to the posterior compartments of the segmented ectoderm is sufficient to promote svp expression. The Hh morphogen needs to be secreted and to freely diffuse from the ectoderm to the underlying mesoderm, as judged from the loss of svp expression in the cardioblasts when a membrane-bound form of Hh is expressed in the same genetic background in place of endogenous Hh. The existence of a specific mechanism to constrain diffusion of the secreted morphogen to the cardioblasts of the P-domain can thus be postulated. Further investigation of this mechanism will provide insight into how specificity of morphogen signaling is achieved across embryonic germ layers (Ponzielli, 2002).
Based on gene expression patterns, Hh signaling is likely to be instrumental in the specification of tin- and svp-cardioblasts by inducing the expression of svp in cardioblasts which, in turn, leads to the repression of tin. Such a repressive action of svp has already been reported, although a direct interaction between tin regulatory sequences and svp has not been demonstrated (Ponzielli, 2002).
Similar relationships between homologs of hh, svp and tin have been described in vertebrate cardiogenesis. A homolog of svp, COUP-TFII is expressed in the posterior region of the mouse primitive heart tube where it is required for heart development; furthermore the expression of COUP-TFII is induced by Sonic hedgehog. Shh (and Indian hedgehog) participates in mouse cardiac morphogenesis but, in contrast to the situation in Drosophila, induces rather than represses the expression of the tin homolog, NKx2.5. It must be concluded, from these remarks, that the genetic networks can be differently interpreted and utilized in invertebrates and vertebrates. Further studies should give better insights into the conservation of the genetic programs at work in heart development (Ponzielli, 2002).
The establishment of planar cell polarity in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it
as R4. The spalt zinc-finger transcription factors
(spalt major and spalt-related) are part of the molecular
mechanisms regulating R3/R4 specification and planar cell polarity
establishment. In mosaic analysis, spalt genes have been shown to be
specifically required in R3 for the establishment of correct ommatidial
polarity. In addition, spalt genes are required for
proper localization of Flamingo in the equatorial side of R3 and R4, and for
the upregulation of Delta in R3. These requirements are very similar
to those of frizzled during R3/R4 specification. spalt genes are required cell-autonomously for the expression of
seven-up in R3 and R4, and seven-up is downstream of
spalt genes in the genetic hierarchy of R3/R4 specification. Thus,
spalt and seven-up are necessary for the correct
interpretation of the Frizzled-mediated polarity signal in R3. Finally, it has been
shown that, posterior to row seven, seven-up represses spalt
in R3/R4 in order to maintain the R3/R4 identity and to inhibit the
transformation of these cells to the R7 cell fate (Domingos, 2004).
Several pieces of evidence demonstrate that sal is required
upstream of svp for R3/R4 specification: (1) sal is required
for svp expression in R3/R4; (2) both sal and svp are required in R3 for the establishment of proper ommatidial chirality; (3) in
both sal and svp mutants Fmi is not asymmetrically localized
in R3/R4 and mdelta0.5-lacZ expression is lost in R4, and (4) exogenous expression of svp in R3/R4 (sev-svp) can rescue the
expression of mdelta0.5-lacZ in
sal– clones (Domingos, 2004).
In addition, posterior to row seven, svp is required
to repress sal expression in R3/R4 , and sal is
responsible for the transformation of R3/R4 into R7 in svp mutants. Based on these results, which demonstrate that sal is both necessary
and sufficient for R7 differentiation posterior to row seven, a model for the action of sal and svp during R3/R4
specification: from
rows three to seven, sal is required for svp expression in
R3/R4 and for R3/R4 specification: posterior to rows seven to nine, repression
of sal by svp in R3/R4 is necessary for the maintenance of
R3/R4 identity and the inhibition of R7 fate. This dual regulation between
sal and svp helps to understand the complex
sal- phenotype in R3/R4. Strikingly, although
svp expression is lost in sal- R3/R4, these
cells do not get transformed into R7, but keep an outer PR identity. Thus, in
the absence of sal, the presumptive R3/R4 remain as outer PRs with an
unspecified subtype identity (Domingos, 2004).
In conclusion, these results demonstrate that sal is required in R3
to allow normal Fz/PCP signaling to specify the R3 and R4 cell fates.
Ommatidia mutant for sal show defects that are very similar to those
observed in fz and dsh mutants, as judged by the loss of
asymmetric Fmi localization at the equatorial side of the R3/R4 precursors,
and by the lack of Dl and E(spl)mdelta upregulation within
the R3/R4 pair. In addition, sal is required upstream of svp
for normal R3/R4 specification. Finally, these results show that, posterior to
row seven, svp represses sal in R3/R4 in order to maintain R3/R4 identity and to inhibit transformation of these cells to the R7 cell fate (Domingos, 2004).
Many different intercellular signaling pathways are known but, for most, it is unclear whether they can generate oscillating cell behaviors. Time-lapse analysis of Drosophila embryogenesis has been used to show that oenocytes delaminate from the ectoderm in discrete bursts of three. This pulsatile process has a 1 hour period, occurs without cell division, and requires a localized EGF receptor (EGFR) response. High-threshold EGFR targets are sequentially activated in rings of three cells, prefiguring the temporal pattern of delamination. Surprisingly, widespread misexpression of the relevant activating ligand, Spitz, is compatible with robust delamination pulses.
A single chordotonal organ precursor (called C1) and its progeny provide the source of secreted Spi relevant for oenocyte induction.
Although Spitz ligand becomes limiting after only two pulses, artificially prolonging its secretion generates up to six additional cycles, revealing a rhythmic underlying mechanism. These findings illustrate how intercellular signaling and cell movements can generate multiple cycles of a cell behavior, despite individual cells experiencing only one cycle of receptor activation (Brodu, 2004).
The induction of larval oenocytes in Drosophila has been used as a simple model system for investigating the developmental regulation of EGFR signaling. Oenocytes are induced from the dorsal ectoderm of abdominal segments by a fixed and highly restricted source of Spi. This triggers a local EGFR response within a ring of overlying dorsal ectodermal cells, termed a whorl, leading to the upregulation of numerous oenocyte-specification genes and subsequent cell delamination. The simple cell geometry of the oenocyte whorl, together with time-lapse microscopy, was used to explore the timing of Spi secretion, EGFR-target activation, early cell induction, and later cell delamination. These studies reveal that oenocytes delaminate in bursts of three and identify the cell-counting mechanism as an EGFR-dependent pulse generator converting the time window of Spi secretion into final oenocyte number. This represents the first example of a rhythmic cell behavior other than the cell cycle to be reported in the Drosophila embryo (Brodu, 2004).
Using a panel of markers for double- and single-ring stages, it was possible to place gene expression 'snapshots' in temporal order with the cell movements recorded in movies. Three generic EGFR targets (activated Rolled/ERK, Yan, and argos) and three oenocyte-specific EGFR targets (Sal, svplacZ, and svplacZΔ18) were analyzed. In wild-type embryos, the high-threshold EGFR outputs of argos and svplacZ expression, detectable Rolled activation, and strong Yan downregulation are all inner ring specific, whereas lower-threshold outputs such as Sal upregulation and svplacZΔ18 expression are present in both precursor rings. Delamination itself also appears to be a high-threshold EGFR response and is thus confined to the inner ring (Brodu, 2004).
Stage 12 marks the beginning of early fat-cell differentiation. Loss of svp function results in the loss of the terminal fat cell differentiation marker Adh, and a reduction of expression of Dcg1, another fat cell differentiation marker specific to the fat body. SVP plays a role in fat-body-specific expression of at least two terminal fat-cell
differentiation genes. The fat-cell lineage is traceable to nine bilateral clusters of cells
within the mesoderm at germ-band extension. These cells represent the progenitor fat cells. Cell clusters can be identified within the mesoderm internal to nautilus-expressing cells at stage 12. These data suggest that the precursor fat cells may be derived from the inner mesoderm, or spanchnopleura (Hoshizaki, 1994).
The pipsqueak (psq) gene is expressed at high
levels in the R3/R4 precursors during eye development. Such expression depends on seven-up. Strong psq alleles are dominant suppressors of mutant svp induced cone
cell transformation phenotype. The gene is a
member of the maternal posterior group of genes, but the effect of strong semilethal alleles
demonstrate an additional specific requirement for psq function downstream of svp for the development of
photoreceptors R3/R4. Interestingly, all viable alleles with a maternal posterior group phenotype cluster
around one specific 5' exon, while all semilethal alleles have lesions which map to a different
alternative 5' exon (Weber, 1995).
tinman is essential for dorsal vessel (heart) formation and is structurally and functionally conserved in vertebrates. In
the mature embryonic dorsal vessel, tinman is expressed in four of the six pairs of cardioblasts in each segment. Evidence that
seven-up, which is homologous to the vertebrate COUP-TF transcription factor and is expressed in the non-Tinman-expressing cardioblasts,
represses tinman in these cells. Loss of function seven-up mutations derepress tinman expression in these cardioblasts while ectopic
expression of seven-up represses tinman in the cardioblasts that normally express tinman. These changes are correlated with alterations in the
expression of additional molecular markers for each of these two types of cardioblasts, such as the novel T-box-containing gene Tb66F2 and
the potassium channel-encoding gene sur. These observations suggest that seven-up has a role in diversifying cardioblast identities within
each segment. The tinman cis sequences that mediate tinman repression by seven-up are described and whether Seven-up can
bind these sequences to directly inhibit tinman was examined. It seems that neither Svp isoform is capable of directly and
specifically binding to the 3' half of the tinC element that
confers segmental repression of tinman. It is still possible that Seven-up
could bind the tinC element in combination with one or
more other proteins in order to repress tinman, or Seven-up may indirectly repress tinman in svp cardioblasts through the regulation of downstream genes that affect tinman expression (Lo, 2001).
In late embryonic dorsal vessels, ß-Gal expression from ß-Gal is seen in two adjacent pairs of the six bilaterally symmetrical pairs of cardioblasts per segment. At these stages, Tinman is present in only four of
the six contiguous pairs of cardioblasts per segment, and
double-staining for ß-Gal and Tinman demonstrate that
the svp-lacZ-expressing cardioblasts correspond to the two
pairs of non-tinman-expressing cardioblasts. In order to extend this observation and better characterize seven-up expression in the entire dorsal vessel and its relationship to tinman expression, the pattern of ß-Gal and Tinman staining during the development of this
organ was examined in embryos from the svp-lacZ line
AE127. Tinman protein expression during early dorsal
vessel development (stage 11) includes all the heart progenitors, whereas in later stages it becomes restricted to the four pairs of cardioblasts per segment and to a subset of pericardical cells referred to as the tin pericardial cells. In AE127 embryos, the earliest detected expression of ß-
Gal in the Tinman-expressing heart progenitors is at mid-
stage 11 in a small subset of these cells, when it appears that
seven-up is simultaneously expressed in at least two heart
progenitor cells in each hemisegment. These cells are irregularly arranged and are first seen in the posterior half of the embryo. By early stage 12, a cluster of four strongly ß-Gal-expressing heart progenitors per hemisegment is seen in seven posterior segments of the embryo, with an additional pair of these cells situated caudally to these clusters. This pattern is maintained from stage 13 until stage 15, when strong ß-Gal expression is observed in two adjacent pairs of cardioblasts in seven segments, plus another single pair located immediately posterior to the last double pair of cardioblasts. The ß-Gal and Tinman double-staining of these late stage dorsal vessels clearly shows that the two pairs of seven-up-expressing cardioblasts correspond to the two pairs of cardioblasts per segment that are not expressing Tinman, such that the two patterns of expression are complementary. The late stage cardioblasts that exclusively express either tinman or svp-lacZ are referred to as the tin and svp cardioblasts, respectively. In addition to the svp cardioblasts, there is also strong expression of svp-lacZ at the anterior end of the dorsal vessel in two bilaterally symmetrical masses of cells that will later fuse at the dorsal midline to form the corpus allatum of the ring gland, an endocrine organ of complex origin (Lo, 2001).
Located laterally to each pair of svp cardioblasts per hemisegment is a pair of cells with much weaker expression of svp-lacZ, which do not express Tinman. Based on their lateral position relative to the cardioblasts and since they appear to arise from dividing svp-lacZ heart progenitors,
these cells are considered to be pericardial cells. Since they
do not express tinman, these are not tin pericardial cells and
thus constitute a novel subtype of pericardial cells. Prior to stage 13, all svp-expressing heart progenitors are
positive for Tinman protein (Lo, 2001).
Molecular characterization of the seven-up gene has identified two different transcripts, svp1 and
svp2, that are derived from this gene. The svp1 cDNA encodes a 543 amino acid protein that is the Drosophila homolog of the vertebrate COUP-TF subfamily of steroid/nuclear hormone receptors and
contains a characteristic N-terminal DNA-binding domain
and C-terminal ligand-binding domain. The svp2 cDNA
encodes a related 746 amino acid protein that is identical
in sequence to the svp1-encoded protein until it completely
diverges in the middle of the ligand-binding domain. Since
the pattern of ß-Gal expression in the AE127 svp-lacZ line
does not differentiate between these two transcripts and may
not reflect endogenous mRNA patterns in the heart and its
progenitors, in situ hybridizations were performed using
probes specific for the unique 3' UTR of each cDNA.
The svp1 in situ hybridization pattern in the dorsal vessel
during its development appears to be identical to the ß-Gal
expression observed in AE127 embryos. svp1 message is
first detected cytoplasmically in a portion of the heart
progenitors in stage 11 embryos, and in later stages the pattern of expression in the non-Tinman-expressing cardioblasts and the corpus allatum is
the same as observed with svp-lacZ.
However, while the pattern of dorsal vessel cells expressing
svp2 during embryogenesis appears to be identical to svp1,
the staining is not cytoplasmic but is instead concentrated in
one or occasionally two speckles per nucleus of those cells.
In a stage 11 embryo, the svp2 transcript is clearly seen as
speckles in the nuclei of a subset of heart progenitors. This speckled intranuclear localization is also seen in
several other tissues expressing the svp2 transcript, e.g. the
dorsal somatic muscles, but not in other tissues such as the
CNS, where it is cytoplasmic, indicating that the intracellular localization of the svp2 transcript is tissue-specific. In the dorsal vessel of stage 15 or older embryos, a pair of adjacent speckles is observed in
between the four tin cardioblasts of each hemisegment, with
each speckle associated with one svp cardioblast.
In addition, there is svp2 expression in the corpus allatum,
seen as two larger clusters of speckles at the anterior end of the dorsal vessel (Lo, 2001).
While the pattern of expression of both svp1 and svp2 in
the svp cardioblasts and ring gland is the same as that seen
with svp-lacZ, expression of neither transcript was observed
in the pericardial cells that were seen to weakly stain for ß-Gal in AE127 embryos. Thus it is proposed that the pericardial cells do not actively express svp and the weak ß-Gal staining seen in the AE127 pericardial cells may be due to the perdurance of ß-Gal originally expressed in the heart progenitor cells from which the pericardial cells are derived (Lo, 2001).
These studies have identified a novel function of seven-up in
determining one of two major cell fates in the cardioblasts, as
defined by expression of Tb66F2 versus tinman, through
repression of the alternative fate. As in the developing retina,
seven-up is expressed in a subset of cells in the dorsal vessel,
specifically, these cells are the double pairs of non-Tinman-expressing cardioblasts in each segment of the dorsal vessel.
The possibility that these two cardioblast types are functionally different in the mature embryonic heart is suggested first
by the exclusive expression in the tin cardioblasts of the sur
gene, which codes for the Drosophila homolog of the sulfonylurea receptor subunit of the vertebrate ATP-sensitive potassium ion channel. While the vertebrate sulfonylurea receptor has no intrinsic potassium ion channel activity, the Drosophila sur gene has additional sequences not present in the vertebrate SUR genes that endow it with this activity. The presence of the sur gene product in tin cardioblasts could conceivably result in a difference in the electrophysiological properties of these cardioblasts relative
to the svp cardioblasts, perhaps in the generation, propagation, and/or control of heartbeat in the dorsal vessel, since it is known that potassium ions are required for proper heartbeat function in Drosophila (Lo, 2001).
Two other differences of the svp cardioblasts from the tin
cardioblasts have previously been noted in a study of the embryonic dorsal vessel; the present study utilized as a marker the P-lacZ insertion line E2-3-9 that
has recently been identified as a svp-lacZ line. The first difference is that the svp cardioblasts are the cardioblasts initially contacted by the alary muscle cells; this is consistent with the alignment of these cells
with the alary muscles in mature embryonic dorsal vessels
as seen in the study. However, this morphological feature
is not visibly disrupted in svp mutant embryos. The
second difference is that in larval stages, these cardioblasts
still maintain their compact and rounded shape while the
other cardioblasts become larger and flattened. It has been
speculated that these svp cardioblasts may be involved in
the formation of the ostia -- segmentally repeated openings present in the larval and adult heart that have a valve-like function in allowing the inflow of
hemolymph into the heart during diastole. These differences
between the tin and svp cardioblasts suggest that the proper
specification of these two different cardioblast cell fates during embryogenesis may be crucial for correct dorsal vessel function (Lo, 2001).
MADS-box gene MEF2 is expressed in all cardioblasts. Because expression of mef2 in all cardioblasts has been shown to depend on tin,
it may seem paradoxical that removal of tin from cardioblasts upon ectopic expression of svp does not cause any loss of mef2 expression in these cells. However, these results can be reconciled in light of the analysis of
sequences controlling mef2 expression in the dorsal vessel; they
show that mef2 is differentially regulated in cardioblasts. In particular, one enhancer element has been defined that directs expression of mef2 specifically in the
four pairs of Tinman-expressing cardioblasts in each
segment, while another drives mef2 expression only in the svp cardioblasts. Consequently, mef2 expression in the presence of
ectopic svp in the entire dorsal vessel probably reflects the
situation which normally occurs in the two pairs of svp
cardioblasts per segment, i.e. it depends on svp (or a svp
downstream gene) but not on tin. The observed complete
absence of cardioblast-specific mef2 expression in tin
mutants is presumably due to an earlier activity of tin,
when it is required to promote the formation of all cardioblasts as well as for svp and late tin expression in subsets of these cells (Lo, 2001).
Recent studies on the svp cardioblasts have determined
that they are derived by asymmetric cell divisions of two
heart progenitor cells, each of which gives rise to a svp
cardioblast and a sibling pericardial cell which expresses
svp-lacZ and odd-skipped. Mutations in genes that control asymmetric cell divisions such as numb and sanpodo affect the number of svp cardioblasts and Odd pericardial cells. It appears that after the svp-expressing
heart progenitor divides asymmetrically, the svp cardioblast
daughter cell continues to express seven-up while its pericardial sibling down-regulates it and initiates odd-skipped expression (Lo, 2001).
Seven-up is a member of the steroid/nuclear receptor
superfamily of proteins and is most related to the vertebrate
COUP-TF family of orphan nuclear receptors. The members of this family are most homologous
in the DNA- and ligand-binding domains characteristic of
all steroid/nuclear receptors. In the case of Seven-up
(derived from the svp1 transcript), it is roughly 95 and
90% homologous to the DNA- and ligand-binding domains,
respectively, of the prototypical human COUP-TFI/Ear3
protein. The COUP-TFs have been
shown to regulate the transcription of a wide range of genes,
and generally act as transcriptional repressors. This repression occurs by several mechanisms, including competition
for binding to hormone response elements as homodimers or
heterodimers with other nuclear receptors, by sequestering
the retinoid X receptor (RXR), which is the universal heterodimeric binding partner for several other nuclear receptors,
and by recruiting corepressors such as SMRT or N-CoR to
actively silence transcription (Lo, 2001).
Drosophila neuronal stem cell neuroblasts (NB) constantly change character upon
division, to produce a different type of progeny at the next division.
Transcription factors Hunchback (Hb), Kruppel (Kr), Pdm, and Castor are
expressed sequentially in each NB and act as determinants of birth-order
identity. How any NB switches its expression profile from one transcription factor
to the next is poorly understood. The Hb-to-Kr switch is directed
by the nuclear receptor Seven-up (Svp). Svp expression is confined to a
temporally restricted subsection within the NB's lineage. Loss of Svp function
causes an increase in the number of Hb-positive cells within several NB
lineages, whereas misexpression of svp leads to the loss of these early-born
neurons. Lineage analysis provides evidence that svp is required to switch off HB at the proper time. Thus, svp modifies the self-renewal stem cell program to allow chronological change of cell fates, thereby generating neuronal diversity (Kanai, 2005).
The expression profile of Svp in the CNS is extremely dynamic. For example, at stage 11, Svp is expressed in NB2-4 but not in NB7-3 just after NB2-4
formation. After NB7-3 has divided, Svp is expressed in NB7-3
and in the GMC that it has generated but Svp is no longer detectable in NB2-4.
Thus, the expression of Svp is confined to temporally restricted
subsections of the NB lineage. While Svp is expressed in many NB and GMCs, only
a small number of neurons are Svp positive. This indicates
that, unlike Hb and Kr, the expression profile of Svp in the NBs is not
maintained in their neuronal progeny (Kanai, 2005).
In the svp mutant, NB7-3 does not switch
its expression pattern from the Hb, Kr double-positive state to the Kr
single-positive state until one division after the normal transition period.
This prolonged expression of Hb results in overproduction of Hb-positive
neurons exhibiting characteristics of early-born neurons. The timing of the
expression of Svp protein in NB7-3 coincides with the transition in the
expression of Hb to Kr, and precocious expression of Svp causes the loss of Hb
expression within the lineage. These results indicate that Svp has an
instructive role in determining the period of Hb expression in the NB and the
proper generation of neuronal diversity. While this work places Svp upstream of
Hb, how the expression of Svp itself is regulated is not well understood. In
hb mutant embryos, Svp is still expressed transiently at the time that
NB7-3 produces its first GMC. Thus,
it is unlikely that the temporal delay of Svp expression with respect to Hb is
due to a negative feedback loop in which Hb induces its own repressor (Kanai, 2005).
Svp is a well-conserved nuclear receptor whose human homolog, COUP, has been shown to
act as a transcriptional repressor. Because a reporter gene that
contains only an enhancer element of the hb gene also responded to Svp,
Svp can affect hb expression at the level of its transcription. It is
thus possible that Svp directly represses hb transcription by binding to
its cis-element. Interestingly, misexpression of Svp in postmitotic
neurons does not affect their Hb expression, consistent with the observation that
the regulatory mechanism of Hb expression differs between the NBs and their
progeny. The repressor activity of Svp on hb expression likely requires other factors that are present in precursor cells of neurons (Kanai, 2005).
In svp mutant embryo, augumented expression of Hb was seen in many NBs, resulting in overproduction of early-born neurons in at least three NB lineages. This suggests that Svp may have a common function in many NB lineages regulating hb expression. However, of 30 NBs within each hemisegment, four do not express svp. Indeed, in an
svp-negative NB1-1 lineage, the number of the early-born neurons aCC and
pCC in svp mutant embryo is normal. How do these NBs
generate birth-order-dependent progeny without svp expression? Since some
NBs are known to start their lineage without expressing Hb, they may not need
Svp to regulate Hb expression. Indeed, svp-negative NB6-1,
which expresses Cas at the time of formation never expresses Hb. It
is also possible that there are other factors or mechanisms to regulate
hb expression. In the nematode C. elegans, hb homolog
lin57/hbl-1 (which controls developmental timing as a
heterochronic gene) is regulated by a micro RNA that binds its 3'UTR. Since Drosophila hb 3' UTR contains putative micro RNA binding sites, transcription
factor switching in Drosophila NBs might also be regulated
posttranscriptionally by micro RNAs (Kanai, 2005).
While the overproduction of Hb-positive
neurons is consistent with the idea that prolonged expression of Hb in
svp mutant NBs causes production of supernumerary GMC-1s, examination of
postmitotic neurons reveals that the number of neurons with particular identity
does not always correspond to duplicated GMC-1s. In the NB7-3 lineage, GMC-1
divides to produce two neurons, EW1 and GW, whereas GMC-2 gives rise to EW2
neuron and its sibling which undergoes programmed cell death. In svp
mutant, two EW1 neurons are present consistent with duplicated GMC-1, but only one
GW-like neuron is observed. Likewise, when Hb is misexpressed in the
NB7-3 lineage, not all GMCs that were transformed toward GMC-1 produced GW
neurons. These
data suggest that the fate of postmitotic progeny from GMCs is dependent not
only on the birth-order identity of GMCs determined by transcription factors
such as Hb and Kr, but is also influenced by signals that come from outside of
the NB lineage. Since the decision for the sibling of the EW2 neuron to undergo
cell death depends on the activation of Notch signaling, it is possible that
signals for Notch activation originate outside the NB7-3 lineage, and are not
affected by genetic manipulations altering the birth-order identity of the GMCs (Kanai, 2005).
In addition to the increase in
the number of early born neurons, svp mutant embryos display another
phenotype, the reduction of late-born neurons that express Zfh-2 (NB7-3), Runt,
and Cas (NB7-1). This phenotype is dramatically enhanced when Kr is
inactivated, freezing the lineage such that only Hb-positive cells are
produced. One interpretation of this phenotype is that Svp somehow cooperates
with Kr to generate the late part of the lineage. In fact,
this seems to be the only known genetic situation in which loss of gene function
eliminates the late born neurons. However, because this phenotype is completely
suppressed by removing Hb, the idea is favored that Svp does not have a direct
role in activating the transcription factors that specify the late-born
identity, but rather acts through repressing Hb, which can repress PDM
expression. Thus the apparent requirement of Svp
in the generation of the late-born neurons deduced from the svp
loss-of-function phenotype is due to its primary function in mediating the
Hb-to-Kr switch, whose failure secondarily blocks the initiation of the late
lineage program. The results also show that the late lineage can be produced in
the absence of Hb and Kr (and Svp), suggesting that it may be the
'default' state. It is possible that primitive lineage consisted
only of the late lineage program, to which Svp was recruited to add the early
program involving Hb and Kr, thereby generating the birth-order-dependent
neuronal diversity (Kanai, 2005).
Neural stem cells often generate different cell types in a fixed birth
order as a result of temporal specification of the progenitors. In
Drosophila, the first temporal identity of most neural stem cells
(neuroblasts) in the embryonic ventral nerve cord is specified by the
transient expression of the transcription factor Hunchback. When reaching the
next temporal identity, this expression is switched off in the neuroblasts by
seven up (svp) in a mitosis-dependent manner, but is
maintained in their progeny (ganglion mother cells). svp
mRNA is already expressed in the neuroblasts before this division. After
mitosis, Svp protein accumulates in both cells, but the downregulation of
hunchback (hb) occurs only in the neuroblast. In the
ganglion mother cell, svp is repressed by Prospero, a transcription
factor asymmetrically localised to this cell during mitosis. Thus, the
differential regulation of hb between the neuroblasts and the
ganglion mother cells is achieved by a mechanism that integrates information
created by the asymmetric distribution of a cell-fate determinant upon mitosis
(Prospero) and a transcriptional repressor present in both cells (Seven-up).
Strikingly, although the complete downregulation of hb is mitosis
dependent, the lineage-specific timing of svp upregulation is
not (Mettler, 2006).
The up- and down-regulation of Hb in the NBs is regulated on the transcriptional level, and it is switched off by the activity of Svp, a member of the orphan receptor family of zinc finger transcription factors. However, svp mRNA expression has already started before the NB divides, after which hb expression is terminated. As a result, both progeny, the NB and the GMC, inherit svp mRNA and produce Svp protein, although the GMC continues to express hb. This suggests that one or more GMC-specific factors are able to suppress the Svp-mediated repression of hb within the GMC. Good candidates for such factors are the asymmetrically segregating cell-fate determinants Numb and Prospero (Pros). Both of these proteins form a basal crescent within the NB prior to division, and both are inherited only by the newly formed GMC. Therefore, hb expression was analyzed in loss-of-function alleles of these genes. Although no obvious difference was found in the number of Hb-positive (Hb+) cells in the absence of Numb function, there was a strong reduction in the number of these cells in pros mutant embryos (Mettler, 2006).
pros codes for a homeodomain transcription factor that enters the
nucleus of the GMC after mitosis, subsequently regulating GMC-specific gene
expression. To confirm that the observed
reduction of Hb+ cells is indeed due to a lack of hb
maintenance within the GMCs and their progeny, the timing of
hb expression was compared within different lineages between wild type and the pros loss-of-function alleles prosC7 and
pros17. The lineages of the thoracic NB2-4T
and NB6-4T, as well as the abdominal NB7-3, were analyzed. As in wild type, NB7-3 in both pros alleles is initially Hb+ and generates a
Hb+ GMC (GMCa) after its first division. At early stage
12, the NB is Hb-negative (Hb-) and generates a second GMC (GMCb)
that is Hb- too. At this stage, GMCa maintains hb
expression in wild type, whereas this is reduced or already undetectable in
pros mutants. In stage 14 pros mutant embryos, 100% of the NB7-3
derived cell clusters do not show any Hb+ cells,
whereas there are two cells in wild type, the EW1 and GW neurons. To rule out
that the lack of Hb+ cells in NB7-3 is due to a loss of these cells
by programmed cell death, prosC7 was recombined with the
deficiency H99 to prevent apoptosis. Again,
only Hb- progeny of NB7-3 were found in later stages,
confirming that the phenotype is indeed due to lack of hb
maintenance. Consistent with its role as a repressor of hb, the opposite phenotype is seen in svp mutants: here the NB stays
Hb+ after its first division and produces at least one additional
Hb+ GMC before becoming Hb- (Mettler, 2006).
A similar result was obtained for the thoracic NB6-4T lineage. This NB is
special because its first division produces a glial precursor instead of a GMC
that gives rise to three glial cells. Again,
the glial precursor and its progeny normally maintain hb expression,
whereas it is switched off in the parental NB. As is expected in
pros mutants, the hb expression in the glial cells is not
maintained, whereas there is a considerable delay in switching off
hb in svp mutants. This lack of svp function leads to one additional Hb+ glial cell in 56% of the hemineuromeres. Concomitantly, a reduction is observed of the number of NB6-4T-derived neurons in almost all hemineuromeres in svp mutants (Mettler, 2006).
Because NB7-3 and NB6-4T terminate hb expression after their first
division, it was next asked whether pros is also necessary for
hb maintenance in NBs that produce two Hb+ GMCs. NB7-1
generates such a lineage and it has already been shown that it also produces
additional Hb+ progeny in svp mutant embryos.
Unfortunately this lineage could not be analyzed in pros mutants
because the expression of even skipped (eve), which is
needed as a marker for the detection of the first NB7-1 progeny, is itself
pros dependent. Therefore, NB2-4T, which was also found to be a
neuroblast generating two Hb+ GMCs leading to four Hb+
neurons, was analyzed. In this lineage too, hb expression stays switched on longer in svp-mutant embryos, and as a result there are about five to eight
Hb+ cells in 86% of the analysed thoracic hemineuromeres. In
pros mutants, the hb expression within the NB2-4T lineage
seems initially to be normal, but at stage 14, in about 53%, there are only two
Hb+ neurons detectable. Thus, in all lineages analysed Pros seems to counteract the hb-downregulating activity of Svp (Mettler, 2006).
To test whether Pros is not only necessary but also sufficient for
hb maintenance, use was made of the GAL4/UAS-system to express
pros ectopically within the NBs. engrailed-GAL4 (en-GAL4) was used to drive pros expression within NB7-3 and its progeny. Ectopic Pros caused a precocious stop in cell divisions within this lineage in all hemineuromeres analysed. This was expected, since Pros has been shown to activate dacapo, which subsequently inhibits further mitotic divisions. Nevertheless, in some hemineuromeres three NB7-3-derived cells could be identified. In most of these cases, all three cells were Hb+. This shows that Pros
activity is indeed sufficient for maintaining hb expression, because
one of these cells must be the NB that has divided at least once (Mettler, 2006).
The opposite phenotypes of svp and pros mutants suggest
that hb maintenance in the GMC is due to Pros activity, which
inhibits the repressive function of svp. If this is the case, a
concomitant loss of Pros and Svp function should show a svp-like
phenotype. To test this, a svpe22,
prosC7 double mutant was generated and the embryonic CNS was stained for hb expression. Generally more Hb+ cells were found,
which is similar to the phenotype in svp single mutant embryos. This
was also confirmed on the lineage level: in the NB7-3 derived cluster of stage
14 svp-mutant embryos, there were three or four Hb+ cells
in 75% of the hemineuromeres. This is similar to the double
mutants, which showed this in 67% of the hemineuromeres, thus supporting the
hypothesis that Pros antagonises Svp activity in the GMC. But on which level
does this occur? One possibility is that svp transcription, which is
initiated before mitosis, is suppressed by Pros in the GMC after division.
Alternatively, Pros could suppress the activity of the Svp protein. To
distinguish between these two possibilities, the dynamics of
svp mRNA expression was analysed in the NB7-3 lineage in wild-type and
pros mutant embryos. In both genotypes, svp expression in
the NB starts before its first division and svp
mRNA is still present in the NB after mitosis. However, when svp mRNA expression was examined in GMCa before the NB divides again, a difference was found between the wild-type and pros mutant embryos. In
wild type, 70% of these GMCs were negative for svp mRNA. In contrast to
that, all GMCs examined in pros mutants expressed svp mRNA,
although on a lower level than the NBs did. After the birth of
GMCb, there is detectable svp mRNA in only eight out of 21 cases in
GMCa in wild type, whereas in pros mutants 13 out of 20 are still positive for this transcript. This suggests that Pros might participate in the GMC-specific transcriptional downregulation of svp. However, overexpression of pros could not eliminate svp expression within the NBs (Mettler, 2006).
Interestingly, the observed difference in svp mRNA expression
between wild-type and pros mutant NB7-3 lineages was not seen in the
Svp protein distribution; there was no or only a weak level of Svp protein found in
NB7-3 before division in both genotypes. Likewise, after
division both cells are always Svp+. After the second
neuroblast division, GMCa remains positive for Svp protein in nearly all
cases in wild type, as well as in pros mutant embryos. At
this stage, Hb protein normally has completely vanished from the NB but is
maintained in GMCa despite the presence of Svp protein. Taken together, this
suggests that Pros acts on both a transcriptional and a post-transcriptional
level to downregulate Svp activity in the GMC (Mettler, 2006).
It has been shown that hb downregulation in the NB is mitosis
dependent, because Hb is maintained in string (stg) mutant
NBs, where mitosis is blocked at the G2/M transition. However, in NB7-3, svp mRNA begins to be expressed
prior to the division that leads to hb downregulation. This timing of svp expression seems to be a general feature,
since this is also seen in other lineages. NB6-4T, which generates only one
hb-positive progeny, switches on svp expression before its
first division, whereas NB2-4T and NB7-1, which both generate two
Hb+ GMCs, start svp expression before the Hb+
GMCb is born. This suggests that either there is no
svp mRNA expression in stg mutant NBs, or that the
svp-mediated hb-repressing activity is
post-transcriptionally upregulated after division (Mettler, 2006).
To distinguish between these two possibilities, svp
mRNA expression was analyzed in stg mutant embryos in Eg-positive NBs at different developmental time points. Z normal onset of svp expression was found within NB2-4T and NB7-3, showing that lack of hb-downregulation in
stg mutants in these NBs is not due to a lack of svp
transcription. To test whether the regulation could be on the level of protein
translation, stg mutant embryos were examined for the presence of Svp
protein in the NBs. Indeed, only a low or undetectable amount of this
protein was found in these cells up to early stage 12, suggesting that the translation of the svp mRNA is very low. The reason for this might be the unusual localisation of the svp mRNA: when comparing the distribution of
hb and svp mRNAs, it was realised that almost all of the visible
svp mRNA is localised in the nucleus, whereas the hb mRNA is
enriched in the cytoplasm. This nuclear localisation of the
svp mRNA is also evident in the in situ hybridisation for
svp mRNA combined with the antibody staining for Hb protein in
stg mutant embryos. It is assumed that this localisation might
prevent efficient translation of the Svp protein, which takes place in the
cytoplasm. However, some of the svp mRNA molecules seem to escape
from the nucleus, since a low level of Svp protein was detected in NBs from
around stage 12 onwards. This seems to lead to a reduction of
hb expression because the amount of hb mRNA and protein in
the svp-expressing NBs is lower than in the other cells (Mettler, 2006).
The observation that, in stg mutants, not only NB6-4T and NB7-3
but also NB2-4T expressed svp mRNA was unexpected because, in this
NB, svp mRNA is normally only detectable after the birth of the first
GMC. The generation of this cell is obviously not necessary for svp upregulation because otherwise NB2-4T would remain svp mRNA negative in stg mutant embryos. The same was observed for the En+ NB7-1; although normally becoming svp positive after the birth of its first GMC at the beginning of stage 10, svp expression started at exactly the same time in stg mutants, despite of lack of cell division. Thus, in contrast to hb downregulation, the timing of svp upregulation is mitosis independent in the analysed lineages (Mettler, 2006).
Common to all genes of the temporal specification cascade is the fact that
after division they are downregulated within the NB but remain expressed in
the newly generated GMCs and their progeny. For hb, this downregulation is dependent on Svp, whose mRNA is already
expressed within the neuroblast before the generation of the Hb+
GMC and is symmetrically distributed to both cells after NB division. Why then does svp downregulate hb only within the NB
and not in the GMC? This is due to the activity of Pros,
a homeodomain transcription factor that is asymmetrically distributed only to
the GMC. Earlier work by other groups has suggested that Pros is involved in the regulation of GMC-specific gene activity. In
principle this is also true for the function of pros in the context
of hb regulation, because it inhibits the NB-specific
svp-mediated downregulation of hb. How is this antagonistic
activity of Svp and Pros achieved at the molecular level? In one case, the
data suggest that Pros downregulates svp transcription, because the
svp mRNA in the first GMC of NB7-3 is present longer in
pros mutants than in wild type. In the other, it seems likely
that Pros also inhibits Svp activity, because the Hb+ GMC often
possesses Svp protein even after the parental NB is Hb-. An
attractive model for this would be that Pros neutralises Svp repressor
function by binding to the same regulatory region of hb. In fact, an evolutionarily conserved enrichment of putative Svp-binding sites was found in
the vicinity of a potential Pros-binding site, within a
regulatory region that is necessary for neural hb regulation (J.
Margolis, PhD thesis, University of California at San Diego, 1992, cited in Mettler, 2006). Whether these sequences are indeed functional in the
proposed context is currently being studied (Mettler, 2006).
Because blocking the transition between the G2 and M phase prevents
hb from being downregulated, the repressing activity of svp must
somehow depend on mitosis. This regulation cannot be at the level of the
transcriptional activation of svp because its mRNA is already present
before the NBs enter the decisive M-phase. Moreover, in stg mutant
embryos, where the G2/M transition is blocked, co-expression of
svp mRNA and Hb is found for several hours, although at later stages the
average amount of Hb molecules seems to be generally lower than in cells
without svp expression. At the protein level the situation is
somewhat different: in wild-type embryos hardly any Svp protein is seen
before the NB divides, suggesting that the svp mRNA cannot be
efficiently translated before mitosis occurred. This might be due to a low
translation rate, because in stg mutants only a slowly
increasing Svp protein level was found in the NBs despite a permanently strong
svp mRNA expression. One reason for this might be the nuclear
localisation of the svp mRNA, which was found in NBs of stg mutant embryos as well as in wild type. This localisation might be able to largely prevent svp mRNA from becoming translated before the cell divides. Clearly, further work is needed to test this interesting hypothesis (Mettler, 2006).
The fact that Svp protein is found in NBs in stg mutants that
reduces but does not switch off hb expression might offer an
explanation as to how the different fates of two Hb+ GMCs might be
determined. A well-studied case is NB7-1, where the first GMC gives rise to
the Zfh2-negative U1 neuron, whereas the second generates a Zfh2-positive U2
neuron. Earlier work provided evidence that U2 is specified by a reduction of hb activity within the NB or GMC. Thus, it is possible that a low level of Svp protein present in the NB before the second GMC is born might be responsible for this. Indeed, when svp is expressed in NB7-1 prematurely before the birth
of the first GMC, there is no U1 neuron and the chain of U neurons often
starts with only one Hb+ neuron, which has a U2 identity. According to the hypothesis, this would be due to a reduced Hb activity caused by the premature svp expression. Likewise, in the absence of svp function the NB first produces many additional Hb+ U1 neurons before it eventually generates the other U neurons starting with U2. In this case, hb expression level might initially remain high resulting in the production of several U1 neurons before it drops down leading to the specification of a U2 neuron (Mettler, 2006).
It has been shown that the lineage-specific timing of the switching on of
svp expression defines the end of the Hb+ time window, and
thereby the number of the progeny generated during this phase. How is this timing regulated? In one group of NBs, the
expression of svp already starts before its first division (e.g.,
NB7-3 and NB6-4T). This could be directly dependent on the activity of
proneural genes. Indeed, the early expression of svp within the
developing Malphigian tubules has been shown to be regulated by these genes. In
this context, it is interesting to note that, in Drosophila, svp
expression in certain NB lineages has already begun in their proneural
clusters within the neuroectoderm. A second group of NBs show svp
upregulation after the generation of their first GMCs (e.g. NB2-4T and NB7-1),
suggesting that the mitotic division is the trigger for this event. Surprisingly, this is not the case: in stg mutant embryos, NB7-1
upregulates svp at the same time as in wild type, although no
division has occurred. The same was found for NB2-4T. Thus, lineage-specific
timing of svp expression is independent of the number of cell
divisions. However, currently it cannot be ruled out that earlier stages of the
cell cycle, like the S-Phase, could be the trigger instead. Interestingly, the
sequential transitions of the temporal specification genes acting after
hb expression have recently been shown to occur independently of the
cell cycle. According to the current results, this might be also true for the
timing of svp expression (Mettler, 2006).
The regulatory interactions between hb, svp and pros are the first example where mitosis-dependent gene activity acts together with an asymmetric cell fate determinant to regulate differential gene expression in space and time. It is currently not known whether such a regulation also exists in other organisms. Interestingly, Svp shows a high homology with COUP-TF orphan receptors from vertebrates, which are also necessary for CNS development. Prox1, the vertebrate homologue of Pros is not asymmetrically distributed during division but is expressed and needed during neurogenesis. During retinal development, Prox1 is involved in the specification of the fate of the early born horizontal neurons. Future investigations will show whether during vertebrate CNS development these homologous factors play a role comparable to Svp and Pros in Drosophila (Mettler, 2006).
Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).
To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).
Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).
Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).
Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).
A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).
The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).
Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).
In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).
The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.
The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).
Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).
To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).
To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).
In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).
Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).
The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).
The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).
Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).
S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).
To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), Worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).
Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).
Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).
Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).
Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).
Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).
The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).
It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).
Drosophila embryonic neuroblasts generate different cell types at different time points. This is controlled by a temporal cascade of Hb->Kr->Pdm->Cas->Grh, which acts to dictate distinct competence windows sequentially. In addition, Seven up (Svp), a member of the nuclear hormone receptor family, acts early in the temporal cascade, to ensure the transition from Hb to Kr, and has been referred to as a 'switching factor'. However, Svp is also expressed in a second wave within the developing CNS, but here, the possible role of Svp has not been previously addressed. In a genetic screen for mutants affecting the last-born cell in the embryonic NB5-6T lineage, the Ap4/FMRFamide neuron, a novel allele of svp was isolated. Expression analysis shows that Svp is expressed in two distinct pulses in NB5-6T, and mutant analysis reveals that svp plays two distinct roles. In the first pulse, svp acts to ensure proper downregulation of Hb. In the second pulse, which occurs in a Cas/Grh double-positive window, svp acts to ensure proper sub-division of this window. These studies show that a temporal factor may play dual roles, acting at two different stages during the development of one neural lineage (Benito-Sipos, 2011).
This study has found that Svp is expressed in two pulses and plays two different roles in the NB5-6T lineage. Initially, Svp is expressed briefly in the early part of this lineage, where it acts to control the downregulation of the first temporal factor, Hb. Subsequently, Svp is expressed in the late part of this lineage, in the Ap window, in a highly dynamic fashion: initiated in all four Ap neurons, to be downregulated in the first- and last-born Ap cells. In the second expression phase, Svp acts to suppress Col and Dimm, thereby preventing the first-born Ap neuron fate, Ap1/Nplp1, from being established in the subsequently born Ap2 and Ap3 neurons. Misexpression studies further indicate that Svp also suppresses the last-born Ap neuron fate, Ap4/FMRFa, from being established in Ap2/3 (Benito-Sipos, 2011).
Previous studies of Svp demonstrated that it is expressed in a brief pulse in the majority of early embryonic neuroblasts, where it acts to suppress Hb, thereby allowing for the switch to the next stage of temporal competence. Recently, studies have identified additional factors involved in the downregulation of Hb: the pipsqueak-domain proteins Distal antenna and Distal antenna-related (herein referred to collectively as 'Dan'). Dan is expressed somewhat earlier than Svp, and is also maintained in a longer pulse. svp and dan do not regulate each other, and although they can be activated by ectopic hb expression, neither Svp nor Dan expression is lost in hb mutants. This raises the intriguing questions of how Svp and Dan are activated during early stages of lineage progression, and how they become downregulated at the appropriate stage (Benito-Sipos, 2011).
Another interesting complexity with respect to Svp expression and function pertains to the fact that the Hb window is of different size in different lineages. For example, in NB6-4T and NB7-3, Hb is downregulated in the neuroblast immediately after the first division, whereas in NB5-6T, Hb expression is evident during three divisions. In line with this, no Svp expression is observed in NB5-6T until stage 10, when the neuroblast has already gone through two rounds of division. How the on- and offset of Svp, and perhaps Dan, expression is matched to the specific lineage progression of each unique neuroblast lineage, to thereby allow for differing Hb window sizes, is an interesting topic for future studies (Benito-Sipos, 2011).
Svp is re-expressed in the NB5-6T lineage in a second pulse. In contrast to the early pulse of Svp expression, where there is no evidence for temporal genes controlling Svp, it was found that the second pulse of Svp expression is regulated by the temporal genes cas and grh. However, it was not found that svp is important for the expression of Cas or Grh. Instead, svp participates in the sub-division of the Cas/Grh temporal window, i.e. the Ap window. Based upon the idea that Svp is regulated by temporal genes, and acts to sub-divide a broader temporal window, it could be referred to as a 'sub-temporal' factor in the latter part of the NB5-6T lineage (Benito-Sipos, 2011).
The expression of Svp is dynamic also in the second pulse of expression, commencing in the neuroblast at stage 14 -- after the three first Ap neurons are born -- and being maintained in the neuroblast until it exits the cell cycle at stage 15. At late stage 14 and 15, Svp expression becomes evident in all four Ap neurons, but it is rapidly downregulated from Ap1 and Ap4 during stages 16 and 17. Svp is, however, maintained in the Ap2 and Ap3 neurons into late embryogenesis. The role of svp in the Ap window appears to be to ensure proper specification of the Ap2/3 interneurons, generated in the middle of the Ap window. This is achieved by svp suppressing the first- and last-born Ap neuron fates: the Ap1/Nplp1 and Ap4/FMRFa fates. With regard to the suppression of the Ap1 fate, one important role for svp is to suppress Col expression in Ap2/3. Importantly, the temporal delay in Svp expression when compared to Col -- commencing two stages after Col in the Ap neurons -- allows for col to play its critical early role in Ap neuron specification: activating ap and eya. The timely suppression of Col in Ap2/3 is mediated also by sqz and nab, and the loss of Nab expression in svp mutants may be a contributing factor to the failure of Col downregulation in svp. However, the potent function of svp in suppressing Ap1/Nplp1 fate when misexpressed postmitotically from apGal4 does not appear to require Nab, as Nab is not ectopically expressed in these experiments. Thus, svp may act via several routes to prevent Ap1/Nplp1 fate from being established in the Ap2/3 cells: by suppressing Col and by activating Nab (Benito-Sipos, 2011).
Regarding the second role of svp in the Ap window -- the suppression of the Ap4/FMRFa fate -- it is less clear what the target(s) may be. However, a common denominator for both the Ap1/Nplp1 and the Ap4/FMRFa neurons is the expression of Dimm. Dimm, a basic-helix-loop-helix protein, is a critical determinant of the neuropeptidergic cell fate, and also controls high-level neuropeptide expression in many neuropeptide neurons. Both svp loss and gain of function results in robust effects upon Dimm expression in the NB5-6T lineage, indicating that Dimm is an important target for svp. However, dimm mutants show only reduced levels of FMRFa expression, and thus svp is likely to regulate additional targets to prevent the Ap4/FMRFa cell fate in the Ap2/3 neurons (Benito-Sipos, 2011).
Another interesting phenotype in svp mutants, pertaining to the second pulse of Svp expression in the NB5-6T lineage, is the finding of one to two extra Ap neurons. This indicates that the NB5-6T neuroblast undergoes one to two extra rounds of division, and that the expression of Svp in the neuroblast during stage age 14-16 is important for precise cell cycle exit. Interestingly, the other temporal (cas and grh) and sub-temporal (sqz and nab) genes acting in the latter part of the NB5-6T lineage also play roles in controlling cell cycle exit. Moreover, studies of neuroblast cell cycle exit in other neuroblasts, both embryonic and postembryonic, have also shown roles for grh and svp in these decisions. Thus, a picture is emerging in which late temporal and sub-temporal genes may be broadly involved in controlling timely cell cycle exit of many neuroblasts (Benito-Sipos, 2011).
The early role of svp, in its first expression pulse, is to suppress Hb expression. Svp is expressed transiently by most if not all neuroblasts, and the regulation of Hb also appears to be a global event. Similarly, the second pulse of Svp expression has been observed in many lineages, although the role for svp in this later pulse was hitherto unknown. The findings of a role for svp as a sub-temporal gene in the latter part of the NB5-6T lineage indicates that svp may play such roles in many lineages. However, it should be noted that global changes in Col, Dimm and Eya expression in the embryonic central nervous system (CNS) are not seen. Thus, unlike the more universal role of svp in regulating Hb during the first pulse, the putative sub-temporal function of the second pulse of svp expression in other lineages must be highly context-dependent and involve other targets (Benito-Sipos, 2011).
In mammals, the svp orthologues COUP-TFI and -II are expressed dynamically in the developing CNS. Functional studies reveal a number of important roles for COUP-TFI/II during nervous system development, and mutant mice display aberrant neuro- and gliogenesis, accompanied by axon pathfinding defects. Intriguingly, recent studies have revealed that COUP-TFI/II acts in a temporal manner to control the timing of generation of sub-classes of neurons and glia in the developing mouse brain. Given that the other genes described in this study are also conserved, it is tempting to speculate that temporal and sub-temporal cascades similar to those outlined in this study are also used in the mammalian CNS during development (Benito-Sipos, 2011).
A fundamental question in brain development is how precursor cells generate a diverse group of neural progeny in an ordered manner. Drosophila neuroblasts sequentially express the transcription factors Hunchback (Hb), Krüppel (Kr), Pdm1/Pdm2 (Pdm) and Castor (Cas). Hb is necessary and sufficient to specify early-born temporal identity and, thus, Hb downregulation is essential for specification of later-born progeny. This study shows that distal antenna (dan) and distal antenna-related (danr), encoding pipsqueak motif DNA-binding domain protein family members, are detected in all neuroblasts during the Hb-to-Cas expression window. dan and danr were identified in a forward genetic screen of ~100 second and third chromosomal deficiency lines for mutants that had altered numbers of Even-skipped (Eve)+ early-born neurons. Dan and Danr are required for timely downregulation of Hb in neuroblasts and for limiting the number of early-born neurons. Dan and Danr function independently of Seven-up (Svp), an orphan nuclear receptor known to repress Hb expression in neuroblasts, because Dan, Danr and Svp do not regulate each other and dan danr svp triple mutants have increased early-born neurons compared with either dan danr or svp mutants. Interestingly, misexpression of Hb can induce Dan and Svp expression in neuroblasts, suggesting that Hb might activate a negative feedback loop to limit its own expression. It is concluded that Dan/Danr and Svp act in parallel pathways to limit Hb expression and allow neuroblasts to transition from making early-born neurons to late-born neurons at the proper time (Kohwi, 2011).
Dan and Danr are required to limit Hb expression in neuroblasts and restrict the number of early-born neurons generated in multiple neuroblast lineages. The orphan nuclear hormone receptor protein Svp also functions to limit Hb expression in neuroblasts, and the current data strongly suggest that Dan/Danr and Svp function in parallel pathways that are each independently required. First, the temporal expression patterns of Dan and Danr versus Svp do not suggest their coordinated activity: Dan and Danr are expressed from the time of neuroblast formation (stage 9), beyond Hb downregulation (stage 10), until the time of strong Castor expression (stage 12). By contrast, Svp protein is very transiently detected in neuroblasts only at the onset of Hb downregulation. Second, Dan/Danr and Svp are not in a linear transcriptional hierarchy: neither mutant affects expression of the other gene. Third, dan danr and svp mutants have distinct phenotypes: for example, compared with the dan danr mutant, the svp mutant has many more early-born neurons in the NB7-1 lineage, whereas it does not have any extra early-born neurons in the NB1-1 lineage. Fourth, the dan danr svp null triple mutant has the summed phenotypes of the dan danr double mutant and the svp single mutant. Fifth, misexpression of Svp, but not Dan, can repress hb transcription in neuroblasts. The fact that neither one appears to have an effect on cell fate when misexpressed in postmitotic neurons suggest that both Svp and Dan function at the level of the mitotic precursors. Taken together, it appears that Dan/Danr and Svp are each required to downregulate hb expression in neuroblasts, but do so using separate mechanisms. The data are consistent with Svp directly repressing neuroblast hb transcription (although this has not been shown) whereas Dan and Danr act more indirectly (Kohwi, 2011).
Do Dan, Danr and Svp have lineage-specific functions? Despite the widespread expression of Dan and Danr in early neuroblasts and the widespread transient expression of Svp in most neuroblasts, it is likely that each has lineage-specific functions. For example, in the NB1-1 lineage, ectopic early-born neurons are generated in dan danr mutants, but not in svp mutants. Further comparing Dan versus Danr in this lineage, it appears that Danr is more important than Dan, because the danrex35 single mutant phenocopies the dan danrex56 double mutant in the number of ectopic aCC/pCC neurons generated and the number of hemisegments affected per embryo. In contrast to the NB1-1 lineage, Dan and Danr each appear to be required for limiting the number of early-born neurons in the NB7-1 lineage, as danrex35 single mutants had a weaker phenotype than the dan danr double mutant. Additionally, there are more early-born neurons in the NB7-1 lineage in svp mutants than in dan danr mutants, highlighting their lineage-specific differences. These differences might be due to different levels or functions of each protein in distinct neuroblasts. For example, there is variability in dan and danr mRNA levels between neuroblasts, suggesting that distinct neuroblasts might have different levels of Dan and/or Danr protein (although Dan protein levels appear constant between newly formed neuroblasts), or that they express Dan and/or Danr protein for different durations. Alternatively, or in addition, the lineage-specific variation might be due to unique cofactors present in different neuroblasts. This seems likely, as Hb misexpression in all neuroblasts has varying effects within different lineages. For example, NB1-1 generates only one to three ectopic early-born neurons in response to Hb misexpression, whereas NB7-1 generates ~20 ectopic early-born neurons. Consistent with the notion that co-factors can alter the functional output of transcriptional regulators, recent evidence shows that the co-regulator CtBP forms complexes with distinct eye specification factors, including Dan and Danr, to regulate proliferation versus differentiation during eye development in Drosophila (Kohwi, 2011).
Do Dan and Danr function redundantly? This hypothesis could not be rigorously tested owing to the lack of a dan null single mutant, but the available evidence suggests that they do have redundant functions. First, they have nearly identical expression patterns. But most crucially, overexpression of Dan in the dan danr double mutant can nearly completely rescue the CNS phenotype, suggesting that high enough levels of Dan can compensate for loss of Danr. However, a danr null single mutant shows a strong phenotype in the NB1-1 lineage and a partial phenotype in the NB7-1 lineage, suggesting that endogenous levels of Dan are insufficient for normal CNS development. The most parsimonious explanation is that each protein has equivalent function, but that both genes are required to generate sufficient levels of Dan/Danr protein (Kohwi, 2011).
Hb overexpression can activate expression of both Dan and Svp. What is the significance of this activation? Previous work has shown that Hb can function both as a transcriptional activator and a repressor. Although its repressive functions are required for the neuroblast to specify early-born fates and maintain neuroblast competence, its activator functions remain elusive. One possibility is that Hb-mediated activation of Svp, and the subsequent Svp-mediated downregulation of Hb, create a negative feedback loop to ensure timely progression of the neuroblast to later temporal fates. This is not unlike what has been observed for Cas, which activates feed-forward and feed-back transcriptional cascades to regulate temporal identity in the NB5-6 lineage. By contrast, Hb activation of Dan expression might be part of the mechanism by which Hb maintains neuroblast competence, because Dan is unlikely to repress hb expression directly (Kohwi, 2011).
What might be the mechanism by which Dan and Danr function to restrict the duration of Hb expression in neuroblasts? Some clues might come from the fact that Dan and Danr are found in a subgroup of pipsqueak-domain containing nuclear proteins that have been proposed to regulate higher order chromatin structure by targeting distal DNA elements. Pipsqueak, the founding member of the family, has been shown to recruit Polycomb group complexes to specific regions of the genome to mediate gene silencing. Perhaps Dan and Danr modify chromatin structure through recruitment of chromatin remodeling complexes, which indirectly affects hb transcription by changing the accessibility of the hb locus to other transcriptional regulators. Such a function in modulating chromatin architecture might not be restricted to regulating just hb expression, but can extend to other temporal identity factors as well. Indeed, in NB7-1, the initial Hb->Cas 'competence window', during which the U1-U5 motor neurons are generated, matches nearly exactly the window of Dan and Danr expression. This raises the possibility that Dan and Danr might have a more global role in NB temporal progression by stabilizing 'transition states' between successive temporal identity factors (e.g. Hb->Kr, Kr->Pdm or Pdm->Cas). Such a function might explain the low frequency misregulation of later-born neuron numbers in several lineages (7-1, 3-1, 7-3), in addition to the extra early-born neurons phenotypes. Future experiments that address the role of Dan and Danr in later temporal identity transitions will provide a better understanding of the mechanisms that control the progression of temporal identity in neuroblasts (Kohwi, 2011).
During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).
During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).
Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).
Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).
Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several RasGTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of RasGTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).
The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active PointedP1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).
This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).
In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of RasGTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).
In the developing compound eye of Drosophila, neuronal differentiation of the R7 photoreceptor cell is induced by the interaction of the receptor tyrosine kinase Sevenless with its ligand, Bride of sevenless (Boss), which is expressed on the surface of the neighboring R8 cell. Boss is an unusual ligand for a receptor tyrosine kinase: it is composed of a large extracellular domain, a transmembrane domain with seven membrane-spanning segments and a cytoplasmic tail. Expression of a monomeric, secreted form of the extracellular domain of Boss is not sufficient for Sevenless activation, and instead acts as a weak antagonist. Because oligomerization appears to be a critical step in the activation of receptor tyrosine kinases, oligomerized forms of the Boss extracellular domain were used to test their ability to bind to Sevenless in vivo and restore R7 induction in vivo. Oligomerization is achieved by fusion to the leucine zipper of the yeast transcription factor GCN4 or to the tetramerization helix of the Lac repressor. Binding of these multivalent proteins to Sevenless can be detected in vitro by immunoprecipitation of cross-linked ligand/receptor complexes and in vivo by receptor-dependent ligand localization. However, neither R8-specific nor ubiquitous expression of multivalent extracellular Boss (Exboss) ligands rescues the boss phenotype. Instead, these ligands acted as competitive inhibitors for wild-type Boss protein and thereby suppressed R7 induction. Therefore the role of the transmembrane or cytoplasmic domains of Boss in the activation of the Sev receptor cannot be replaced by oligomerization (Sevrioukov, 1998).
Why do the oligomeric forms of Exboss not activate Sev? Given that oligomerization actually enhances receptor binding, it does not appear likely that the tight association of the leucine zippers occludes the interface of Exboss, which is required for Sev binding. A potential problem could be the specific spatial arrangement of the Exboss subunits in the Exboss-GCN dimers or Exboss-Lac oligomers. To address this issue a test was performed on three versions of Exboss-GCN, which differ only in the orientation of their subunits relative to one another. Given the similarity of the effects of the three rotated Exboss-GCN forms and of Exboss-Lac, which uses a different oligomerization unit, it seems unlikely that a specific spatial arrangement of the Exboss subunits is the cause of their dominant-negative effects. The most straightforward explanation for the data is that the 7TM and cytoplasmic domains of Boss have an additional, different function in R7 induction. The structure of the Boss protein is reminiscent of seven transmembrane receptors, although no sequence homology has yet been identified. This similarity raises the possibility that Boss may function not only as a ligand but also as a receptor. One possible function for Boss as a receptor would be to act indirectly in R7 induction. However, constitutively active Sevenless receptor results in R7 induction even in the absence of Boss, arguing against a role for Boss as a receptor in the induction of R7 cell fate. A second possible function for the Boss protein as a receptor is to affect R8 development. An R8-specific Rhodopsin, Rh5, is expressed in a subset of R8 cells that is paired with R7 cells expressing Rh3. Interestingly, in eye discs lacking R7 cells, R8 cells no longer express the Rh5 opsin (Chou, 1996 and Papatsenko, 1997). These recent findings constitute the first indication that a signal from R7 influences gene expression in R8, and provide an assay to test the possibility that Boss acts as a receptor as well as a ligand (Sevrioukov, 1998).
An active Ras pathway is essential for the function of both SVP isoforms. Loss-of-function mutations in components of the Ras signal transduction cascade
act as dominant suppressors of the cone cell transformation, while loss-of-function
mutations in negative regulators of Ras-activity act as dominant enhancers. Furthermore,
svp-mediated transformation of cone cells to outer photoreceptors, reminiscent of its
wild-type function in specifying R3/4 and R1/6 identity, requires an activated Ras pathway in
the same cells, or alternatively dramatic increase in ectopic SVP protein levels. The nature of the requirement for the Ras pathway for SVP function is unknown. There are several possiblities for this requirement. For instance, SVP could partner with a target of the Ras pathway, or SVP itself could be modified by members of the Ras pathway (Begemann, 1995).
SVP, like COUP-TF (chicken ovalbumin upstream transcription factor), can modulate Ultraspiracle-based hormonal signaling. Seven-up can inhibit
ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex
of USP and the Ecdysone receptor. This repression depends on the dose of SVP and occurs with two
different Drosophila ecdysone response elements. Ectopic expression of svp in vivo induces
lethality during early metamorphosis, the time of maximal ecdysone responsiveness. Concomitant
overexpression of usp rescues the larvae from the lethal effects of svp. DNA binding studies show
that SVP can bind to various direct repeats of the sequence AGGTCA but cannot bind to one of
the ecdysone response elements used in the transient transfection assays.
SVP-mediated repression can occur by both DNA binding competition and protein-protein
interactions (Zelhof, 1995a).
Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the
hormone. Corepressors, which bind to the receptor's silencing domain, are involved in this repression. Hormone binding leads to
dissociation of corepressors and binding of coactivators, which in turn mediate gene activation.
Alien (Drosophila homolog: Alien) is a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of
Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation
experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows
that Alien is localized in the cell nucleus. Alien is a highly conserved, showing 90% identity between human and Drosophila proteins. Drosophila Alien shows similar
activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homolog of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily (Dressel, 1999).
To gain insight into the mechanism of Alien-mediated gene repression, tests were performed to see whether the silencing function of Alien
is based on complex formation with the known corepressors SMRT and N-CoR or with SIN3A. The ability of full-length human Alien (h-Alien) to interact with
either full-length SMRT, the C terminus of N-CoR, or mouse SIN3A was tested in the yeast two-hybrid system. No interaction of h-Alien with the
SMRT/N-CoR class of corepressors was detected. Interestingly, a strong interaction of Alien with SIN3A, a protein shown to be part of a deacetylase
complex, was detected. A specific Alien-SIN3A interaction was observed in GST pull-down experiments with GST-h-Alien and in vitro-translated SIN3A. To verify this interaction, coimmunoprecipitations were performed. The anti-Alien antibody immunoprecipitates SIN3A from HeLa extracts. This indicates that Alien is mediating silencing, at least in part, by recruiting a factor known to be involved in deacetylase activity (Dressel, 1999).
To further investigate the association of Alien with a deacetylase activity, trichostatin A (TSA), a specific inhibitor of histone deacetylases, was used. CV1 cells were transfected with Gal-h-Alien or Gal-N-CoR and treated with TSA for 8 h. Addition of TSA reduces silencing by both Alien and N-CoR, a protein known to repress transcription by recruitment of deacetylase activity. Taken together, this supports a role for deacetylase activity in Alien-mediated silencing. Since the SMRT/N-CoR class of corepressor also interacts with SIN3A, the observed TR-Alien-SIN3A interaction may strengthen the recruitment of a deacetylase complex to genes regulated by selected NHRs. Thus, these data indicate that one mechanism by which Alien confers silencing may, at least to some extent, be based on recruitment of deacetylase activity via interaction with SIN3A (Dressel, 1999).
The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is
required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the activator function AF-2 domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo (Beckstead, 2001).
Database searches have revealed that bon encodes the only Drosophila homolog of mammalian TIF1s. Bon exhibits 29% identity with mouse TIF1alpha and mouse TIF1beta, and 26% identity with human TIF1gamma. The overall identity between Bon and TIF1s is similar to the identity observed between the TIF1 members. A higher degree of identity is seen in the N- and C-terminal regions spanning the conserved domains. At the N terminus, a C3HC4 zinc-finger motif or RING finger is followed by two cysteine-rich zinc binding regions (B-boxes) and a coiled coil domain forming a tripartite motif designated RBCC. At the C terminus, a bromodomain is preceded by a C4HC3 zinc-finger motif or PHD finger (Beckstead, 2001 and references therein).
Northern analysis demonstrates that bon produces one predominant 6 kb transcript and two 4 kb transcripts, which each encode a protein of ~140 kDa. The two 4 kb transcripts are only present in 0-3 hr embryos and adult females. It is therefore possible that the 4kb mRNAs are maternal components. bon is expressed throughout embryogenesis and in first instars. Its levels increase in 9-12 hr embryos and are low during the second instar stage. bon is upregulated in late third instar larvae. The upregulation of bon during midembryogenesis and prior to pupariation correlates well with known high titer pulses of ecdysone (Beckstead, 2001).
Immunohistochemical staining of numerous tissues show that Bon is a nuclear protein expressed in most and possibly all cells during embryogenesis, in fat body, imaginal discs, salivary glands, brain, gut, Malpighian tubules, and trachea. Bon is a chromatin-associated protein that localizes to ~10%-15% of the polytene chromosome bands. This pattern is highly reproducible (Beckstead, 2001).
To test whether Bon interacts with betaFTZ-F1 as well as other Drosophila nuclear receptors in vitro, binding assays were performed using purified recombinant proteins. Glutathione-S transferase (GST)-fused betaFTZ-F1, alphaFTZ-F1 (amino acids 154-1029), Seven-up (SVP), DHR3, USP, and EcR were immobilized on glutathione-Sepharose and incubated with purified N-terminally His-tagged Bon (His-Bon). His-Bon binds to GST-betaFTZ-F1, GST-alphaFTZ-F1, GST-DHR3, GST-SVP, GST-USP, and GST-EcR, but not to GST alone. Thus, Bon can bind directly to many members of the nuclear receptor family in vitro (Beckstead, 2001).
To determine whether Bon is able to repress transcription, the coding sequence of Bon was fused to the yeast GAL4 DNA binding domain. The resulting fusion protein was tested for its ability to repress transcription activated by ER(C)-VP16, a chimeric activator containing the DBD of ERalpha fused to VP16. GAL4-Bon and ER(C)-VP16 were transiently transfected into S2 cells with a reporter containing a GAL4 binding site (17M) and an estrogen response element (ERE) in front of a thymidine kinase (tk) promoter-CAT fusion (17M-ERE-tk-CAT). GAL4-Bon efficiently represses transcription in a dose-dependent manner. In contrast, coexpression of Bon without the GAL4 DNA binding domain causes a reproducible increase in CAT activity, indicating that repression by Bon is entirely dependent on DNA binding (Beckstead, 2001).
To map the domain of Bon responsible for transcriptional repression, a set of N- and C-terminally truncated derivatives were assayed for their ability to repress VP16-activated transcription in S2 cells. In the absence of the RBCC motif, the GAL4-Bon fusion protein, GAL4-Bon [471-1133]) fails to repress transcription, indicating that the N-terminal region of Bon is required for repression. However, this region is not sufficient for full repression. Consistent with this, a C-terminal truncation, GAL4-Bon (1-890), is a less potent repressor, indicating that the C-terminal residues of the protein including the PHD finger and the bromodomain also contribute to the repression potential of Bon. However, this domain on its own exhibits little repression. A 3- to 4-fold increase in CAT activity is observed with the central region between the coiled-coil and the PHD finger, suggesting that Bon may also contain a 'masked' activation domain. Note, however, that no significant activation was observed with GAL4-Bon (471-890) tested in the absence of ER(C)-VP16. Taken together, these results indicate that most of the repression activity of Bon resides within the N-terminal RBCC domain (Beckstead, 2001).
Bon and TIF1s contain an N-terminal RBCC (RING finger/B boxes/coiled coil) motif. In the absence of the RBCC motif, the GAL4-Bon protein, unlike the full-length protein, fails to repress transcription. The TIF1beta RBCC domain has been shown to be necessary for the oligomerization of TIF1beta and KRAB binding. Because Bon is able to homodimerize, this domain may be involved in formation of protein complexes (Beckstead, 2001).
The PHD finger and bromodomain are characteristic features of nuclear proteins known to be associated with chromatin and/or to function at the chromatin level. For instance, the chromosomal proteins Trithorax and Polycomb-like contain multiple PHD fingers, while the histone acetyltransferases CBP and GCN5 as well as the chromatin-remodeling factor SWI2/SNF2 are also bromodomain containing proteins. Bromodomains have been shown to bind to acetyl-lysine and specifically interact with the amino-terminal tails of histones H3 and H4, suggesting a chromatin-targeting function for this highly evolutionarily conserved domain. Because Bon is localized to hundreds of chromatin bands on Drosophila polytene chromosomes, it is probably involved in chromatin-mediated regulation of transcription of numerous genes (Beckstead, 2001).
Bon can repress both basal and activated transcription when recruited to the promoter region of a target gene, similar to TIF1alpha, -beta, and -gamma. For TIF1alpha and TIF1beta, a link between silencing and histone modification has been established, and TIF1beta is part of a large multiprotein complex that possesses histone deacetylase activity. Moreover, TIF1beta was also reported to colocalize and interact directly with members of the heterochromatin protein 1 (HP1) family. Similar to TIF1beta, TIF1alpha can bind the HP1 proteins in vitro. However, TIF1alpha-mediated repression in transfected cells does not require the integrity of the HP1 interaction domain, nor is there any significant subnuclear colocalization of HP1alpha and TIF1alpha. No interactions were observed between Bon and HP1 in a yeast two-hybrid assay, nor was any evidence found for genetic interactions. However, in a yeast two-hybrid screen, Bon interacted with members of the Polycomb group, suggesting that Bon may also be part of heterochromatin-like complexes and/or may require some of the members of the Polycomb group genes to repress transcription. This would imply that Bon has a dual role, similar to some members of the Polycomb group family: transcriptional repression and heterochromatin formation. Both of these roles may be required in transcriptional repression (Beckstead, 2001).
The interaction of Bon with nuclear receptors is similar to TIF1alpha but unlike TIF1beta and TIF1gamma. This interaction requires the integrity of the nuclear receptor AF-2 activation domain and is mediated by the Bon/TIF1alpha LxxLL motif. These observations suggest that Drosophila nuclear receptors and Bon have co-evolved to maintain their interaction. It is therefore likely that the biological role of this interaction has been conserved in mammals (Beckstead, 2001).
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Rather than delaminating from the ectoderm in a continuous stream, oenocyte precursors segregate in discrete well-separated bursts of three cells. Genetic backgrounds affecting the pattern of cell segregation but not early fate specification were used to show how these pulses are regulated by EGFR signaling. The signaling parameters regulating the time of onset, time of cessation, and in particular, the cyclical nature of cell delamination of oenocytes are discussed (Brodu, 2004).
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