Transcriptional Regulation

There is no expression of prospero in embryos that lack the proneural gene daughterless (Vaessin, 1994).

prospero gene becomes transcriptionally activated at a low level in all Sevenless-competent cells prior to Sevenless signaling, and this requires the activities of Ras1 and two Ras1/MAP kinase-response ETS transcription factors, Yan and Pointed. Activation of pros transcription in all cells within the R7 equivalence group requires the down-regulation of Yan activity through phosphorylation by Rolled in R7 and cone cell precursors. Loss of pointed results in a reduction in the number of pros expressing cells. Two other nuclear factors, Seven in absentia (SINA) and Phyllopod are required for R7 determination, but are not absolutely for pros expression. However, the presence of phyl in cells is sufficient to induce them to express elevated levels of pros. phyl requires sina activity to stimulate pros expression. SINA protein can be shown to form a complex with PHYL (Kauffmann, 1996)

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.

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. svp expression in this region is negatively regulated by Lz. Lz and Pnt both been shown to directly bind to the pros promoter/enhancer region and pros expression occurs only when Pnt and Lz have bound simultaneously to the pros enhancer/promoter. In wild type, Lz is expressed prior to pros expression in rows 4-7; subsequent to Ras1 ubiquitous activation, pros expression takes place in this region. Thus, in all wild-type progenitors situated in rows 4-7, Lz may bind to the pros enhancer/promoter so as to impart progenitor cells with pros expression potential. In wild type, pros expression first becomes apparent in R7 precursors at row 8. The absence of pros expression in rows 4-7 in wild type may then be accounted for by the possible absence of Ras1 signal activity. This possibly may be an oversimplification since, for instance, this does not explain why pros is repressed in R1/R6 photoreceptors which also arise from Lz-positive progenitor cells, or why pros is not induced efficiently on Ras1 activation in row 10 and more posterior regions (Hayashi, 2001).

The former might be caused by the absence of the strong N signal in R1/R6 precursors, but another unknown mechanism may be required to explain the latter. Therefore, more remains to be discovered about pros regulation, but what is known is nonetheless an excellent model for understanding the manner in which cooperative action of prepattern genes and differentiation signals give rise to specific cell fates from common progenitors (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).

In undifferentiated cells of the larval eye imaginal disc, the transcriptional repressor Yan outcompetes the transcriptional activator Pointed for ETS binding sites on the prospero enhancer. During differentiation, the Ras signaling cascade alters the Yan/Pointed dynamic through protein phosphorylation, effecting a developmental switch. In this way, Yan and Pointed are essential for prospero regulation. Hyperstable YanACT cannot be phosphorylated and blocks prospero expression. Lozenge is expressed in undifferentiated cells, and is required for prospero regulation. The eye-specific enhancer of lozenge has been sequenced in three Drosophila species spanning 17 million years of evolution and complete conservation of three ETS consensus binding sites was found. lozenge expression increases as cells differentiate, and YanACT blocks this upregulation at the level of transcription. Expression of Lozenge via an alternate enhancer alters the temporal expression of Prospero, and is sufficient to rescue Prospero expression in the presence of YanACT. These results suggest that Lozenge is involved in the Yan/Pointed dynamic in a Ras-dependent manner. It is proposed that upregulated Lozenge acts as a cofactor to alter Pointed affinity, by a mechanism that is recapitulated in mammalian development (Behan, 2002).

A genetic approach was used to examine Yan/Lz interactions; Yan is shown to temper lz expression. The degree of regulation is dependent upon the presence of the eye-specific enhancer. The complete conservation of three Ets binding sites across 17 million years of evolution is strong supporting evidence that this regulation is direct. At this time, however, the possibility that this regulation is indirect cannot be ruled out (Behan, 2002).

Separate inputs by Ets factors and Lz have been shown to be required for regulation of prospero and D-Pax2. The current data must be interpreted in this context. The lzgal4 reporter system was used to show that hyperstable YanACT is able to block lz expression at the level of transcription. The GMR and Sev ectopic expression systems have been used to tease out Yan control of lz apart from control of other genes (Behan, 2002).

A model is here proposed for prospero regulation by Prospero and Yan, with the added information that lz is also a target of Yan. Yan tempers Lz expression. In the undifferentiated cell, Yan represses prospero by directly binding to Ets sites. The transcriptional activator Pointed competes for the same DNA but with much less affinity. Lozenge transcription is tempered by Yan, but not entirely repressed. Upon activation of Egfr and Sevenless by their respective ligands Spitz and BOSS, Ras1 is stimulated. Ultimately, Yan and Pointed are phosphorylated, downstream of Ras1 but with opposite effects. Phosphorylated Yan is targeted for degradation. Phosphorylated Pointed binds DNA with a higher affinity. Yan repression of Lz is alleviated, and becomes upregulated by some other mechanism. Upregulated Lz binds with Pointed to mediate prospero transcription (Behan, 2002).

This begs the question, why the double level of control, i.e., with both Lozenge and Pointed required for prospero regulation? It is hypothesized that Lz, functioning as a transcription factor, is involved directly in the Ets developmental switch, acting as a cofactor to enhance the ability of Pointed to compete with Yan for Ets sites. What follows supports this argument. The developmental potential of R7 in the presence of one dose of sev-yanACT has been analyzed. In this mutant background, Pointed is phosphorylated normally by MAP kinase, but is unable to compete with the mutant hyperstable Yan. The result is that Prospero expression is never upregulated: Runt expression is not turned on, and R7 differentiation fails. Coexpression of GMR-[lz-c3.5] rescues Prospero expression. Furthermore, the ectopic R7 cells that develop in the GMR-[lz-c3.5] background follow their normal developmental pathway. Unlike the native R7 precursors, these ectopic R7 precursors express both upregulated Prospero and Runt. This may indicate a difference in the level of expression of the sev-yanACT transgene between the two cell types, or some other cell-specific factors involved in regulation. The results in both the endogenous and ectopic R7 cells indicate that the presence of Lz effects a change in the dynamic between Yan and Pointed (Behan, 2002).

Although this could be accomplished by a number of mechanisms, the hypothesis is favored that Lz induces a change in the ability of Pointed to bind to DNA. This is based on a mammalian paradigm. The mammalian homologs of Lz and Pointed are RUNX1 and Ets-1. Lz is 71% identical to RUNX1 in its homologous domains. Pointed is 95% identical to Ets-1 in the Ets DNA binding domain; Pointed and Ets-1 proteins are functionally homologous and can replace each other in vitro and in vivo. It has been shown that RUNX1 and Ets-1 bind cooperatively to separate, but nearby, DNA sites on the T cell receptor, and that this cooperativity can exist even when these sites are as far apart as 33 base pairs. Notably, RUNX1 and Ets-1 can not substitute for each other. Both inputs are required for stable ternary complex development. The presence of RUNX1 enhances Ets-1 DNA binding affinity by as much as 20 times in vitro. Regions of RUNX1 and Ets-1 outside of the DNA binding domains that are necessary for cooperative DNA binding, and similar regions exist in both Lz and Pointed proteins. Furthermore, it has been speculated that this type of cooperativity may exist in Drosophila eye development (Behan, 2002 and references therein).

Strikingly, in the prospero enhancer one Ets binding site is only 7 base pairs away from a Lz binding site. The genetic results reported in this study are consistent with Lz and Pointed acting cooperatively on the prospero enhancer. The double input of Lz and Pointed effectively competes with YanACT. Clearly a dynamic exists between the three factors Lz, Yan and Pointed. Work in flies and mammals has shown that this dynamic is influenced by phosphorylation, competition, transcriptional regulation and cofactor availability (Behan, 2002).

The Drosophila gene dead ringer (dri) [also known as retained (retn)] encodes a nuclear protein with a conserved DNA-binding domain termed the ARID domain (AT-rich interaction domain). dri is expressed in a subset of longitudinal glia in the Drosophila embryonic central nervous system and dri forms part of the transcriptional regulatory cascade required for normal development of these cells. Analysis of mutant embryos reveals a role for dri in formation of the normal embryonic CNS. Longitudinal glia arise normally in dri mutant embryos, but they fail to migrate to their final destinations. Disruption of the spatial organization of the dri-expressing longitudinal glia accounts for the mild defects in axon fasciculation observed in the mutant embryos. The axon phenotype includes incorrectly bundled and routed connectives, and axons that sometimes join the wrong bundle or cross from one tract to another. Consistent with the late phenotypes observed, expression of the glial cells missing (gcm) and reversed polarity (repo) genes was found to be normal in dri mutant embryos. However, from stage 15 of embryogenesis, expression of locomotion defects (loco) and prospero (pros) was found to be missing in a subset of LG. This suggests that loco and pros are targets of Dri transcriptional activation in some LG. It is concluded that dri is an important regulator of the late development of longitudinal glia (Shandala, 2003).

What is the molecular basis of the mutant phenotype found in dri mutants? Dri is a transcription factor, so the link between loss of dri function and the failure to differentiate properly is likely to be indirect, mediated through misregulation of dri targets required for normal longitudinal glial development. The most informative data came from an analysis of the position of dri in the glial transcriptional regulatory cascade. In general terms, dri activity was found to be downstream of gcm and repo, and independent of pnt and cut. It was also found to be upstream of two genes, loco and pros, which are essential for normal development of some glial cells. In this developmental context dri acts as an activator of downstream targets (Shandala, 2003).

The brain tumor gene, acting upstream of pros, negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila

Brain development in Drosophila is characterized by two neurogenic periods, one during embryogenesis and a second during larval life. Although much is known about embryonic neurogenesis, little is known about the genetic control of postembryonic brain development. This study used mosaic analysis with a repressible cell marker (MARCM) to study the role of the brain tumor (brat) gene in neural proliferation control and tumour suppression in postembryonic brain development of Drosophila. The findings indicate that overproliferation in brat mutants is due to loss of proliferation control in the larval central brain and not in the optic lobe. Clonal analysis indicates that the brat mutation affects cell proliferation in a cell-autonomous manner and cell cycle marker expression shows that cells of brat mutant clones show uncontrolled proliferation, which persists into adulthood. Analysis of the expression of molecular markers, which characterize cell types in wild-type neural lineages, indicates that brat mutant clones comprise an excessive number of cells, which have molecular features of undifferentiated progenitor cells that lack nuclear Prospero (Pros). pros mutant clones phenocopy brat mutant clones in the larval central brain, and targeted expression of wild-type pros in brat mutant clones promotes cell cycle exit and differentiation of brat mutant cells, thereby abrogating brain tumour formation. Taken together, these results provide evidence that the tumour suppressor brat negatively regulates cell proliferation during larval central brain development of Drosophila, and suggest that Prospero acts as a key downstream effector of brat in cell fate specification and proliferation control (Bello, 2006).

Previous studies suggested that brat loss-of-function mutants lead to massive cellular overgrowth and tumour formation in larval optic lobes of Drosophila. These studies also indicated an embryonic requirement for brat to suppress tumour formation. By contrast, the current analysis showed that the brat overproliferation phenotype is due to loss of proliferation control in the larval central brain; the optic lobes initially appear wild-type-like but subsequently are overgrown by neoplastic central brain brat mutant tissue. This conclusion is further supported by MARCM clonal analysis which demonstrated that loss of brat function causes overproliferation in the larval central brain only (Bello, 2006).

In vivo mosaic analysis reveals a cell-autonomous, larval requirement for brat to limit cell proliferation in the brain. Although brat is expressed in all parts of the nervous system both in the embryo, induction of brat mutant clones in the first larval instar is sufficient to cause massive overproliferation in the central brain but not the ventral ganglia. This may suggest that either unknown compensatory mechanisms actively suppress a brat mutant phenotype in the larval ventral ganglia, or that this reflects region-specific differences in cell cycle control. Indeed, transcriptional activity of the mitotic regulator string/Cdc25 is regulated by a plethora of cis-acting elements, most of which are devoted to differential control of cell proliferation during embryonic and larval neurogenesis (Bello, 2006).

During postembryonic neurogenesis, intense proliferation takes place in the brain. This analysis shows that central brain brat mutant clones display sustained cell cycle marker expression, indicating that mutant cells are unable to withdraw from the cell cycle. This is further supported by the presence of enormous brat mutant clones with pronounced proliferative activity even in 3-week-old adult brains, an observation that contrasts with the postmitotic adult wild-type brain. Previous studies have shown that cessation of proliferation in the developing Drosophila brain occurs during metamorphosis, although the underlying genetic mechanisms are currently unknown. The elevated and aberrant cell cycle activity of central brain brat mutant cells suggests that these cells are either able to escape or that they lack cell cycle termination signals (Bello, 2006).

Mosaic analysis demonstrates that enlarged brat mutant clones comprise cells that display sustained expression of neural progenitor cell markers, and simultaneously lack marker gene expression specific for differentiating ganglion cells. Indeed, lack of axonal processes suggests that brat mutant clones comprise an excessive number of mutant cells that are unable to exit the cell cycle and hence do not differentiate into ganglion cells but rather continue to proliferate. These data indicate that brat mutation impairs proliferation control of neural progenitor cells, namely either neuroblasts and GMCs or only one of these progenitors, since in the wild-type central brain only these two cell types are actively engaged in the cell cycle. Based on this analysis it is not possible to distinguish unambiguously between the two possibilities and the underlying mechanisms. The possibility that differentiating ganglion cells de-differentiate due to brat mutation was excluded, because lack of differentiation was consistently observed right after clone induction and also at any later stages of mutant clone development. This was especially exemplified by the lack of nuclear Pros expression, which in the wild type is unambiguously detectable in differentiating progeny of larval neuroblast lineages, namely GMCs as well as ganglion cells (Bello, 2006).

Moreover, loss-of-function analysis indicates that brat mutant MARCM clones lack Pros and also phenocopy pros mutant clones. Thus, enlarged pros mutant clones consist of cells that are devoid of Elav expression, that lack axonal processes but display sustained expression of Grh and Mira as well as cell cycle markers such as CycE, CycB and PH3. These data suggest that mutant clones are essentially devoid of terminally differentiating postmitotic ganglion cells, indicating that Pros functions like Brat in terminating neural progenitor cell proliferation and inducing ganglion cell differentiation. In the embryonic CNS, Pros functions to terminate cell proliferation by repression of cell-cycle activators and simultaneously to induce a differentiation program, effectively coupling the two events. This Pros function appears to be warranted by its localization in the basal cortex of asymmetrically dividing neuroblasts and hence its distribution to only one daughter cell, the GMC. Upon completion of mitosis, Pros translocates from cytoplasm into the nucleus where it executes its transcriptional program ensuring both terminal division of the GMC and cell differentiation of its progeny. In the larval CNS nuclear localisation of Pros is observed in GMCs and ganglion cells but not in the neuroblast, suggesting that Pros has comparable functional features in larval central brain neurogenesis (Bello, 2006).

In addition, the results provide evidence that Pros acts downstream of Brat in neural proliferation control. The following points support this notion: (1) brat mutant clones lack nuclear Pros; (2) brat and pros mutant clones are indistinguishable both at the morphological and at the molecular level; (3) Brat expression is unaltered in pros mutant clones, which together with point no. 1 strongly suggests that Brat is epistatic over Pros; and (4) trans-activation of wild-type pros in brat mutant clones is sufficient to promote both cell cycle exit and differentiation. The experiments, however, do not provide any evidence about the direct or indirect nature of their interaction. Since overexpressed Pros is detected specifically in brat mutant clones in a wild-type-like pattern, the possibility that brat acts as a translational repressor of Pros, comparable to its role in hunchback repression during embryonic abdominal segmentation, is excluded. In addition, brat mutation apparently does not affect pros transcription, since pros RNA in situ hybridization in zygotic brat mutants produced a pattern indistinguishable from wild-type controls. Thus, Brat and Pros may act indirectly in the same pathway, regulating progenitor cell proliferation control in the brain. Alternatively, Brat may act in a process required to cargo Pros, comparable to the function of its mammalian homolog BERP (Bello, 2006).

In vivo mosaic analysis demonstrates that a single mutation in either brat or pros is sufficient to cause brain tumour formation in a cell-autonomous manner, suggesting that indefinite proliferation of brat and pros mutant cells is a cell intrinsic property. GFP-labelled MARCM cells each derive from a common precursor cell, implying that brat and pros mutant cells all descend from individual tumour cells of origin and hence lead to brain tumour formation in a clonally related manner. Moreover, the data indicate that pros and brat mutant clones in the larval central brain are composed of an excessive number of mutant progenitor cells that are unable to differentiate into ganglion cells but rather continue to proliferate. In this sense the results provide in vivo support for the notion that the initiating event in the formation of a malignant tumour is an error in the process of normal differentiation (Bello, 2006).

In addition, the unlimited capacity to generate undifferentiated, proliferating progeny suggests that cells mutant for brat or pros retain self-renewing capacities. In human, brain cancers are thought to arise either from normal stem cells or from progenitor cells in which self-renewal pathways have become activated, however the underlying mechanisms are elusive. The results in Drosophila may therefore provide a rationale and genetic model for the origin of brain cancer stem cells. Although parallels to human tumour formation are speculative, it is noteworthy that TRIM3, a human homolog of brat is located on chromosome 11p15, a region frequently deleted in brain tumours. Moreover, functional studies have shown that the pros homologue Prox1 regulates proliferation and differentiation of neural progenitor cells in the mammalian retina. These data may indicate that brat and pros function in cell differentiation and tumour suppression in an evolutionarily conserved manner (Bello, 2006).

The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for glial subtype-specific prospero expression

The development, organization and function of central nervous systems depend on interactions between neurons and glial cells. However, the molecular signals that regulate neuron-glial communication remain elusive. In the ventral nerve cord of Drosophila, the close association of the longitudinal glia (LG) with the neuropil provides an excellent opportunity to identify and characterize neuron-glial signals in vivo. This study found that the activity and restricted expression of the glycosyltransferase Fringe (Fng) renders a subset of LG sensitive to activation of signaling through the Notch (N) receptor. This is the first report showing that modulation of N signaling by Fng is important for CNS development in any organism. In each hemisegment of the nerve cord the transcription factor Prospero (Pros) is selectively expressed in the six most anterior LG. Pros expression is specifically reduced in fng mutants, and is blocked by antagonism of the N pathway. The N ligand Delta (Dl), which is expressed by a subset of neurons, cooperates with Fng for N signaling in the anterior LG, leading to subtype-specific expression of Pros. Furthermore, ectopic Pros expression in posterior LG can be triggered by Fng, and by Dl derived from neurons but not glia. This effect can be mimicked by direct activation of the N pathway within glia. These genetic studies suggest that Fng sensitizes N on glia to axon-derived Dl and that enhanced neuron-glial communication through this ligand-receptor pair is required for the proper molecular diversity of glial cell subtypes in the developing nervous system (Thomas, 2007).

This study identified Fng as a means by which a specific subtype of glia, the anterior LG, are made sensitive to N activation, evidence was provided that Dl, expressed on axons, activates N signaling in these glia leading to subtype-specific gene expression. Fng is required for maintenance of Pros expression in the anterior LG, which can also be blocked by antagonism of the N pathway with no effect on their survival or positioning. This is in contrast with studies of pros mutants, which found a role for Pros earlier in CNS development in establishing glial cell number. The role of Pros in mature LG is poorly understood, but it has been proposed to retain mitotic potential in these cells for use in repair or remodeling of the nervous system in subsequent larval or adult stages. It will be important to determine the consequences of lost Pros expression from mature anterior LG, and whether additional features and functions of the anterior LG are controlled by N signaling from axons (Thomas, 2007).

The importance of glycosylation for N function has been demonstrated in vivo. The addition of O-linked fucose to EGF repeats in the N extracellular domain is essential for all N activities and is mediated by O-fucosyltransferase-1 (O-fut1). By contrast, Fng is selectively used in specific developmental contexts, and has been best studied in the formation of borders among cells in developing imaginal tissues. Fng catalyzes the addition of GlcNac to O-linked fucose, to which galactose is then added. The resulting trisaccharide is the minimal O-fucose glycan to support Fng modulation of Notch signaling. Fng activity reduces the sensitivity of N for the ligand Ser but increases its sensitivity for Dl. By contrast with imaginal discs, in which modulation of N sensitivity to both ligands appears to be important, loss of Fng in LG resulted in reduced N activation only, consistent with reduced response to Dl. Expression of Pros in LG can be triggered by Dl derived from neurons but not glia, and this effect can be mimicked by direct activation of the N pathway within glia. Genetic experiments implicate neuron-derived Dl as the relevant N ligand for Pros expression in anterior LG, consistent with the ability of Fng to sensitize N to signaling by Dl. Enriched Fng expression in the anterior LG probably renders them differentially sensitive to sustained N signaling from Dl-expressing axons (Thomas, 2007).

The final divisions of the six LG precursors that give rise to 12 LG are thought to be symmetric, with low levels of Pros first distributed evenly between sibling cells after division. However, Pros is maintained and in fact upregulated in the anterior LG, and downregulated in sibling LG that migrate posteriorly. fng transcripts first appear to be expressed in all LG, then become enriched in the anterior LG and reduced in the posterior LG. It is speculated that refinement of fng expression may involve a positive feedback mechanism to consolidate and enhance N signaling in the anterior LG, since preliminary evidence suggests that N signaling can positively influence fng expression in the LG (Thomas, 2007).

Like Pros, Glutamine synthetase 2 (Gs2) is specifically expressed in the anterior LG but not posterior LG, indicating that these are functionally distinct glial subtypes with respect to their ability to recycle the neurotransmitter glutamate. The specificity of N signaling for Pros but not Gs2 indicates that N signaling is unlikely to influence cell fate decisions in the LG lineage and that Fng is unlikely to be the primary determinant of anterior versus posterior LG identity. Rather, Fng probably serves to consolidate this distinction through sustained N signaling (Thomas, 2007).

NICD is a potent activator of Pros expression in the posterior LG. This leads to a consideration of what factors limit Pros expression to the anterior LG in wild-type animals, since posterior LG are indeed capable of expressing Pros in response to constitutive N activity. (1) Based on analysis of fng mutants and Fng misexpression, it is proposed that Fng is a major determinant. The finding that misexpression of Fng causes ectopic Pros in posterior LG supports the argument that Dl-expressing axons do not contact the anterior LG only. It is likely that they make contact with at least some of the posterior LG. Therefore, in wild-type animals, in which Fng is reduced on posterior LG, contact from the subset of Dl axons is alone not sufficient to drive Pros expression. (2) Misexpression of Dl in all postmitotic neurons led to ectopic expression of Pros in posterior LG, indicating that the restricted expression of Dl on a subset of neurons also limits N activation. (3) N appears to be expressed in most or all LG, though it was also found that overexpression of full-length N caused ectopic expression of Pros. From these data a threshold model is proposed for N activation in LG that invokes a combination of factors, including Fng-regulated N sensitivity, exposure of N to ligand, N expression levels, and perhaps others. Increasing any of these factors can provide sufficient signaling for ectopic Pros induction in posterior LG. In wild-type embryos, these factors are also likely to combine with one another in the anterior LG to achieve supra-threshold N signaling and sustained Pros expression during normal development (Thomas, 2007).

Signaling through N is important for glial cell development in Drosophila, although it is context-dependent. Both an embryonic sensory lineage and the subperineurial CNS glial lineage utilize N activation to promote Gcm expression and glial fate. By contrast, in the sensory organ of adult flies, antagonism of N leads to Gcm expression in the glial precursor cell. In vertebrates, signaling through Notch receptors promotes the differentiation of peripheral glia, Müller glia, radial glia and mature oligodendrocytes. A Fng ortholog, lunatic fringe, is expressed in the developing mouse brain in a pattern consistent with glial progenitors. It will be interesting to determine whether Fng-related proteins in vertebrates have a role in glial cell differentiation, and whether they too can modulate N sensitivity and the context of N signaling between neurons and glia (Thomas, 2007).

A conserved nuclear receptor, Tailless, is required for efficient proliferation and prolonged maintenance of mushroom body progenitors in the Drosophila brain: Ectopic expression of Tll represses Pros

The intrinsic neurons of mushroom bodies (MBs), centers of olfactory learning in the Drosophila brain, are generated by a specific set of neuroblasts (Nbs) that are born in the embryonic stage and exhibit uninterrupted proliferation till the end of the pupal stage. Whereas MB provides a unique model to study proliferation of neural progenitors, the underlying mechanism that controls persistent activity of MB-Nbs is poorly understood. This study shows that Tailless (Tll), a conserved orphan nuclear receptor, is required for optimum proliferation activity and prolonged maintenance of MB-Nbs and ganglion mother cells (GMCs). Mutations of tll progressively impair cell cycle in MB-Nbs and cause premature loss of MB-Nbs in the early pupal stage. Tll is also expressed in MB-GMCs to prevent apoptosis and promote cell cycling. In addition, it was shown that ectopic expression of tll leads to brain tumors, in which Prospero, a key regulator of progenitor proliferation and differentiation, is suppressed whereas localization of molecular components involved in asymmetric Nb division is unaffected. These results as a whole uncover a distinct regulatory mechanism of self-renewal and differentiation of the MB progenitors that is different from the mechanisms found in other progenitors (Kurusu, 2009).

tll was expressed in the dividing MB-Nbs and GMCs, but not in the postmitotic neurons, through the stages of MB development. Tll expression is initially found in almost all procephalic neuroblasts, but became largely restricted to anterior cells by stage 16. Double immunostaining with an anti-Dac antibody, which labels MB neurons, confirmed that they were MB-Nbs and GMCs. In the larval stages, Tll is expressed in the MB-Nbs and GMCs as well as in lamina precursor cells. While the expression in lamina precursor cells disappears by the end of the larval stage, Tll expression in the MB progenitors is maintained during the pupal stages. In newly eclosed flies, Tll expression was found in a few GMC-like cells in the middle of the MB cell clusters, although their exact identity is unknown (Kurusu, 2009).

Several lines of evidence indicate that Tll is cell autonomously required for efficient proliferation activity MB-Nbs. BrdU labeling experiments demonstrate that DNA synthesis is partially suppressed in tll mutant Nbs in both the larval and the pupal stages. Cell cycle defects in the mutant MB-Nbs are not evident in the larval stage but confirmed by marked suppression of PH3 and Cyc B activity at 20 h APF before the disappearance of mutant Nbs. As a whole, these data suggest that Tll is required to maintain efficient cell cycle progression in MB-Nbs, particularly in the pupal stage. In contrast, although the premature loss of the mutant Nbs might be a consequence of cell cycle exit as has been suggested with other Nbs, the exact mechanism of the disappearance of mutant MB-Nbs in the early pupal stage is unknown. It is also plausible that mutant Nbs are removed by apoptosis, as is the case with mutant GMCs, although TUNEL signals for MB-Nbs were not detected at 20 h APF, shortly before their disappearance whereas cell death signals in GMCs are evident at both the larval and pupal stages (Kurusu, 2009).

Despite marginal reduction in cell division activity of MB-Nbs at the larval stage, loss of tll activity results in significant reduction of the larval MB clones. Instead, the results demonstrate that cell cycle progression is impaired in larval MB-GMCs. Moreover, the majority of the MB-GMCs are lost by cell death. The molecular mechanism underlying these GMC defects is yet to be investigated, but it is unlikely that they are mediated by altered Pros expression since Pros is co-expressed with Tll in wild-type MB-GMCs, and its expression is unaltered in mutant GMCs. In addition, the results demonstrating that neither p35 nor Diap1 rescues GMC death suggest that Tll might be involved in suppression of an unconventional cell death pathway (Kurusu, 2009).

What is the molecular function of Tll in the regulation of MB progenitors? The fact that Tll is a transcription factor localized in the nucleus suggests that Tll might specify neuronal identity of MB progenitors by regulating cell-type specific genes. However, unlike other regulatory factors that confer either spatial or temporal identity, Tll is expressed only in Nbs and GMCs, and mutant neurons exhibit wild-type like dendritic and axonal wiring patterns even in the adult stage, in which perdurance of wild-type tll activity in the mutant clones is unlikely. Rather, Tll might provide MB progenitors with cellular identity that specify a distinctive proliferation pattern, either by promoting cell cycle or by preventing apoptosis or by both in parallel. In any case, such identity cannot be determined by Tll on its own because Tll is expressed in other neuronal progenitors such as lamina precursor cells in the optic lobes. Instead, it is presumed that the proliferation identity of MB progenitors may be specified in combination with other regulatory factors such as Eyeless, which is expressed in MB-Nbs, GMCs and postmitotic neurons to control MB development (Kurusu, 2009).

In the course of MB proliferation, Tll might downregulate key regulatory genes involved in cell-cycle exit and differentiation, particularly given the fact that Tll functions mostly as a repressor in the early embryogenesis. One such candidate gene is pros. Pros is detected in MB-GMCs, but not MB-Nbs. However, loss of pros causes neither tumorous transformation of MB progenitors nor suppression of tll phenotype in pros tll double mutant clones. Moreover, Pros is not upregulated in tll mutant clones. Thus, these data argue against the involvement of pros in the regulation of MB progenitors although they do not exclude a redundant mechanism involving Pros cooperating with other factors. Alternatively, Tll could indirectly control cell cycle progression by downregulating genes that suppress progenitor division. In support of this, it is noteworthy that the mammalian homolog Tlx (NR2E1) represses a tumor suppressor gene, Pten, via consensus Tll/TLX binding sites located in the pten promoter, and thereby indirectly stimulates the expression of various cell cycle genes including Cyclin D1, p57 kip2, and p27 kip1 (Kurusu, 2009).

Studies on Drosophila neural progenitors reveal heterogeneity among the brain Nbs in terms of temporal windows of cell division, patterns of self-renewal, and susceptibility to mutations that regulate proliferation and termination of progenitors. Among the Nbs in the Drosophila brain, MB-Nbs exhibit a highly unique proliferation pattern. Most Nbs pause cell division between the late embryonic and the early first instar stages, and cease proliferation by the early pupal stage. By contrast, MB-Nbs divide continuously from the embryonic stage till the end of pupal stage, generating diverse identities of neurons by temporal order. In house cricket and moth, proliferation activity of MB-Nbs further extends beyond the pupal stage to exhibit persistent neurogenesis during adult life (Kurusu, 2009).

Although the data clearly indicate a pivotal function of Tll for persistent proliferation and maintenance of MB-Nbs, the mechanism that determines the exit from cell cycling at the end of pupal stage remains elusive. Neither extension of Tll expression beyond the end of the pupal period nor blocking cell death program, by p35 or Diap1, prolonged MB-Nb proliferation beyond the pupal stage, suggesting existence of other mechanisms that schedule the end of MB-Nb activity. In most brain Nbs, a burst of Pros in the nucleus at around 120 h after larval hatching (24 h APF) induces cell cycle exit to regulate generation of postmitotic progeny in the brain. However, no burst of nuclear Pros is detected for MB-Nbs at the end of the pupal stage when they finally exit from cell cycling, although the data demonstrate that, as is the case with other Nbs in the brain, Pros indeed has such regulatory potential in larval MBs that its overexpression results in partial loss of the MB-Nbs. Moreover, MB clones lacking pros activity, which exhibit normal growth, cease cell division by the end of the pupal stage (Kurusu, 2009).

During asymmetric cell division of Drosophila Nbs, Pros is kept inactive by tethering to the cell cortex by MIRA. At telophase of Nb cell cycle, Pros is segregated into GMC, where it enters the nucleus to trigger cell cycle exit and promote differentiation of post mitotic progeny that are generated by the division of GMC. Accordingly, nuclear Pros is expressed at high levels in postmitotic neurons and at moderate levels in GMCs. However, whereas this partition pattern of Pros in the post-embryonic brain is shared between MB and non-MB progenies, Pros seems dispensable for cell-cycle control of MB-GMCs. In non-MB lineages, loss of pros activity in GMCs leads to failure of cell-cycle exit and transforms of GMCs into Nbs. However, loss of pros activity never causes transformation of MB-GMCs although mutant MB neurons exhibit considerable dendritic defects. In contrast, Tll is expressed and required for MB-GMCs to suppress apoptosis and maintain active cell cycling. Intriguingly, whereas Pros is suppressed by Tll in non-MB progenitors, both proteins are coexpressed in MB-GMCs, clearly suggesting that, as compared to the progenitors of non-MB lineages, a different mechanism may operate in MB progenitors to control the expression of regulatory factors that are important for cell division and differentiation (Kurusu, 2009).

The brain hyperplasia produced by Tll overexpression is reminiscent of brain tumors caused by mislocalization of asymmetric determinants. Aberrant Nb divisions that disrupt the positioning of such factors generate brain tumors. Brain tissues from pins, mira, numb, or pros mutants generate tumors when transplanted in the wild-type abdomen. In double mutants of pins and lgl, mislocalization of aPKC in the basal cortex results in the generation of supernumerary Nbs at the expense of GMCs, and thus, neurons. BRAT is required for the asymmetric positioning of Pros, which in turn suppresses self-renewal of GMC and promotes cell differentiation by transcriptional control. Mutant clones of either brat or pros are highly tumorigenic, forming a large number of MIRA-positive Nbs (Kurusu, 2009).

While recapitulating the tumor phenotype, ectopic expression of Tll does not affect asymmetric localization of aPKC, PINS, and BRAT. Instead, Tll downregulates Pros in hyperplasic brains and in overexpression clones, suggesting that the tumorigenesis phenotype caused by Tll expression is mediated by Pros downregulation in GMCs. This notion is further supported by the fact that coexpression of Pros with Tll suppresses brain hyperplasia. Notably, the cis-regulatory region of pros harbors a consensus Tll binding site within 500 base pairs from the transcriptional initiation site, consistent with the idea that Tll might repress transcription of pros via direct DNA binding (Kurusu, 2009).

Recently, atypical large Nb lineages in the dorsomedial part of the larval brain have been described and designated as Posterior Asense-Negative (PAN) Nbs. Nbs of such lineages divide asymmetrically to self renew, but, unlike other Nbs, generate smaller intermediate progenitors that express Nb markers. The fact that these atypical Nbs are MIRA-positive and Pros negative raises a possibility that tumor clones induced by Tll could either correspond to or originate from them. As with other Nbs, clones of the PAN-Nb lineages accompany only a single large Nb, with their progeny arranged regularly in a columnar order. By contrast, clones generated by Tll overexpression harbor several large to intermediate-sized Nbs, exhibiting irregular morphology, which is typical of tumors. PAN-Nbs are the Nb subpopulation that exhibits overgrowth in brat mutants. However, it is also unlikely that Tll induced overgrowth originates from overgrowth of PAN Nbs, which correspond to eight Nbs in the DPM group among the ~90 Nbs per hemisphere. On the contrary, Tll induces clonal tumors not only in DPM but also in CM and BLP lineages. Indeed, Tll overgrowth phenotype is not localized to a specific location of the hemisphere, but broadly detectable in the brain including the optic lobe. Moreover, Tll overgrowth phenotype is also induced in the embryonic CNS, arguing against the involvement of larval PAN-Nbs (Kurusu, 2009).

The Drosophila Tll and the vertebrate homolog TLX (NR2E1) share high sequence similarity in the DNA binding domain. Tlx mutant mice exhibit a reduction of rhinencephalon and limbic structures with emotional and learning defects. Notably, Tlx mutant mice exhibit reduction of neuron numbers in cortical upper layers. Postnatally, TLX is localized to the adult neurogenic regions including the subgranular layer of the dentate gyrus to maintain stem cells in a proliferative and undifferentiated state. Recent behavioral studies have shown that such TLX-positive neural stem cells actually contribute to animal's spatial learning. Thus, combined with the current results, these studies highlight a functional commonality of the tll/Tlx homologs between flies and mammals, and imply an intriguing evolutionary conservation of the genetic programs underlying neural progenitor controls in crucial brain structures involved in memory and other cognitive functions (Kurusu, 2009).

Intriguingly, the mammalian pros homolog Prox1 promotes cell cycle exit and differentiation of the neural progenitors in the developing subventricular zone and the retina, the neural tissues in which Tlx functions antagonistically to control progenitor proliferation. Based on the tll GOF phenotypes in Drosophila, it is predicted that deregulation of Tlx in the developing brain may cause suppression of Prox1 and could result in severe neurological tumors in humans. On the other hand, consistent with the loss-of-function phenotypes in flies, several mutations have been identified in the regulatory regions of Tlx in humans with microcephary. Given the commonality in progenitor control, further studies of the Drosophila MB-Nbs may shed light on the molecular basis of the proliferation and differentiation of neural progenitors, and would provide important cues for understanding progenitor disorders in the human brain (Kurusu, 2009).

dFezf/Earmuff maintains the restricted developmental potential of intermediate neural progenitors in Drosophila; Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation

To ensure normal development and maintenance of homeostasis, the extensive developmental potential of stem cells must be functionally distinguished from the limited developmental potential of transit amplifying cells. Yet the mechanisms that restrict the developmental potential of transit amplifying cells are poorly understood. This study shows that the evolutionarily conserved transcription factor dFezf/Earmuff (Erm) functions cell-autonomously to maintain the restricted developmental potential of the intermediate neural progenitors generated by type II neuroblasts in Drosophila larval brains. Although erm mutant intermediate neural progenitors are correctly specified and show normal apical-basal cortical polarity, they can dedifferentiate back into a neuroblast state, functionally indistinguishable from normal type II neuroblasts. Erm restricts the potential of intermediate neural progenitors by activating Prospero to limit proliferation and by antagonizing Notch signaling to prevent dedifferentiation. It is concluded that Erm dependence functionally distinguishes intermediate neural progenitors from neuroblasts in the Drosophila larval brain, balancing neurogenesis with stem cell maintenance (Weng, 2010).

Tissue development and homeostasis often require stem cells to transiently expand the progenitor pool by producing transit amplifying cells. Yet the developmental potential of transit amplifying cells must be tightly restricted to ensure generation of differentiated progeny and to prevent unrestrained proliferation that might lead to tumorigenesis. Transit amplifying cells are defined by their limited developmental capacity, a feature specified during fate determination. It is unknown whether an active mechanism is required to maintain restricted developmental potential in transit amplifying cells after specification. This study used intermediate neural progenitors (INPs) in developing Drosophila larval brains as a genetic model to investigate how restricted developmental potential is regulated in transit amplifying cells (Weng, 2010).

A fly larval brain hemisphere contains eight type II neuroblasts that undergo repeated asymmetric divisions to self-renew and to generate immature INPs. Immature INPs are unstable in nature and are mitotically inactive, and they lack the expression of Deadpan (Dpn) and Asense (Ase). Immature INPs commit to the INP fate through maturation, a differentiation process necessary for specification of the INP identity. INPs express Dpn and Ase, and undergo 8-10 rounds of asymmetric divisions to self-renew and to produce ganglion mother cells (GMCs) that typically generate two neurons. While 5-6 immature INPs and 1-2 young INPs are always in direct contact with their parental neuroblasts, the older INPs become progressively displaced from their parental neuroblasts over time (Weng, 2010).

During asymmetric divisions of type II neuroblasts, the basal proteins Brain tumor and Numb are exclusively segregated into immature INPs, and function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate. brain tumor or numb mutant type II neuroblasts generate immature INPs that fail to mature and do not commit to the INP fate. Instead, brain tumor or numb mutant immature INPs adopt their parental neuroblast fate, leading to supernumerary type II neuroblasts. Thus, brain tumor and numb specify the INP fate, and the ectopic expansion of type II neuroblasts in these mutant genetic backgrounds occurs due to failure to properly specify the INP fate. Although Brain tumor is also asymmetrically segregated into GMCs during asymmetric divisions of INPs, the mosaic clones in brain tumor mutant INPs contain only differentiated neurons. This result indicates that Brain tumor is dispensable for maintaining the restricted developmental potential of INPs. How restricted developmental potential is maintained in INPs is currently unknown (Weng, 2010).

To identify genes that regulate self-renewal of neuroblasts, a genetic screen was conducted for mutants exhibiting ectopic larval brain neuroblasts. One mutation, l(2)5138, specifically resulted in massive expansion of neuroblasts in the brain but did not affect neuroblasts on the ventral nerve cord. The l(2)5138 mutation mapped to the 22B4-7 chromosomal interval that contains the earmuff (erm) gene (Pfeiffer, 2008). The erm transcripts are first detected at embryonic stage 4-6 in the specific domain preceding formation of the embryonic brain and remain highly expressed in the brain throughout development. Tbis study reports that Erm functions to restrict the developmental potential of INPs by promoting Prospero-dependent termination of proliferation and suppressing Notch-mediated dedifferentiation. By restricting their developmental potential, Erm ensures that INPs generate only differentiated neurons during Drosophila neurogenesis (Weng, 2010).

All neuroblasts in l(2)5138 homozygous mutant brains were proliferative, expressed all known neuroblast markers, and lacked neuronal and glial markers. The l(2)5138 mutation mapped to the erm gene, which encodes a homolog of the vertebrate Forebrain embryonic zinc-finger family (Fezf) transcription factors. The l(2)5138 mutants contained a single A/T nucleotide change in the erm coding region, leading to the substitution of a leucine for a conserved histidine in the third C2H2 zinc-finger domain. Consistent with its predicted molecular function, ectopic expression of Erm transgenic proteins tagged with a HA epitope at the amino- or carboxyl-terminus driven by neuroblast-specific Wor-Gal4 was detected in the nuclei of neuroblasts. However, the expression of the HA-tagged Erm transgenic protein bearing the identical leucine-to-histidine substitution as in the l(2)5138 mutant was undetectable, suggesting that the mutant Erm protein is unstable. It is concluded that l(2)5138 is a mutant allele of erm (Weng, 2010).

To determine whether erm mutant brains have ectopic type I and/or type II neuroblasts, the expression pattern was examined of Ase and Prospero (Pros), which are only expressed in type I neuroblasts. It was found that erm mutant brains contained over 20-fold more type II neuroblasts (Dpn+Ase-) than wild-type brains, with no significant change in the number of type I neuroblasts (Dpn+Ase+). Next, the localization of Prospero was examined in mitotic neuroblasts in larval brains expressing GFP induced by Ase-Gal4 (Ase > GFP), which mimicked the expression pattern of the endogenous Ase protein. In erm mutant larval brains, all mitotic type I neuroblasts (GFP+) showed formation of basal Prospero crescents, but none of the mitotic type II neuroblasts (GFP-) showed the expression of Prospero. Furthermore, GFP-marked erm mutant type II neuroblast clones consistently contained multiple type II neuroblasts, whereas erm mutant type I neuroblast clones always contained single type I neuroblasts and neurons. It is concluded that erm mutant brains exhibit an abnormal expansion of type II neuroblasts (Weng, 2010).

To determine the cellular origin of ectopic type II neuroblasts in erm mutant brains, the identity of cells in the GFP-marked clones derived from wild-type or erm mutant type II neuroblasts was examined using specific cell fate markers. At 30 hr after clone induction, wild-type and erm mutant neuroblast clones appeared indistinguishable, containing single parental neuroblasts (Dpn+Ase-; R10 mm) in direct contact with 5-6 immature INPs (Dpn-Ase-), while most of the INPs (Dpn+Ase+; R6 mm) were 1 cell or more away from the parental neuroblasts. At 48 hr after clone induction, the overall size of both wild-type and erm mutant neuroblast clones increased significantly due to an increase in cell number, reflecting continuous asymmetric divisions of the parental neuroblasts. In both wildtype and erm mutant clones, the parental neuroblasts remained surrounded by 5-6 immature INPs, while INPs and differentiated neurons (Dpn-Ase-Pros+) were found several cells away from the parental neuroblasts. However, erm mutant clones contained fewer INPs than the wild-type clones. Importantly, erm mutant clones consistently contained 4-6 smaller ectopic type II neuroblasts (Dpn+Ase-; 6-8 mm in diameter). Thus, Erm is dispensable for both the generation and maturation of immature INPs (Weng, 2010).

Ectopic type II neuroblasts in 48 hr erm mutant clones were always several cells away from the parental neuroblasts. This result strongly suggests that ectopic type II neuroblasts in erm mutant clones likely originate from INPs and Erm likely functions in INPs. However, it was not possible to assess the spatial expression pattern of the endogenous Erm protein in larval brains due to lack of a specific antibody and low signals by fluorescent RNA in situ. Alternatively, the expression of the R9D series of Gal4 transgenes was analyzed, in which Gal4 is expressed under the control of overlapping erm promoter fragments (Pfeiffer, 2008). The expression of R9D11-Gal4 was clearly detected in INPs, but was undetectable in type II neuroblasts and immature INPs even when two copies of the UAS-mCD8-GFP transgenes were driven by two copies of R9D11-Gal4 at 32°C for 72 hr after larval hatching. Consistently, the expression of Erm-Gal4 was virtually undetectable in brain tumor mutant brains that contain thousands of type II neuroblasts and immature INPs. While the expression of UAS-erm induced by the neuroblast-specific Wor-Gal4 driver led to premature loss of type II neuroblasts, expression of UAS-erm driven by Erm-Gal4 failed to exert any effect on type II neuroblasts. Importantly, targeted expression of the fly Erm or mouse Fezf1 or Fezf2 transgenic protein driven by R9D11-Gal4 restored the function of Erm and efficiently rescued the ectopic neuroblast phenotype in erm mutant brains. Therefore, R9D11-Gal4 (Erm-Gal4) contains the enhancer element sufficient to restore the Erm function in INPs leading to suppression of ectopic type II neuroblasts in erm mutant brains (Weng, 2010).

Mutant clonal analyses and overexpression studies strongly suggest that Erm functions to suppress reversion of INPs back into a neuroblast state. This study directly tested whether INPs in erm mutant brains can dedifferentiate back into type II neuroblasts. βgal-marked lineage clones originating exclusively from INPs were induced via FRT-mediated recombination. A short pulse of flipase (FLP) expression was targeted in INPs by heat-shocking larvae carrying a UAS-flp transgene under the control of Erm- Gal4 and tub-Gal80ts at 30°C for 1 hr. At 72 hr after heat shock, INP clones in wildtype brains contained only differentiated neurons (Dpn-Ase-). In contrast, INP clones in erm mutant brains contained one or more type II neuroblasts as well as immature INPs, INPs, GMCs, and neurons. This result indicates that while INPs in wild-type larval brains can give rise to only neurons, INPs in erm mutant brains can dedifferentiate into type II neuroblasts that can give rise to all cell types found in a normal type II neuroblast lineage. It is concluded that Erm functions to maintain the restricted developmental potential of INPs and prevents them from dedifferentiating back into a neuroblast state (Weng, 2010).

To determine how Erm maintains the restricted developmental potential of INPs, microarray analyses was performed, and prospero mRNA was found to be drastically reduced in erm mutant brains compared to the control brains. It was confirmed that the relative level of prospero mRNA was indeed reduced by 60%-70% in erm mutant brain extracts by using real-time PCR. These data supported that Erm is necessary for proper transcription of prospero, and prompted a test to see if overexpression of Erm might be sufficient to induce ectopic Prospero expression. A short pulse of Erm expression in brain neuroblasts was induced by shifting larvae carrying a UAS-erm transgene under the control of Wor- Gal4 and tub-Gal80ts to from 25°C to 30°C. A 3.5 hr pulse of Erm expression was sufficient to induce nuclear localization of Prospero in larval brain neuroblasts. Consistent with nuclear Prospero promoting termination of neuroblast proliferation, ectopic expression of Erm induced by Wor-Gal4 resulted in decreased neuroblasts compared to wild-type brains (Figure 5B). Thus, it is concluded that overexpression of Erm can restrict neuroblast proliferation by triggering nuclear localization of Pros (Weng, 2010).

The data suggest that Erm might restrict the developmental potential of INPs in part by limiting their proliferation by activating Prospero-dependent cell cycle exit. If so, it was predicted that overexpression of Erm should induce ectopic nuclear Prospero in INPs and overexpression of Prospero should suppress ectopic neuroblasts in erm mutant brains. In wild-type brains, 9.6% of INPs (32/325) showed nuclear localization of Prospero. However, overexpression of Erm driven by Erm-Gal4 led to nuclear localization of Prospero in 41.5% of INPs (105/253), likely restricting their proliferation potential and resulting in some parental type II neuroblasts surrounded only by differentiated neurons. Importantly, ectopic expression of Prospero induced by Erm-Gal4 efficiently suppressed ectopic neuroblasts and restored neuronal differentiation in erm mutant brains. Thus, Erm likely restricts the proliferation of INPs by promoting nuclear localization of Prospero. To confirm that Prospero indeed functions downstream of Erm to restrict the proliferation of INPs, genetic epistatic analyses were performed. Consistent with previously published results, prospero mutant type I neuroblast clones contained ectopic type I neuroblasts. In contrast, prospero mutant type II neuroblast clones exhibited accumulation of ectopic INPs while maintaining single parental neuroblasts. Furthermore, overexpression of Erm failed to suppress ectopic INPs in prospero mutant type II neuroblast clones, consistent with Prospero functioning downstream of Erm. These results indicate that blocking differentiation is not sufficient to trigger the dedifferentiation of INPs back into type II neuroblasts. Thus, Erm’s restriction on the proliferation of INPs is dependent on Prospero function, but its suppression of the dedifferentiation of INPs is independent of Prospero (Weng, 2010).

Previous studies showed that overexpression of constitutively active Notch (Notchintra) in both type I and II neuroblasts is sufficient to trigger ectopic neuroblasts. This study tested whether Erm suppresses the dedifferentiation of INPs by inhibiting Notch signaling. Indeed, knockdown of Notch function by RNAi in erm mutant brains led to a dramatic reduction in ectopic type II neuroblasts compared to erm mutant brains alone. Complementarily, ectopic expression of constitutively active Notch (Notchintra) induced by Erm-Gal4 transforms INPs into ectopic type II neuroblasts. Thus, reduced Notch function suppresses the dedifferentiation of INPs in erm mutant brains whereas ectopic activation of Notch induces the dedifferentiation of INPs. Next, whether Erm suppresses the dedifferentiation of INPs by antagonizing a Notch-activated mechanism was tested. Coexpression of Erm under the control of Erm-Gal4 is sufficient to suppress ectopic neuroblasts induced by the expression of Notchintra. Thus, it is concluded that Erm can suppress the dedifferentiation of INPs by negatively regulating a Notch-activated signaling mechanism (Weng, 2010).

This study has reported a mechanism that actively maintains the restricted developmental potential of transit amplifying cells after specification of their identity. The evolutionarily conserved transcription factor Erm/Fezf functions to maintain the restricted developmental potential of INPs by limiting their proliferation potential and suppressing their dedifferentiation capacity. Combining proper specification of the transit amplifying cell identity and active maintenance of their restricted developmental potential ensures the generation of differentiated progeny and prevents aberrant expansion of stem cells (Weng, 2010).

The lineage clones derived from single INPs in erm1/erm2 mutant brains contain dedifferentiated neuroblasts, immature INPs, INPs, GMCs, and neurons. Several mechanisms could lead to the diversity of cells within the clones. First, INPs in erm mutant brains might generate GMCs and neurons initially due to the presence of maternally deposited Erm. However, erm transcripts are undetectable in both adult male and female germlines by microarray analyses and in stage 1-3 embryos by RNA in situ. Furthermore, the erm1/erm2 allelic combination resulted in little to no zygotic Erm in the brain because the erm1 mutation likely leads to the production of an unstable Erm protein, whereas the erm2 mutation deletes the entire erm open reading frame. Additionally, the ectopic neuroblast phenotype in erm1/erm2 mutant brains can be observed as early as 36-48 hr after larval hatching. Thus, generation of GMCs and differentiated neurons by INPs in erm1/erm2 mutant brains is unlikely due to the maternal effect. Alternatively, erm may promote GMC differentiation in the type II neuroblast lineage, and in erm mutant brains, GMCs might dedifferentiate back into neuroblasts. If so, an ectopic accumulation of INPs would be predicted in similarly staged mosaic clones derived from erm mutant type II neuroblasts as compared to wild-type clones. However, 48 hr erm mutant single neuroblast clones consistently contained fewer INPs when compared to the wild-type clones. In addition, blocking GMC differentiation by removing Prospero function resulted in ectopic accumulation of INPs but did not lead to ectopic neuroblast formation. Therefore, the diversity of cells within erm mutant clones is also unlikely due to blocking GMC differentiation. The interpretation is favored that erm mutant INPs dedifferentiate into apparently normal neuroblasts that can give rise to all cell types found in a type II neuroblast lineage. Consistently, the dedifferentiated neuroblasts in erm mutant brains exhibited normal cortical polarity and proliferation potential. Furthermore, the dedifferentiated neuroblasts in erm mutant brains also lost the expression of Pros-Gal4 and Erm-Gal4 and established ectopic type II neuroblast lineages encapsulated by the cortex glial membrane.Thus, it is concluded that Erm likely restricts the developmental potential of INPs by limiting proliferation and suppressing dedifferentiation (Weng, 2010).

Although mutations in erm, brain tumor, and numb genes all lead to ectopic type II neuroblasts, the proteins appear to regulate INPs at distinct steps in the type II neuroblast lineage. Numb and Brain tumor function cooperatively, but nonredundantly, to ensure that immature INPs undergo maturation and commit to the INP fate (Boone, 2008; Bowman, 2008). While ectopic expression of Numb induces premature differentiation of type II neuroblasts and immature INPs, overexpression of Numb is not sufficient to suppress ectopic neuroblasts in brain tumor mutant brains. Thus, Numb likely promotes differentiation of immature INPs whereas Brain tumor likely prevents immature INPs, which are unstable in nature, from adopting their parental neuroblast fate. More studies will be necessary to discern whether ectopic neuroblasts in brain tumor mutant brains arise from dedifferentiation of partially differentiated immature INPs or failure of immature INPs to initiate differentiation. In contrast, immature INPs in erm mutant brains mature into functional INPs that exhibit normal cortical polarity and proliferation potential and can generate GMCs and neurons. Additionally, overexpression of Brain tumor or Numb in INPs was not sufficient to suppress ectopic neuroblasts in erm mutant brains. Finally, lineage clones derived from single INPs in erm mutant brains always contain ectopic type II neuroblasts, multiple immature INPs, INPs, GMCs, and neurons. These results indicate that Erm is dispensable for maturation of immature INPs and is not within the genetic hierarchy specifying the INP identity. Instead, Erm maintains the restricted developmental potential of INPs after specification of their identity (Weng, 2010).

Prospero encodes a homeodomain transcription factor, and nuclear Prospero has been shown to trigger cell cycle exit and GMC differentiation. In the wild-type brain, 9.6% of INPs showed nuclear Prospero and were likely undergoing differentiation. prospero mutant type II neuroblast clones showed ectopic accumulation of INPs but contained single neuroblasts, indicating that blocking differentiation is not sufficient to trigger the dedifferentiation of INPs. Thus, Prospero restricts the proliferation potential of INPs but does not suppress dedifferentiation of INPs (Weng, 2010).

While ectopic expression of Prospero in INPs can restore neuronal differentiation in erm mutant brains, targeted expression of Erm in neuroblasts or INPs was sufficient to induce rapid nuclear localization of Prospero in these cells and terminate their proliferation. In wild-type brains, Prospero is sequestered in a basal crescent by the adaptor protein Miranda in mitotic neural progenitors. Interestingly, mitotic neural progenitors including neuroblasts and INPs transiently overexpressing Erm also showed basal localization and segregation of Miranda and Prospero. As such, Erm likely restricts the proliferation potential of INPs by indirectly promoting nuclear localization of Prospero. Therefore, Prospero does not localize in the nuclei of mitotically active INPs, which express Miranda, but does localize in the nuclei of GMCs that do not express Miranda (Weng, 2010).

How does Erm suppress the dedifferentiation of INPs? The results show that reduced Notch function can efficiently suppress ectopic neuroblasts in erm mutant brains while constitutive activation of Notch signaling induced the dedifferentiation of INPs. Importantly, coexpression of Erm is sufficient to suppress the dedifferentiation of INPs triggered by expression of constitutively active Notchintra. Together, these results strongly suggest that Erm prevents the dedifferentiation of INPs by antagonizing a Notch-activated mechanism through interfering with the assembly of the Notch transcriptional activator complex or inhibiting the expression of Notch targets. Intriguingly, the amino terminus of all Fezf proteins contains an engrailed homology 1 domain. This domain can mediate direct interaction with the conserved transcriptional corepressor Groucho that can function as a corepressor of Notch signaling. Additional experiments will be needed to discern how Erm antagonizes Notch-activated dedifferentiation of INPs (Weng, 2010).

Histone deacetylase Rpd3 regulates olfactory projection neuron dendrite targeting via the transcription factor Prospero

Compared to the mechanisms of axon guidance, relatively little is known about the transcriptional control of dendrite guidance. The Drosophila olfactory system with its stereotyped organization provides an excellent model to study the transcriptional control of dendrite wiring specificity. Each projection neuron (PN) targets its dendrites to a specific glomerulus in the antennal lobe and its axon stereotypically to higher brain centers. Using a forward genetic screen, a mutation in Rpd3 was identified that disrupts PN targeting specificity. Rpd3 encodes a class I histone deacetylase (HDAC) homologous to mammalian HDAC1 and HDAC2. Rpd3−/− PN dendrites that normally target to a dorsolateral glomerulus mistarget to medial glomeruli in the antennal lobe, and axons exhibit a severe overbranching phenotype. These phenotypes can be rescued by postmitotic expression of Rpd3 but not HDAC3, the only other class I HDAC in Drosophila. Furthermore, disruption of the atypical homeodomain transcription factor Prospero (Pros) yields similar phenotypes, which can be rescued by Pros expression in postmitotic neurons. Strikingly, overexpression of Pros can suppress Rpd3−/− phenotypes. This study suggests a specific function for the general chromatin remodeling factor Rpd3 in regulating dendrite targeting in neurons, largely through the postmitotic action of the Pros transcription factor (Tea, 2010).

To identify genes that are essential for dendrite wiring specificity in Drosophila olfactory projection neurons (PNs), a MARCM-based forward genetic screen was performed using ethyl methanesulfonate as mutagen. MARCM allows visualization and genetic manipulation of single cell or neuroblast clones in an otherwise heterozygous animal, allowing the study of essential genes in mosaic animals. GH146-GAL4 was used to label a single PN born at newly hatched larva, which in wild-type animals always projects its dendrites to the dorsolateral glomerulus DL1 in the antennal lobe. A mutant, called 12-37, was identified in which DL1 PNs mistargeted towards dorsomedial or ventromedial regions of the antennal lobe. SNP and deletion mapping identified the causal gene to be Rpd3, encoding a homolog of mammalian HDAC1 and HDAC2. The mutant 12-37 causes a G136R missense mutation in the catalytic domain; the glycine at this position is conserved from yeast to humans (Tea, 2010).

The mistargeting phenotype was conserved to be caused by the mutation in Rpd3 using the following two criteria. First, MARCM single cell clones of two previously existing Rpd3 mutants gave similar mistargeting phenotypes. Second, MARCM expression of UAS-Rpd3 in DL1 single cell clones using GH146-GAL4 significantly rescued the phenotype for all three Rpd3 alleles, especially at higher temperatures where GAL4 activity increases. Because GH146-GAL4 expresses UAS-Rpd3 only in postmitotic PNs, it was conclude that Rpd3 plays an essential role in postmitotic PNs to regulate dendrite targeting (Tea, 2010).

Three lines of evidence indicate that Pros expression and function in the PN lineage differs from its function in the embryonic lineage. First, Pros is clearly present in postmitotic PNs. Immunostaining for Pros at 24hAPF shows that wild-type postmitotic PNs express varying amounts of Pros protein; this variable expression persists throughout development and adulthood. Wild-type DL1 PNs express an intermediate level of Pros protein. pros−/− DL1 or neuroblast clone PNs have an undetectable level of Pros protein in the cell body, confirming both antibody specificity and the nature of the loss-of-function mutation. Pros immunoreactivity can be restored by MARCM expression of UAS-pros in postmitotic PNs (Tea, 2010).

Second, the pros−/− single cell clone phenotype indicates that pros mRNA must be transcribed in postmitotic neurons and/or ganglion mother cells; it cannot be transcribed only in neuroblasts. There are two scenarios in which a single cell MARCM clone can be produced. The mitotic recombination could occur right before the ganglion mother cell divides to give rise to two postmitotic cells, and therefore only the postmitotic DL1 PN is pros−/−. Alternatively, because the sibling of the DL1 PN dies during development, the mitotic recombination could also occur in the neuroblast; in this case, the ganglion mother cell giving rise to the DL1 PN is pros−/−. In either case, the neuroblast remains pros+/−. Therefore pros must be transcribed either in the postmitotic PN or the ganglion mother cell to account for a single cell phenotype (Tea, 2010).

Third, postmitotic UAS-pros expression can rescue the mistargeting defect of pros−/− DL1 dendrites as well as overbranching of DL1 axons, indicating that Pros functions in postmitotic neurons to regulate PN dendrite targeting and axon branching (Tea, 2010).

It has long been thought that histone deacetylases play a general role in chromatin remodeling and transcriptional control, and many studies have examined genome-wide patterns of histone modifications. Yet recent studies have also suggested that 'general' chromatin remodeling factors can have very specific roles. This study has shown a new function for Rpd3, a ubiquitously expressed protein that is the major histone deacetylase in Drosophila olfactory projection neurons. Rpd3 plays a specific role in controlling dendrite targeting and axon terminal branching, and this function cannot be replaced by the only other class I HDAC. Furthermore, this study has shown that the majority of its function in regulating dendrite targeting and a portion of its function in regulating axon branching are likely carried out through the downstream transcription factor Prospero (Tea, 2010).

Although the possibility cannot be ruled that Rpd3 and Pros act in parallel pathways to regulate dendrite targeting and axon branching, several lines of evidence support the notion that Rpd3 acts via Pros to regulate these events. First, loss-of-function mutations in single cell clones of Rpd3 and pros give similar dendrite targeting and axon branching phenotypes. Second, overexpression of Pros can largely suppress the dendrite mistargeting phenotype of Rpd3−/− and can partially suppress the axon overbranching phenotype of Rpd3−/−. This suppression is specific, as overexpressing Pros does not cause defects in wild-type cells nor does it suppress mistargeting phenotypes due to a few other mutations. Conversely, overexpression of Rpd3 does not suppress pros mutant phenotypes. Because the suppression is more robust for dendrite mistargeting defects compared to axon overbranching defects, it is possible that Pros function accounts for more of Rpd3’s activity in regulating dendrite targeting than axon branching (Tea, 2010).

To address the mechanism by which Rpd3 and Pros function together in regulating PN development, several models of their interactions were tested. One model is that Rpd3 functions to upregulate the expression of Pros, which predicts that Rpd3−/− would lead to a decrease in Pros protein. However, anti-Pros immunoreactivity in Rpd3−/− DL1 PN clones was not decreased compared to wild-type DL1 PN clones. A second model is that Rpd3 and Pros directly bind and work together to regulate the transcription of Pros target genes. Yet in wild-type embryos or in S2 cell culture, no complex between Rpd3 and Pros could be detected via immunoprecipitation. A third model is that Rpd3 directly deacetylates the Pros protein to affect its function, but Pros acetylation could not be detected in the presence of HDAC inhibitors by immunostaining or mass spectrometry. Together, these data suggest that Rpd3 indirectly affects the function of Pros. This effect may be through posttranslational modification of Pros to modify its activity. For example, Pros has previously been shown to be phosphorylated. If posttranslational modification increases Pros activity, then Rpd3−/− would result in reduced Pros activity. Overexpression of Pros in Rpd3−/− may compensate for the reduced activity of unmodified Pros, and therefore suppress the Rpd3−/− phenotype (Tea, 2010).

Future studies will determine how Rpd3 regulates Prospero, how these factors act together with other transcription factors, and what transcriptional target genes they regulate in order to orchestrate the developmental program for precise wiring of the olfactory circuit (Tea, 2010).

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRING) and (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

The role of Dichaete in transcriptional regulation during Drosophila embryonic development

Group B Sox domain transcription factors play conserved roles in the specification and development of the nervous system in higher metazoans. However, comparatively little is known about how these transcription factors regulate gene expression, and the analysis of Sox gene function in vertebrates is confounded by functional compensation between three closely related family members. In Drosophila, only two group B Sox genes, Dichaete and SoxN, have been shown to function during embryonic CNS development, providing a simpler system for understanding the functions of this important class of regulators. Using a combination of transcriptional profiling and genome-wide binding analysis this study conservatively identified over 1000 high confidence direct Dichaete target genes in the Drosophila genome. Dichaete is shown to play key roles in CNS development, regulating aspects of the temporal transcription factor sequence that confer neuroblast identity. Dichaete also shows a complex interaction with Prospero in the pathway controlling the switch from stem cell self-renewal to neural differentiation. Dichaete potentially regulates many more genes in the Drosophila genome and was found to be associated with over 2000 mapped regulatory elements. This analysis suggests that Dichaete acts as a transcriptional hub, controlling multiple regulatory pathways during CNS development. These include a set of core CNS expressed genes that are also bound by the related Sox2 gene during mammalian CNS development. Furthermore, Dichaete was identified as one of the transcription factors involved in the neural stem cell transcriptional network, with evidence supporting the view that Dichaete is involved in controlling the temporal series of divisions regulating neuroblast identity (Aleksic, 2013).

The core Dichaete binding intervals identified in this study are enriched for Sox binding motifs but significant overrepresentation was also found of binding motifs for Vfl (Zelda), the GAGA-binding factor Trl, and the JAK-STAT pathway transcription factor Stat92E. All three of these factors have been identified as key elements in the regulatory programme that drives the onset of zygotic gene expression in the blastoderm embryo. Dichaete also plays a key role in early zygotic gene expression, regulating the correct expression of pair rule genes, and this study found overlapping Vfl/Dichaete binding at eve, h, and run stripe enhancers. While most of the work on Vfl has focused on understanding its function during the maternal to zygotic transition, the gene is expressed more widely after cellularisation, particularly in the CNS. Indeed recent work has shown a specific role for Vfl in the CNS midline, a tissue where Dichaete is known to be active, and this study found overlapping Vfl/Dichaete binding associated with slit and comm, known Dichaete midline targets. Post cellularisation functions for Trl and Stat92E are well established (Aleksic, 2013).

These three factors, particularly Vfl and Trl, have been strongly associated with enhancer activity driven by Highly Occupied Target (HOT) regions. HOT regions have been identified in large scale studies of the Drosophila, C. elegans and human genomes, and represent genomic sites where many functionally unrelated transcription factors bind, frequently in the absence of specific binding motifs. The finding that Dichaete binding locations are marked by overrepresentation of binding motifs for factors defining HOT regions, coupled with the widespread gene expression effects of Dichaete mutations, suggests that Dichaete may also play a role in regulatory interactions at HOT enhancers. It is notable that Dichaete, in common with all other characterised Sox proteins, is known to bend DNA upon binding. It is possible that Dichaete activity at HOT regions is mediated by this bending activity, helping to bring together complexes of other regulators. In this view, Dichaete would assist binding of factors at non-canonical target sites by favouring protein-protein interactions. In one of the bona fide Dichaete regulatory elements that have been studied in detail, the slit midline enhancer, Dichaete helps coordinate interactions between the POU factor Vvl and a Sim/Tango heterodimer (Aleksic, 2013).

Aside from a proposed role at HOT regions, this analysis indicated Dichaete binds to and is active at many characterised regulatory elements. Almost half the enhancers catalogued by RedFly and a substantial fraction of neural enhancers identified by the FlyLight project show evidence of Dichaete regulation. Along with this, an association between Dichaete binding and transcriptional start sites was observed, suggesting one of two possibilities. Either Dichaete directly engages with core promoter elements or looping interactions between Dichaete bound enhancers and the transcriptional machinery results in ChIP or DamID assays capturing these interactions. In this respect it is noted that Dichaete binds in the minor groove of DNA, perhaps making it more likely to capture indirect interactions (Aleksic, 2013).

Whether Dichaete acts at defined tissue-specific enhancers, HOT regions, core promoters, or all three, this analysis uncovered widespread involvement in specific developmental processes in the embryo. For example, previous studies highlighted a role for Dichaete in hindgut morphogenesis and identified dpp as a likely target gene, since targeted dpp expression in the hindgut of Dichaete mutants was able to partially rescue the phenotype. This new analysis implicates Dichaete in the regulation of many of the key factors responsible for hindgut specification and morphogenesis, with most of the characterised transcription factors or signalling pathway components known to be important for hindgut development bound and regulated by Dichaete. This further emphasises the view that Dichaete plays a hub-like role in controlling regulatory networks. It is noted that hindgut phenotypes and gene expression are unlikely to be functionally compensated by other Sox factors. While the group E gene Sox100B is also expressed in the embryonic hindgut, these is no evidence for synergistic interactions between Dichaete and Sox100B mutants and thus functional compensation by Sox100B is less likely. In contrast, the related group B gene Sox21b is expressed in the hindgut and partially overlaps with Dichaete (McKimmie, 2005). Although deletions encompassing Sox21b show no obvious phenotype, assessing possible functional compensation of Dichaete functions is difficult due to the close proximity of the two genes (~40 kb). It has recently been reported that human SOX2 is involved in gut development where it interacts antagonistically with CDX2. Caudal is a Drosophila orthologue of CDX2 and this study found Dichaete binding and associated repression of cad, hinting at further levels of regulatory network conservation across metazoa (Aleksic, 2013).

In common with vertebrate group B genes, Dichaete plays a prominent role in the CNS. Many previous studies focused on single genes have shown that Dichaete is involved in neural specification via the regulation of proneural genes in the Achaete-scute complex and the current analysis provides a genomic perspective on this, identifying extensive Dichaete binding across the complex. Importantly, much of this binding coincides with mapped regulatory elements and this study found changes in the expression of complex genes in Dichaete mutants. Dichaete is involved in the temporal cascade that confers specific identities to neuroblasts and their progeny and this analysis provides considerable insights into this role. Dichaete binding was found associated with all of the characterised genes in the temporal cascade, as well as considerable overlapping binding between Dichaete, Hb and Kr, strongly supporting the idea that cross-regulatory interactions between these genes is important for correct neural specification. For example, maintenance of Hb or loss of Cas, the first and last genes in the cascade, lead to prolonged expression of Dichaete and cells remain in a neuroblast state (Maurange, 2008). This analysis suggests that Dichaete may help maintain the temporal cascade expression in the neuroblast (Aleksic, 2013).

Finally, this analysis uncovered a striking relationship between Dichaete and Pros, with Dichaete negatively regulating pros expression early in neural development. In addition, both proteins show an extensive and highly significant overlap in their binding profiles. The gene expression data indicate that Dichaete and Pros may have antagonistic interactions since genes encoding neuroblast functions (e.g. ase, insc, mira and dpn) were found to be downregulated in Dichaete mutants but upregulated in pros mutants. However, this study also found that genes involved in aspects of neuronal differentiation (e.g. esc, zfh1 and Lim1) are positively regulated by both factors. Taken together it is tempting to speculate that in neuroblasts, when Pros is cytoplasmic, Dichaete positively regulates genes required to maintain the self-renewal state and keeps pros levels down. In the GMC, Dichaete function must be downregulated to allow cells to exit the cell cycle and differentiate, consequently pros expression would be upregulated and the protein translocated to the nucleus by the well-established asymmetric division mechanism, repressing neuroblast genes and promoting differentiation. While Dichaete appears to be uniformly expressed in the neuroectoderm, its expression in neuroblasts is dynamic with many neuroblasts expressing Dichaete transiently. In addition, and related to the subcellular partitioning of Pros, Dichaete is reported to shuttle between cytoplasm and nucleus, at least early in CNS development. Furthermore, Dichaete is dynamically expressed in GMCs and their progeny, consistent with the proposed interaction with Pros (Sanchez-Soriano, 2000). These observations are consistent with the view that control of Dichaete is important for first determining self-renewal versus differentiation, followed by a role in aspects of neuronal differentiation (Aleksic, 2013).

The emerging view from these studies and previous work with Dichaete is of a transcriptional regulator with multifaceted roles in development. Previous studies have shown that mammalian Sox2 can provide Dichaete function, rescuing Dichaete mutant phenotypes. However, the designation of Dichaete as a group B1 protein based on functional arguments is considered by some to be inconsistent with phylogenetic arguments that firmly place Dichaete in the B2 group. In vertebrates, group B2 proteins act as transcriptional repressors, antagonising group B1 functions. Since very few genes are seen to be upregulated in Dichaete mutants, this analysis suggests that Dichaete may be acting primarily as a transcriptional activator. However, this type of mutant expression study is prone to pleiotropic effects, so further investigation of specific targets and tissues is needed. In vertebrates the group B1 proteins play critical roles in the specification and maintenance of neural stem cells, exactly the functions described for Dichaete. The observed correspondence between Dichaete and Sox2 target genes show that these proteins are not only conserved at the functional level when assayed in mutant rescue experiments but also, remarkably, at the level of the gene regulatory networks they control in the fly and mouse nervous system (Aleksic, 2013).

One possible explanation for these disparate findings regarding the classification of Dichaete as a group B1 or B2 protein may be provided by the role of Dichaete in the regulation of proneural genes and its early activity on pros. In these specific cases, Dichaete acts to repress these genes in the neuroectoderm while SoxN acts as an activator. It is therefore possible that, in the last common ancestor of the vertebrates and invertebrates when the B1-B2 split occurred, the ancestral Dichaete gene had an limited B2-like repressor role as well as more prominent B1-like activator role in the CNS. As the lineages diverged the vertebrate B2 genes evolved specialised repressor functions while, in the invertebrates, they maintained more basal activator function. Support for the idea that insect Sox genes represent conserved basal functions of more diverged vertebrate family members comes from experiments replacing the mouse group E gene, Sox10, with the fly Sox100B coding sequence. In these studies the fly gene is able to provide substantial Sox10 function in the developing embryo, more so than the Sox8 gene, which is far closer to Sox10 at the sequence level (Aleksic, 2013).

In summary, this study presents a rigorous analysis of the genomics of the Drosophila group B transcription factor Dichaete, highlighting regulatory input into several key developmental pathways. These studies provide a baseline for more detailed analysis of highly conserved aspects of group B Sox function in neural stem cells and in neuronal differentiation (Aleksic, 2013).

The Sp8 transcription factor Buttonhead prevents premature differentiation of intermediate neural progenitors

Intermediate neural progenitor cells (INPs) need to avoid differentiation and cell cycle exit while maintaining restricted developmental potential, but mechanisms preventing differentiation and cell cycle exit of INPs are not well understood. This study reports that the Drosophila homolog of mammalian Sp8 transcription factor Buttonhead (Btd) prevents premature differentiation and cell cycle exit of INPs in Drosophila larval type II neuroblast (NB) lineages. Loss of Btd leads to elimination of mature INPs due to premature differentiation of INPs into terminally dividing ganglion mother cells. Evidence is provided to demonstrate that Btd prevents the premature differentiation by suppressing the expression of the homeodomain protein Prospero in immature INPs. It was further shown that Btd functions cooperatively with the Ets transcription factor Pointed P1 to promote the generation of INPs. Thus, this work reveals a critical mechanism that prevents premature differentiation and cell cycle exit of Drosophila INPs (Xie, 2014).

This study shows that the Sp family transcription factor Btd is required to prevent the premature differentiation of INPs by suppressing the expression of Pros in immature INPs. Furthermore, evidence is provided to demonstrate that the combination of Btd and PntP1 is sufficient to specify type II NB lineages and promote the generation of INPs. Thus, this work reveals a critical mechanism that regulates INP generation (Xie, 2014).

The most striking phenotype resulting from the loss of Btd is the elimination of mature NPs. In addition, about 40% of btbmutant type II NB lineages ectopically express Ase in the NB and become type I-like NB lineages. However, although forced expression of Ase in type II NBs is sufficient to eliminate INPs in type II NB lineages, the loss of INPs is obviously not primarily due to the ectopic Ase expression or the transformation of type II NB lineages into type I-like NB lineage in that the loss of mature INPs occurs independently of the ectopic Ase expression in most btb mutant or Btd RNAi knockdown type II NB lineages. Instead, this study demonstrates that the loss of mature INPs in the absence of Btd is due to the premature differentiation of Ase+ immature INPs into GMCs. In Btd RNAi knockdown or btb mutant type II NB lineages without the ectopic Ase expression, Ase- immature INPs differentiate into Ase+ immature INPs normally as indicated by the expression of R9D11-mCD8-GFP, Mira, as well as PntP1 in Ase+ daughter cells next to the Ase- immature INPs. However, instead of differentiating into mature INPs, it is argued that Ase+ immature INPs prematurely differentiate into GMCs based on the following two pieces of evidence. First, Ase+ daughter cells eventually undergo terminal divisions as indicated by the positive pH3 staining and the position of the pH3-positive cells. Second, unlike mature INPs, the dividing Ase+ daughter cells do not form basal Mira crescent at metaphase. The terminal division and the lack of Mira crescent during the division are two unique features that distinguish GMCs from INPs in addition to the expression of nuclear Pros. Therefore, the elimination of mature INPs resulting from the loss of Btd is due to the premature differentiation of Ase+ immature INPs into GMCs (Xie, 2014).

Why does the loss of Btd lead to premature differentiation of INPs? The results show that the loss of Btd results in a reduction or loss of PntP1 in type II NBs and immature INPs as well as ectopic expression of Pros in early immature INPs. Previous studies show that PntP1 suppresses Ase in type II NBs and that inhibiting PntP1 activity leads to ectopic expression of Ase in type II NBs and elimination of INPs. Given that the ectopic Ase expression in btb mutant type II NBs is closely associated with the severe reduction or complete loss of PntP1 and that expression of UAS-pntP1 largely suppresses the ectopic Ase expression in btb mutant type II NBs, the severe reduction or loss of PntP1 most likely accounts for the ectopic Ase expression in btb mutant type II NBs. However, although the loss of PntP1 could lead to the loss of INPs, several lines of evidence are provided to demonstrate that the elimination of INPs in btb mutant or Btd RNAi knockdown type II NB lineages is primarily due to the ectopic activation of Pros in immature INPs rather than the reduction or loss of PntP1. First, ectopic nuclear Pros is consistently expressed in Ase- immature INPs when mature INPs are eliminated. Second, the loss of mature INPs can be fully rescued by Pros RNAi knockdown or even just by removing one wild type copy of pros. Third, Pros RNAi knockdown also rescues the reduction of PntP1 and suppresses the ectopic Ase expression in btb mutant type II NBs. In contrast, the expression of UAS-pntP1 fails to rescue mature INPs in most btb mutant type II NB lineages although it largely suppresses the ectopic Ase expression in the NBs. Furthermore, the complete elimination of mature INPs is also observed occasionally in btb mutant type II NB lineages without the reduction of PntP1. Therefore, the elimination of mature INPs resulting from the loss of Btd is primarily due to the ectopic Pros expression, which likely promotes premature differentiation of INPs into GMCs and cell cycle exit. The severe reduction or loss of PntP1 is responsible for the ectopic Ase expression in btb mutant type II NBs and is more likely a secondary effect due to the ectopic Pros expression and/or the loss of INPs. INPs and/or other progeny may provide feedback signals to the NBs as has been demonstrated in other systems (Xie, 2014).

The ectopic expression of Pros in Ase- immature INPs resulting from the loss of Btd suggests that Btd is critical for suppressing Pros expression in Ase- immature INPs. Btd was known as a head gap gene. It has been suggested that gap factors act largely as transcriptional repressors. Btd could directly suppress Pros by binding to the pros promoter as a transcriptional repressor. Alternatively, Btd could suppress Pros indirectly by regulating the expression or antagonizing the activity of factor(s) that activate(s) pros expression. The results show that ectopic/overly expression of Btd in type I NB lineages or mature INPs does not lead to overproliferation of type I NBs as observed in pros mutant type I NB lineages. Instead, ectopic expression of Btd promotes the generation of INP-like cells from type I NBs and transforms some type I NB lineages into type II-like NB lineages. Therefore, it is more likely that Btd suppresses Pros indirectly by regulating the expression or antagonizing the activity of pros activator(s). Previous studies have suggested that Ase, Daughterless, Numb, and Erm could activate pros expression. Since Ase and R9D11-Cd4-tdTomato, which is under the control of erm promoter, are not expressed in Ase- immature INPs in the absence of Btd, it is unlikely they are involved in the activation of pros in immature INPs. It would be interesting to investigate in the future if Numb or Daughterless could activate pros in immature INPs in the absence of Btd (Xie, 2014).

This study has provided several lines of evidence to demonstrate that Btd and PntP1function cooperatively to specify type II NB lineages and promote the generation of INPs. Results from this study as well as a previous study show that ectopic expression of UAS-pntP1 or UAS-btb alone can only promote the generation of INP-like cells in a subset of type I NB lineage, whereas ectopic expression of UAS- pntP1 in Btd-positive type I NB lineages or coexpression of UAS-btb and UAS-pntP1 can promote the generation of INP-like cells in nearly all type I NB lineages and transforms all these lineages into type II-like NB lineages. Consistently, the ability of PntP1 to promote the generation of INP-like cells in btb mutant type I NB lineages is largely impaired. These results suggest that the specification of type II NB lineages and the generation of INPs requires both PntP1 and Btd and that the combinatorial PntP1 and Btd is sufficient to promote the generation of INPs (Xie, 2014).

It is proposed that PntP1 and Btd function cooperatively but through different mechanisms to promote INP generation. PntP1 is responsible for the suppression of Ase in type II NBs. Meanwhile, PntP1 must be regulating the expression of other unknown target gene(s) that are/is essential for the generation of INPs, such as specification of immature INPs, because loss of Ase is not sufficient to promote the generation of INP-like cells in any type I NB lineages. Btd likely acts after PntP1 to mainly prevent premature differentiation of INPs into GMCs by indirectly suppressing pros in immature INPs. The role of Btd in suppressing Ase in type II NBs is minimal if there is any because unlike PntP1, which suppresses ase in nearly all type I NBs when it is ectopically expressed, overexpression of Btd only suppresses Ase in a small subset of type I NBs that produce INP-like cells in larval brains. Furthermore, Ase is expressed in Btd+ type I NBs, indicating Btd does not suppress Ase in type I NBs when it is expressed at normal levels. Studies in mammals as well as in Drosophila suggest that the Btd/Sp8 could functions downstream of Wnt signaling to regulate the expression of Fgf8 as well as Distal-less (Dll) and Headcase (Hdc) during the forebrain patterning as well as limb development. However, inhibiting Wnt signaling alone in type II NB lineages does not have any obvious phenotypes, indicating that Btd unlikely functions downstream of Wnt signaling in type II NB lineages (Xie, 2014).

Whether Fgf8, Dll, or Hdc could function downstream of Btd to regulate INP generation remains to be investigated in the future. In mammals, the Btd homolog Sp8 palys important roles in brain development. In the developing mouse forebrain, Sp8 is expressed in cortical progenitors in a mediolateral gradient across the ventricular zone as well as in the lateral ganglionic eminence (LGE) and medial ganglionic eminence (MGE). In developing human brains, Sp8 is abundantly expressed in the ventricular zone and the outer subventricular zone where RGs and oRGs reside. In addition to its roles in interneuron development and the patterning of developing mammalian brains and spinal cords, it was also shown that loss of Sp8 led to the reduction of the progenitor pool. The current results show that mammalian Sp8 can rescue the loss of mature INPs resulting from the loss of Btd in Drosophila, suggesting that Btd/Sp8 could have conserved functions across different species. It would be interesting to investigate if Sp8 has similar roles in promoting the generation of transient amplifying INPs, such as oRGs, in developing mammalian brains (Xie, 2014).

Targets of Activity

In many organisms, single neural stem cells can generate both neurons and glia. How are these different cell types produced from a common precursor? In Drosophila, glial cells missing is necessary and sufficient to induce glial development in the CNS. GCM mRNA has been reported to be asymmetrically localized to daughter cells during precursor cell division, allowing the daughter cell to produce glia while the precursor cell generates neurons. In this study, it has been shown that (1) GCM mRNA is uniformly distributed during precursor cell divisions; (2) the Prospero transcription factor is asymmetrically localized into the glial-producing daughter cell; (3) Prospero is required to upregulate gcm expression and induce glial development, and (4) mislocalization of Prospero to the precursor cell leads to ectopic gcm expression and the production of extra glia. A model for the separation of glia and neuron fates in mixed lineages is proposed in which the asymmetric localization of Prospero results in upregulation of gcm expression and initiation of glial development in only precursor daughter cells (Freeman, 2001).

In thoracic segments the neural precursor 6-4 generates both glia and neurons, and is referred to as NGB 6-4T; in abdominal segments the 6-4 precursor produces only glia, and so it is called GB 6-4A. The neural precursor 7-4 generates a lineage composed of both neurons and glia in all segments, and is referred to as NGB 7-4 (Freeman, 2001).

NGB 6-4T and GB 6-4A form at early embryonic stage 10 as part of the S3 wave of neuroblasts. The first division of NGB 6-4T is oriented along the apical-basal axis, producing a large apical post-divisional precursor (NGB 6-4T) and a smaller basal daughter cell (G). Gcm is expressed before this first division and both daughter cells inherit Gcm protein, which enters the nucleus in these cells immediately after NGB division. Interestingly, shortly after NGB 6-4T completes this division, the G daughter cell migrates from its basal position to a position just medial to the post-divisional NGB 6-4T, which may explain why this division was previously scored as mediolateral. By the end of the G cell medial migration, Gcm protein is downregulated in NGB 6-4T and maintained only in the G daughter cell. 6-4T continues to divide along the apical-basal axis and subsequently produces neuronal progeny. The G daughter cell maintains high levels of Gcm protein and produces three glial cells. These glia continue to express Gcm protein, activate the glial specific gene reversed polarity (repo), migrate medially, and differentiate into cell body glia (CBGs) (Freeman, 2001).

These results raise the question of how Gcm protein becomes asymmetrically restricted to the G daughter cell after the first division of NGB 6-4T. It has been proposed that GCM mRNA is asymmetrically partitioned into the G daughter cell during mitosis of NGB 6-4T. To test this model, GCM mRNA localization was scored by fluorescent in situ hybridization. Levels of GCM mRNA are observed in the predivisional NGB 6-4T, followed by uniform localization in the mitotic NGB 6-4T, and equal distribution to both NGB 6-4T and G sibling cells. After G cell migration GCM mRNA was observed in the G cell and down-regulation of GCM mRNA was observed in the post-divisional NGB 6-4T. gcm-expressing cells were scored throughout the CNS, and asymmetric localization of GCM mRNA has never been observed in any mitotic precursor cell. It is concluded that GCM mRNA is not asymmetrically localized in the mitotic NGB 6-4T, but rather that it becomes transcriptionally upregulated in the G daughter cell soon after it is born (Freeman, 2001).

Similar to NGB 6-4T, GB 6-4A divides along the apical-basal axis and its basal daughter cell rapidly migrates to a position medial to its apical sibling. In contrast to NGB 6-4T, GB 6-4A expresses high levels of Gcm protein and mRNA before its first division, and both daughter cells maintain gcm expression. These two cells subsequently express repo, migrate medially, and differentiate into cell body glia (CBG) (Freeman, 2001).

NGB 7-4 forms at late stage 8 as the most lateral En-positive S1 neuroblast. The first progeny from NGB 7-4 are Prospero positive and Gcm negative, and differentiate into neurons. At stage 10 (just before the formation of 6-4 neural precursors) NGB 7-4 begins producing several Prospero-positive, Gcm-positive daughter cells that make a total of six to seven glia. At stage 12, NGB 7-4 switches back to making Prospero-positive Gcm-negative daughter cells that develop into neurons. All divisions of NGB 7-4 are along the apico-basal axis; Gcm-positive progeny are budded off the basal surface of NGB 7-4 but then migrate extensively to their final positions; ultimately, two glia migrate along the ventral surface of the CNS and differentiate as a pair of En-positive midline channel glia, three remain on the ventral surface of the CNS near NGB 7-4 and develop into CBGs posterior to the En-positive stripe, and one or two migrate to a position slightly dorsal and lateral to NGB 7-4 and differentiate as lateral subperineurial glia. In contrast to the 6-4 neural precursors, GCM mRNA or protein is not detected in NGB 7-4, only in its glial progeny (Freeman, 2001).

In summary, glia-producing divisions of NGB 6-4T and GB 6-4A occur along the apicobasal axis, and these divisions are followed by medial migrations of glial progeny. Gcm mRNA and protein are present in the predivisional NGB 6-4T, and Gcm protein enters the NGB and daughter cell nuclei immediately after the first division of this NGB. In addition, no evidence is found for asymmetric localization of GCM mRNA in any glial lineage, including NGB 6-4T (Freeman, 2001).

If GCM mRNA and protein are equally distributed into NGB 6-4T and its first-born G daughter cell, how are GCM mRNA and protein levels upregulated in G but not NGB 6-4T? To address this issue, mitotic NGB 6-4T were assayed for proteins known to be asymmetrically localized along the apical-basal axis of neuroblasts. The goal was to identify candidate genes that could differentially regulate gcm expression in the NGB 6-4T lineage. Insc protein marks the apical side of most or all mitotic neuroblasts and is necessary and sufficient for apical-basal spindle orientation. In NGB 6-4T, Insc is localized as an apical crescent at all stages of mitosis and is partitioned into the apically-positioned NGB 6-4T following cytokinesis. The mitotic GB 6-4A also shows apical Insc localization. Because Insc is sufficient to orient the mitotic spindle in all neuroblasts and epithelial cells assayed, the apical localization of Insc in NGB 6-4T and GB 6-4A provides strong confirmation that both cells divide along their apical-basal axis (Freeman, 2001).

Miranda, Prospero, Staufen, and Numb proteins mark the basal side of many or all mitotic neuroblasts and regulate the fate of daughter cells or their neuronal progeny. In NGB 6-4T, Miranda, Prospero, Staufen and Numb all form basal crescents from metaphase through telophase, and are partitioned into the basally positioned G daughter cell of NGB 6-4T after cytokinesis. The mitotic GB 6-4A also shows basal localization of Miranda, Prospero, Staufen and Numb. These results further confirm the apical-basal division axis of NGB 6-4T and GB 6-4A during glial producing divisions, and show that all of the above proteins are candidates for regulating gcm expression in the basal G daughter cell of NGB 6-4T (Freeman, 2001).

To determine if miranda, prospero, staufen or numb are involved in the development of glia in the NGB 6-4T lineage, embryos mutant for each gene were scored for the number and position of mature glia derived from NGB 6-4T. The three glia from NGB 6-4T express repo and have distinctive positions within the CNS: two near the midline and one between NGB 6-4T and the midline. These are the only Repo-positive glia adjacent to the midline at the ventral surface of the CNS, and thus are easy to identify unambiguously. Mutations in staufen and numb have no effect on glial development in the NGB 6-4T lineage. By contrast, prospero mutant embryos show striking loss of NGB 6-4T-derived glia, while miranda mutant embryos have a similar but weaker phenotype. It is concluded that prospero and miranda, but not staufen or numb, are required for normal glial development in the NGB 6-4T lineage (Freeman, 2001).

To determine the earliest aspect of the prospero mutant phenotype in the NGB 6-4T lineage, whether gcm is expressed normally in the G daughter cell was assayed. In wild-type embryos, Gcm protein is detectable in the predivisional NGB 6-4T and in the sibling NGB 6-4T/G cells immediately after cytokinesis; subsequently, Gcm disappears from NGB 6-4T and is upregulated in the G cell and its progeny, which proceed to migrate medially and express repo. In prospero mutant embryos, gcm expression is activated normally in the predivisional NGB 6-4T and is detectable in the immediately post-mitotic NGB 6-4T and G cell. Therefore the early induction of gcm expression in these cells is clearly prospero-independent. However, Gcm protein levels subsequently decline in the G cell and its progeny and these cells fail to migrate to the midline or express repo. These data indicate that Prospero is required in the G daughter cell to maintain or upregulate gcm expression levels, induce medial migration, and activate repo expression. Surprisingly, these Gcm negative, Repo negative cells do not express the neuron-specific elav gene, and thus they appear unable to differentiate as glia or neurons (Freeman, 2001).

In prospero mutant embryos, gcm expression is also greatly reduced in the progeny of NGB 7-4. Low level expression of gcm is detectable in many NGB 7-4 progeny shortly after their birth, indicating that in this lineage (as in NGB 6-4T) the induction of gcm expression can occur in the absence of prospero function. However, gcm expression fades rapidly and these cells never express repo. Thus, prospero is essential for the maintenance of gcm expression and normal glial cell fate induction in both the NGB 6-4T and 7-4 lineages (Freeman, 2001).

prospero is clearly necessary for upregulation of gcm and glial cell fate induction in the 6-4T and 7-4 lineages, but is it sufficient to induce gcm expression in these lineages? In miranda mutant embryos, prospero mRNA and protein are delocalized during neural precursor cell division, resulting in similar concentrations of Prospero segregating to both NGBs and their daughter cells. Interestingly, in miranda mutants ectopic gcm expression is found in NGB6-4T at stage 13, a time when this NGB is normally making neuronal progeny. miranda mutants also show ectopic expression of gcm in NGB 7-4 lineage during its window of glial production. Thus, mislocalization of Prospero to the NGB by removal of miranda function is sufficient to induce ectopic gcm expression in these NGBs (Freeman, 2001).

Does the upregulation of gcm in NGBs drive the production of extra glial progeny? To address this question, Repo expression was assayed in the NGB6-4T lineage in miranda mutants because the entire NGB 6-4T lineage can be identified. In miranda mutants only four Repo positive cells are typically found in the entire NGB 6-4T lineage, but neuronal progeny are completely absent. This phenotype is interpreted to indicate that the G cell produces three glia as usual, but that its sibling NGB differentiates directly into a Repo-positive glia cell, resulting in a termination of the lineage. Thus, it appears that Prospero mislocalized to the NGB can potently activate gcm expression in the NGB and transform it into a glial cell (Freeman, 2001).

Two additional phenotypic classes in miranda mutants are found: (1) a variable number of Repo-positive glia are produced (between two and four) and subsequent neuronal progeny are generated normally (12% of hemisegments); or (2) the wild-type pattern of three Repo-positive glia and neuronal progeny are produced. These phenotypes indicate that low level Prospero in the NGB is not always sufficient to induce a glial fate, and that reduced Prospero in the G cell may lead to fewer glial progeny (Freeman, 2001).

Mislocalization of Prospero to NGB 7-4 by removal of miranda function can also induce Repo expression in this NGB (25% of hemisegments), showing that NGB 7-4 can also be partially transformed towards a glial fate. It is not known if this NGB differentiates as a glial cell (like the Pros-positive NGB 6-4T), generates extra glial progeny, or if it can eventually produce neurons. miranda, prospero double mutants do not show upregulation of gcm in NGBs 6-4T or 7-4, demonstrating that the upregulation of gcm in these NGBs in miranda mutant embryos is due to Prospero protein that is delocalized into the NGB. These results indicate that Miranda, by asymmetrically localizing Prospero to NGB daughter cells, restricts gcm upregulation and induction of the glial developmental program to the progeny of NGBs 6-4T and 7-4 during their phases of glial production (Freeman, 2001).

Previous reports have suggested that GCM mRNA is asymmetrically localized in a medial crescent in the mitotic NGB 6-4T, resulting in the selective partitioning of GCM mRNA into the medial glia-producing progeny of NGB 6-4T. These conclusions are likely to be in error for three main reasons: (1) the mitotic NGB 6-4T contains evenly distributed GCM mRNA, and this mRNA is partitioned equally between NGB 6-4T and its glial-producing G daughter cell after cytokinesis; (2) previous studies did not use a mitosis-specific marker together with probes for GCM mRNA localization to prove that the localization was being scored in mitotic NGBs; (3) NGB 6-4T always divides along the apical-basal axis, therefore a medial localization of GCM mRNA would not result in it being partitioned unequally into one daughter cell. Indeed, in a recent study use was made of more specific markers for mitotic stage and cell orientation. It was found that the first division of NGB 6-4T is not along the mediolateral axis, and that GCM mRNA is inherited by both the NGB and the G daughter cell. It is therefore concluded that the asymmetric localization of GCM mRNA is not the mechanism by which neuronal and glial lineages are separated in the NGB 6-4T lineage (Freeman, 2001).

Gcm protein has been reported to be absent from the predivisional NGB 6-4T, perhaps owing to translational repression of GCM mRNA prior to its first division. This is not the case, since Gcm protein is clearly present in the predivisional NGB 6-4T. In addition, it has been reported that Gcm protein is excluded from the nucleus in the postdivisional NGB 6-4T, and that an unidentified mechanism regulates nuclear entry of Gcm specifically in the G daughter cell. Robust Gcm protein expression, however, has been shown in the nucleus of NGB 6-4T, arguing strongly against the existence of such a mechanism (Freeman, 2001).

This study shows that gcm is induced properly in prospero mutant embryos, but that both the G daughter cell and the post-divisional NGB downregulate gcm expression with a similar timecourse. Thus, the induction of gcm expression in NGB 6-4T is prospero-independent. Interestingly, prospero mutant embryos also fail to upregulate gcm to high levels in NGB 7-4 daughter cells. This is consistent with a previous report that NGB 7-4-derived Repo-positive glia are absent in prospero mutants. Unlike NGB 6-4T, gcm expression was not detected in NGB 7-4, only in its new-born (pre-migration) daughter cells. Low level Gcm expression is still present in prospero mutant embryos; thus, the induction of gcm expression in this lineage also appears to be prospero-independent. It is proposed that prospero functions to upregulate low levels of gcm in these lineages, but is not sufficient to induce gcm expression on its own. Such a mechanism would explain why all neural stem cell progeny in the CNS express high levels of prospero but most never induce gcm and the glial developmental program. In agreement with this model, misexpression of low levels of gcm throughout the CNS requires Prospero to induce glia in a subset of lineages (Freeman, 2001).

How might the Prospero transcription factor upregulate low levels of gcm expression? Prospero is known to act as a co-factor to stimulate transcriptional activity of several DNA-binding proteins. Recent studies show that Gcm can positively autoregulate its own expression in neural tissues. It is possible that Prospero may act together with Gcm to stimulate expression levels of the gcm gene until they become sufficiently high for Gcm to positively autoregulate its own expression. However, embryos homozygous for the gcmN7-4 allele produce non-functional Gcm protein that is upregulated with normal kinetics in the NGB 6-4T and 7-4 lineages, indicating that Gcm function is dispensable for its own upregulation. It is proposed that a lineage-specific co-factor or extrinsic signal converges with Prospero function in these lineages to upregulate gcm expression (Freeman, 2001).

How does a NGB know when to make neurons or glia? For example, the first born daughter cell from NGB 6-4T gives rise to glia while all subsequent progeny are neuronal. By contrast, NGB 7-4 first produces several neuronal progeny, then switches to making glia, and finally switches back to making neurons. Interestingly, the window of developmental time during which these two neural precursors are making glia are strikingly similar: NBG 7-4 begins making glia at stage 10, shortly after this NGB 6-4T is born (late stage 10) and begins making glia; at stage 11 both precursors terminate glial production and switch to making neurons. The coordinate timing of glial production from these lineages may indicate that a temporally regulated extrinsic cue induces gcm expression in these NGBs or their newly born progeny (Freeman, 2001).

In prospero mutant embryos, NGB 6-4T and its progeny only transiently express low levels of gcm. What is the fate of these cells? They never express the glial-specific repo gene, and they also fail to express the neuron-specific elav gene, indicating that neither the glial or neuronal developmental program has been initiated. gcm is thought to transcriptionally activate genes that promote glial fate or repress neuronal fate. It is proposed that NGB 6-4T in prospero mutants produces enough Gcm protein to repress neuron-specific genes, yet insufficient amounts to robustly induce glial-specific genes. This in turn suggests that there may be different gcm thresholds for activating glial development (high threshold) and for repressing neuronal development (low threshold) (Freeman, 2001).

In miranda mutant embryos, Prospero protein is delocalized at mitosis, allowing NGB/daughter cell siblings to inherit equal concentrations of Prospero. In these embryos, ectopically upregulated gcm is frequently seen in NGBs 6-4T and 7-4, and extra glia are derived from NGB 6-4T. These data indicate that prospero is a potent activator of gcm expression in the NGB 6-4T and 7-4 lineages. The extra glia observed could come from an extension of the glial portion of the NGB 6-4T lineage, or from a transformation of this NGB into a purely glial progenitor. The latter model is favored, because neurons are never observed in the NGB 6-4T lineage when extra glia are observed. Moreover, high levels of Gcm are correlated with pure glial lineages such as GB 6-4A and the GP, and gcm is known to positively autoregulate which may commit precursors with high Gcm to a glial-producing fate (Freeman, 2001).

In miranda mutant embryos, the ectopic expression of gcm in NGB 6-4T and 7-4 is in fact due to delocalization of Prospero and not simply the absence of Miranda, because miranda; prospero double mutants fail to upregulate gcm in NGBs. In both the NGB 6-4T and 7-4 lineage, the delocalization of Prospero has relatively little effect on glial production by the daughter cells, presumably because there is sufficient Prospero protein in these daughter cells to upregulate gcm expression. Thus, with respect to glial cell fate induction, the asymmetric localization of Prospero may be more important for removing Prospero from the NGB than for enriching Prospero in the daughter cell (Freeman, 2001).

Activation of fushi tarazu and even-skipped expression in ganglion mother cells requires prospero function. Repression of deadpan and asense in ganglion mother cells requires prospero function (Doe, 1991).

It seems that loss or gain of cousin of atonal function causes neuronal defects. Like cato, embryos mutant for pros show neuronal defects, although the basis of these defects is not known. It was asked whether cato expression depends on pros function. Strikingly, loss of pros function results in the ectopic appearance of cato transcription within the ganglion mother cells (GMCs) and neurons of the developing CNS. This correlates with the wild-type expression of Pros in GMCs, indicating that Pros is normally a transcriptional repressor of cato in the CNS. There are indications that cato derepression begins in neuroblasts, even though pros is not thought to function until GMC formation: although Pros protein is present in the neuroblasts, it becomes localized only in the nucleus in the GMCs. Interestingly, although pros is apparently recessive, a weak derepression of cato is also observed in heterozygote pros/+ embryos. In this case cato expression is observed most clearly as nuclear sites of nascent transcription (Golding, 2000).

lethal (2) giant larvae and discs large are involved in localization of Prospero

Drosophila neuroblasts are a model system for studying asymmetric cell division: they divide unequally to produce an apical neuroblast and a basal ganglion mother cell that differ in size, mitotic activity and developmental potential. During neuroblast mitosis, an apical protein complex orients the mitotic spindle and targets determinants of cell fate to the basal cortex, but the mechanisms of these two processes are unknown. The tumor-suppressor genes lethal (2) giant larvae (lgl) and discs large (dlg) regulate basal protein targeting, but not apical complex formation or spindle orientation, in both embryonic and larval neuroblasts. Dlg protein is apically enriched and is required for maintaining cortical localization of Lgl protein. Basal protein targeting requires microfilament and myosin function, yet the lgl phenotype is strongly suppressed by reducing levels of myosin II. It is concluded that Dlg and Lgl promote, and myosin II inhibits, actomyosin-dependent basal protein targeting in neuroblasts (Peng, 2000).

Embryonic Drosophila neuroblasts develop from an apical/basal polarized epithelium. Individual cells delaminate into the embryo, enlarge to form neuroblasts, and begin a series of asymmetric cell divisions; these divisions result in the production of a large mitotically active apical cell (neuroblast), and a smaller basal cell (ganglion mother cell, GMC) that differentiates into two neurons or glia. A growing number of proteins are known to be asymmetrically localized in mitotic neuroblasts: apically localized proteins include Bazooka (Baz), Inscuteable (Insc) and Partner of Inscuteable (Pins); basally targeted proteins include Miranda, Prospero, Partner of Numb (Pon) and Numb, which are important for GMC development. Miranda and Prospero are apically localized at late interphase before their mitosis-dependent transport to the basal cortex. The Baz/Insc/Pins apical complex is required for both apical/basal spindle orientation and basal protein targeting, but little is known about how this complex regulates either process (Peng, 2000).

To identify genes required for apical/basal protein targeting in neuroblasts, deficiency stocks were screened looking for defects in Prospero basal localization in neuroblasts. This screen identified the lgl gene, which encodes a WD-40 repeat protein with homologues in many species, including the closely related 'Lgl family' genes Lgl1/Lgl2 (human), Lgl1 (mouse), U51993 (Caenorhabditis elegans); the slightly more divergent 'Tomosyn family' genes Tomosyn (rat), KIAA1006 (human), C617762 (Drosophila ), and M01A10 (C. elegans); and recently duplicated genes similar to both families: sro7/sro77 (budding yeast). In Drosophila, lgl mutations affect protein targeting to epithelial apical junctions, epidermal cell-shape changes, and produce tumors of the brain and the imaginal disc. This spectrum of phenotypes has been noted for another tumor-suppressor gene, discs large. This study explores the role of Lgl and Dlg in regulating neuroblast cell polarity (Peng, 2000).

Apical and basal protein targeting are compared in neuroblasts from wild-type embryos and embryos that lack all maternal and zygotic Lgl or Dlg function (called lglGLC or dlgGLC embryos). Wild-type metaphase neuroblasts show apical Insc/Pins localization, and basal Miranda/Prospero/Pon crescents. In addition, Miranda and Prospero proteins can be observed around the apical centrosome and weakly on the mitotic spindle in wild-type neuroblasts. In contrast, all lglGLC and dlgGLC metaphase neuroblasts show cytoplasmic Pon and uniformly cortical and strongly spindle-associated Miranda/Prospero; the apical proteins Insc/Pins are normal or slightly expanded. Although lglGLC and dlgGLC embryos show striking defects in neuroblast basal protein localization, they also show an early loss of embryonic epithelial apical/basal polarity, which could indirectly cause the observed neuroblast defects (Peng, 2000).

To determine the neuroblast-specific function of Lgl and Dlg, Lgl- or Dlg-depleted neuroblasts were studied in embryos or larvae where epithelial development occurs normally. Initially, homozygous null lgl4 embryos were studied, in which maternal Lgl protein allows normal embryonic epithelial development (including Armadillo, Crumbs and Dlg localization). In stage 16-17 lgl4 embryos, mitotic neuroblasts show normal Baz/Insc/Pins apical crescents, and normal spindle orientation, but Miranda/Prospero are delocalized onto the spindle and around the cortex and Pon is cytoplasmic. This phenotype is less severe in early embryos but fully penetrant in older embryos, presumably due to progressive loss of maternal Lgl protein. Next, neuroblasts were assayed in lgl3344 or dlgv55 homozygous larvae -- these larval neuroblasts are persistent embryonic neuroblasts that develop from a normal embryonic epithelium due to maternal Lgl and Dlg protein function. Wild-type larval metaphase neuroblasts have Insc/Pins crescents the opposite of Miranda/Prospero/Pon/Numb crescents, whereas homozygous lgl3344 or dlgv55 larval metaphase neuroblasts show normal Insc/Pins crescents but Miranda/Prospero/Pon proteins are cytoplasmic, uniformly cortical, and weakly spindle-associated. It is concluded that Lgl and Dlg are required specifically in neuroblasts for basal protein targeting, without affecting apical protein localization or spindle orientation (Peng, 2000).

Delocalization of the Prospero and Numb proteins produces defects in the nervous system and other tissues, so lgl mutant embryos were scored for cell fate defects. lglGLC embryos have severe morphological defects that preclude analysis, and lgl4 embryos can only be scored for late embryonic phenotypes, due to persistence of maternal Lgl protein. lgl4 embryos show a decrease in Even-skipped lateral (EL) neuron number at stage 17. A similar but stronger phenotype is seen in numb mutants, suggesting that the lgl phenotype may be due to delocalization of Numb during the GMC divisions that produce the EL neurons. The relatively mild lgl phenotype could be due to 'telophase rescue' of Numb protein in these GMCs, or to maternal Lgl protein (Peng, 2000).

How does Lgl regulate basal protein targeting? Lgl binds non-muscle myosin II in all organisms tested, and sro7/77 and myo1 (encoding Lgl-related proteins and myosin II, respectively) show strong negative genetic interactions in yeast. Tests were performed for genetic interactions between lgl4 and two different null mutations in zipper (encoding myosin II), scoring Miranda basal localization in stage 17 neuroblasts, when maternal Lgl and Myosin II protein levels are lowest. Wild-type and zip embryos have normal basal protein localization, whereas lgl4 embryos show complete delocalization of basal proteins. However, lgl4 embryos lacking one copy of myosin II show a significant increase in basal protein targeting; and lgl4;zip1 mutant embryos show virtually normal basal protein targeting. Thus, reducing myosin II levels strongly suppresses the lgl phenotype, indicating that myosin II can inhibit basal targeting when Lgl levels are low (Peng, 2000).

In addition, the general myosin inhibitor 2,3-butanedione monoxime (BDM) can suppress the lgl phenotype: stage 10 lgl4 embryos treated with BDM show a significant increase in basal protein localization compared with sham-treated stage 10 lgl4 embryos. Wild-type or lgl4 embryos treated with 50 mM BDM show delocalization of Miranda, Prospero and Pon. These data indicate that a myosin that is sensitive to 25 mM BDM inhibits basal protein localization in lgl embryos (probably myosin II), and at least one myosin that is sensitive to 50 mM BDM promotes basal protein targeting in mitotic neuroblasts (Peng, 2000).

Thus, in neuroblasts Lgl and Dlg regulate targeting of all known basal proteins without affecting apical protein localization or spindle orientation. In epithelia, Lgl and Dlg are necessary to restrict proteins to the apical membrane domain. Lgl could promote protein targeting to specific membrane domains in both neuroblasts (basal) and epithelia (apical), similar to the role of Lgl-related proteins in facilitating secretory vesicle fusion at specific membrane domains in yeast and mammals. If so, Lgl must act in neuroblasts via a secretory pathway that is independent of brefeldin A, because it has been shown that treatment with brefeldin A disrupts Golgi, inhibits Wingless secretion, but does not block basal protein targeting. Alternatively, Lgl may actively promote actomyosin-dependent localization of basal proteins and/or function to keep myosin II levels low so that they do not interfere with myosin-dependent basal localization. A general function of the Lgl protein family may be to increase the fidelity of protein targeting to specific domains of the plasma membrane (Peng, 2000).

Specification of motoneuron fate in Drosophila: Integration of positive and negative transcription factor inputs by a minimal eve enhancer: Pros activates eve

The mechanisms that generate neuronal diversity within the Drosophila central nervous system (CNS), and in particular in the development of a single identified motoneuron called RP2, are of great interest. Expression of the homeodomain transcription factor Even-skipped (Eve) is required for RP2 to establish proper connectivity with its muscle target. The mechanisms by which eve is specifically expressed within the RP2 motoneuron lineage have been examined. Within the NB4-2 lineage, expression of eve first occurs in the precursor of RP2, called GMC4-2a. A small 500 base pair eve enhancer has been identified that mediates eve expression in GMC4-2a. Four different transcription factors (Prospero, Huckebein, Fushi tarazu, and Pdm1) are all expressed in GMC4-2a, and are required to activate eve via this minimal enhancer; one transcription factor (Klumpfuss) represses eve expression via this element. All four positively acting transcription factors act independently, regulating eve but not each other. Thus, the eve enhancer integrates multiple positive and negative transcription factor inputs to restrict eve expression to a single precursor cell (GMC4- 2a) and its RP2 motoneuron progeny (McDonald, 2003).

GMC4-2a forms at stage 9, becomes Eve+ at stage 11, and generates the Eve+ RP2/sib neurons at late stage 11. The second-born Eve-negative GMC4-2b forms at stage 10, and generates an unknown pair of neurons. The first transcription factors detected in GMC4-2a are Pros and Hkb, due to inheritance of the proteins from the neuroblast. The next transcription factors detected in GMC4-2a are Ftz and Pdm1. Ftz is first detected at stage 10, and Pdm1 is first detected at stage 11. The de novo expression of Pdm1 is distinct from its inheritance in GMCs produced by Pdm+ neuroblasts during the assignment of temporal identity. The last protein to be detected is Eve, which appears only at late stage 11. Pros, Hkb, Ftz, and Pdm1 are each expressed transiently in the RP2/sib neurons at stage 12, but by stage 16 none of these proteins is detectable in the mature RP2 neuron. It is concluded that there is a temporal sequence of transcription factor expression in GMC4-2a: first Pros and Hkb, then Ftz, then Pdm1, and that Eve is detected only after all of these proteins are present (McDonald, 2003).

GMC4-2b forms at late stage 10, never expresses Eve, and generates two unknown Eve-negative neurons. Three transcription factors that positively regulate eve expression are detected in GMC4-2b: Pros, Ftz, and Hkb. The pattern of Pdm1 expression is too complex to score at the time GMC4-2b is born. The negative regulator Klu is detected in GMC4-2b but not GMC4-2a. It is concluded that GMC4-2b expresses at least three of the four positively acting transcription factors that are required to activate eve (Pros, Ftz, Hkb), and at least one negative regulator of eve expression (Klu). The absence of eve expression is likely due to the presence of Klu, rather than the absence of a positive regulator, because klu mutants can activate eve transcription in GMC4-2b (McDonald, 2003).

The sequential expression of Pros, Hkb, Ftz, Pdm1, and Eve in GMC4-2a raises the possibility that these four transcription factors act in a linear pathway to regulate eve expression. If so, then a mutant in an early-acting gene should lead to loss of expression of all later-acting genes in the pathway. Alternatively, the four transcription factors could all act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present. In this case, mutants in one gene should have no effect on any other gene except eve. To distinguish between these two models, pros, hkb, ftz, and pdm1 mutants were examined for expression of all four transcription factors and eve. Pdm1 is detected in GMC4-2a in all mutant genotypes: Ftz is detected in GMC4-2a in all mutant genotypes: pros, hkb, and pdm1, and Hkb is detected in GMC4-2a in all mutant genotypes. Finally, Pros is observed in GMC4-2a in all mutant genotypes, as expected because Pros is transcribed and translated in neuroblasts and is asymmetrically partitioned into each GMC. Taken together, these data support the model that all four transcription factors act directly to activate eve transcription, with expression of eve occurring only after all transcription factors are present (McDonald, 2003).

To test the model that Pros, Hkb, Ftz, and Pdm1 transcription factors directly regulate eve expression, the eve cis-regulatory DNA that confers regulated expression in the NB4-2 lineage was identified. Eve is expressed in a subset of neurons in the embryonic CNS, including the aCC/pCC neurons derived from NB1-1, the U1-5 neurons derived from NB7-1, the EL neurons derived from NB 3-3, and the RP2/sib neurons derived from NB4-2. An eve cis-regulatory element [R79R92; from ~7.9 and ~9.2 kilobase pair (kb) on the eve genomic map] has been defined that accurately directs lacZ expression to the Eve+ cells within two NB lineages: GMC4-2a and its RP2 progeny and GMC1-1a and its aCC/pCC progeny. The properties of this element are examined in this study in detail. When the R79R92 eve element was truncated to ~7.9 to ~8.6 kb (R79N86), lacZ expression in RP2 and aCC was normal, whereas expression in the pCC neuron was reduced. Truncation of the eve element to ~7.9 to ~8.4 kb (R79S84) almost completely abolished expression of lacZ in pCC, although occasionally expression in pCC was observed at low levels, whereas expression in RP2 and aCC remained high. Further truncation of the left end point to ~8.0 kb (S80S84) resulted in a reduction of expression in both aCC and RP2. Addition of the region ~8.4 to ~8.6 kb to this fragment (S80N86) increased the level of expression. However, because the region ~8.4 to ~9.2 kb (S84R92) did not show any ability to activate lacZ, the region ~8.4 to ~8.6 kb is apparently insufficient on its own to direct expression, and thus serves an auxiliary function. The removal of ~8.2 to ~8.4 kb from P80N86 abolished expression (SNdeltaSC). Together with the fact that each of the fragments ~7.9 to ~8.2 kb (S79C82) and ~8.2 to ~9.2 kb (C82R92) failed to activate lacZ, this indicates that both of the regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are necessary to direct expression, and that neither alone is sufficient. Consistent with this, two tandem copies of ~8.2 to ~8.4 kb failed to activate lacZ (C82S84x2), suggesting that the two regions may provide qualitatively different activities. In summary, the critical eve cis-regulatory element for the GMC4-2a and RP2 lies in a 0.5 kb fragment of genomic DNA between ~7.9 and ~8.4 kb (McDonald, 2003).

Do the genes that activate or repress eve expression in the NB4-2 lineage work through the minimal 500 bp RP2/aCC eve enhancer? Expression of R79S84-lacZ was assayed in pros, ftz, hkb, pdm1, and klu mutant embryos, and whether it was regulated identically to the endogenous eve gene was tested. ftz, pdm1, and hkb mutant embryos show loss of R79S84-lacZ in the RP2 neuron but not the aCC neuron, identical to the pattern of endogenous eve expression in these mutants. pros mutants show loss of eve-lacZ in both RP2 and aCC, identical to the pattern of endogenous eve expression in pros mutants. In embryos lacking klu, R79S84-lacZ is expressed in two cells at the RP2 position, whereas expression in aCC is normal; this matches the pattern of endogenous eve expression in klu mutant embryos. It is concluded that the R79S84 minimal eve cis-regulatory element precisely reproduces the pattern of endogenous eve expression within the NB4-2 lineage, and that transcription factors regulating eve in GMC4-2a can act through this enhancer to activate or repress eve expression (McDonald, 2003).

Expression of eve is not detected in GMC4-2b in wild-type embryos, but mutations in the klu gene result in ectopic expression of eve in GMC4-2b. Klu contains four predicted zinc fingers, one of which is highly homologous to the WT1 zinc finger domain. The consensus binding site for the WT1 zinc finger transcription factor is a ten nucleotide sequence, 5'-(C/G/T)CGTGGG( A/T)(G/T)(T/G)-3', with variable nucleotides shown in parentheses. It was reasoned that if Klu directly binds to the eve enhancer to repress expression in GMC4-2b, one or more WT1 consensus binding sites should be found in the minimal eve enhancer R79S84. Three conserved putative Klu-binding sites were found in the R79S84 sequence: site 1, GGGTGGGGAG at nucleotides ~8066 to ~8075; site 2, GCGTGGGTGA at nucleotides ~8090 to ~8099; and site 3, TCGCCCACCA at ~8262 to ~8271. Based on the fact that altering the C2, G3, G5, G6, and G7 to T or T4 to A in the WT1-consensus binding site abolished WT1 binding, nucleotide substitutions were made in the three putative Klu-binding sites. In sites 1 and 2, As were substituted for T4, G6, and G7. In site 3, which is a reversed binding site, Ts were substituted for C4, C6, and A7. These substitutions were made at all three sites; transgenic lines were constructed expressing the mutant enhancer driving lacZ (eveK123-lacZ), and the pattern of lacZ expression was examined in the CNS of wild-type embryos and embryos misexpressing Klu protein in the NB4-2 lineage (McDonald, 2003).

In wild-type embryos, the eveK123-lacZ transgene is expressed in the aCC and RP2 neurons, similar to the wild-type (R79S84) eve-lacZ transgene. However, in one or two hemisegments per embryo, an extra cell expressing eveK123-lacZ adjacent to the RP2 neuron was observed. This phenotype is very similar to wild-type (R79S84) eve-lacZ expression in klu mutant embryos, although slightly less penetrant. It is concluded that the eveK123-lacZ transgene mimics the klu mutant phenotype, and it is proposed that Klu represses eve expression via direct binding to one or more of these sites (McDonald, 2003).

To further test this hypothesis, gain of function experiments were used to test whether ectopic Klu in GMC4-2a can repress eve-lacZ expression via these sites. Expression of a wild-type (R79S84) eve-lacZ transgene was compared with a transgene containing three mutated Klu consensus binding sites (eveK123-lacZ) in embryos where Scabrous-Gal4 (Sca-Gal4) drives ectopic expression of UAS-klu in all neuroblast lineages. The wild-type (R79S84) eve-lacZ expression is partially repressed by ectopic Klu expression, but the eveK123-lacZ transgene with mutated Klu sites is repressed to a lesser extent. This difference in repression is only observed when the levels of transgene expression are lowered by raising the embryos at 18°C; when the transgenes are more strongly expressed (by raising the embryos at 23°C) no detectable repression was observed. Taken together, Klu loss of function and misexpression studies indicate that Klu acts partly, but not completely, through three predicted Klu-binding sites to repress eve expression in the NB4-2 lineage (McDonald, 2003).

In summary, hkb, ftz, pdm1, and pros are independently required to activate eve expression in GMC4-2a. This suggests that the eve enhancer is capable of integrating the input of all four of these transcription factors to activate transcription. Hb and Ind are also necessary for eve expression in GMC4-2a, but it is not known if they act directly on the eve element or via one of the four transcription factors described in this study. Putative binding sites were found for each of the positively acting transcription factors within the minimal eve element, but mutation of these sites had no effect on expression of the eve-lacZ transgene in embryos (M. Fujioka, J.A. McDonald, and C.Q. Doe, unpublished results reported in McDonald, 2003). It remains to be determined whether Pros, Hkb, Ftz, or Pdm1 activate eve transcription via direct binding to the minimal eve element, or indirectly by activating or facilitating the binding of other transcriptional activators (McDonald, 2003).

Based on functional dissection of the RP2/aCC/pCC eve element, it seems to be composed of three parts. The regions ~7.9 to ~8.2 kb and ~8.2 to ~8.4 kb are each necessary to direct the expression pattern (together they comprise the minimal element for expression in RP2 and aCC), while the region ~8.4 to ~8.6 kb enhances the level of expression. Expression in the pCC neuron is further enhanced by the region extending to ~9.2 kb. The two regions within the minimal element seem to be regulated by different factors, because two copies of ~8.2 to ~8.4 kb (increasing the number of activator binding sites within this region by twofold) could not substitute for the function of the region ~7.9 to ~8.2 kb. This is consistent with the fact that at least four factors are independently required to activate eve in RP2 neurons. How does Klu repress eve expression in GMC4-2b? Negative regulation of eve expression by Klu is due to direct binding to the eve minimal element. (1) It is shown that klu mutants exhibit similar derepression of the eve minimal element transgene and the endogenous eve gene in the NB4-2 lineage; (2) three consensus binding sites are detected for Klu in the eve minimal element (comparison of Drosophila virilis and Drosophila melanogaster shows that the three identified sites are highly conserved); (3) mutation of these sites results in ectopic expression of eve-lacZ in the NB4-2 lineage in wild-type, and (4) mutation of these sites impairs repression of eve-lacZ by ectopic Klu in the NB4-2 lineage. The predicted Klu binding sites (K123) are probably only a subset of relevant Klu binding sites, however, because mutation of the sites gives only partially penetrant phenotypes (McDonald, 2003).

Surprisingly, it was not possible to separate the GMC4-2a/ RP2 element from the GMC1-1a/aCC/pCC element. In both NB 1-1 and NB 4-2 lineages, eve is expressed in the first-born GMC and its neuronal progeny. Both first-born GMCs share expression of several transcription factors, including Pros and Ftz. However, many other transcription factors are differentially expressed, such as the GMC1-1a specific expression of Vnd and Odd-skipped, and the GMC4-2a specific expression of Hkb, Pdm1, and Ind. It is possible that one or more commonly expressed transcription factors are required for expression of eve in both GMC1-1a and GMC4-2a, such as Pros, and this is why the elements cannot be subdivided (McDonald, 2003).

Nerfin-1, a target of Pros, is required for early axon guidance decisions in the developing Drosophila CNS

Nerfin-1 is a nuclear regulator of axon guidance required for a subset of early pathfinding events in the developing Drosophila CNS. Nerfin-1 belongs to a highly conserved subfamily of Zn-finger proteins with cognates identified in nematodes and man. The neural precursor gene prospero is essential for nerfin-1 expression. Unlike nerfin-1 mRNA, which is expressed in many neural precursor cells, the encoded Nerfin-1 protein is only detected in the nuclei of neuronal precursors that will divide just once and then transiently in their nascent neurons. Although nerfin-1 null embryos have no discernible alterations in neural lineage development or in neuronal or glial identities, CNS pioneering neurons require nerfin-1 function for early axon guidance decisions. Furthermore, nerfin-1 is required for the proper development of commissural and connective axon fascicles. Nerfin-1 is essential for the proper expression of robo2, wnt5, derailed, G-oα47A, Lar, and futsch<, genes whose encoded proteins participate in these early navigational events (Kuzin, 2005).

Comparative analysis of Nerfin-1 and Pros expression in the developing nervous system revealed a marked, although not complete, overlap in expression. The nuclear co-localization of these proteins in neuronal precursor cells, and the fact that mutations in nerfin-1 and pros trigger axon guidance defects, raised the possibility that they may regulate the expression of one another. To determine the epistatic relationship between nerfin-1 and pros, their expression dynamics were studied in each other's loss-of-function mutant backgrounds. Pros immunostaining in nerfin-1null embryos did not identify any significant changes in Pros expression. In marked contrast, nerfin-1 mRNA and protein levels in embryos collected from two independent loss-of-function pros mutants revealed that pros is required for wild-type nerfin-1 expression levels. Nerfin-1 expression was significantly reduced throughout the CNS and PNS; however, its expression was not completely ablated in any of the prosnull alleles tested (Kuzin, 2005).

To determine if the axon guidance phenotype observed in prosnull (prosI13) mutant embryos could be explained by a requirement for wild-type nerfin-1 expression, the extent of CNS axon disorganization triggered by single and double mutant combinations were examined. Comparisons demonstrated that loss of pros function resulted in a more severe phenotype than that observed in the nerfin-1null embryos. Although both prosnull and nerfin-1null embryos exhibited disruptions in the longitudinal axon connectives, loss of pros function resulted in an overall greater disruption in commissure organization. In addition, the nerve cord in prosnull embryos was considerably wider when compared to nerfin-1 mutants or wild-type embryos. The axon scaffolding phenotype observed in nerfin-1null; prosI13 double mutant was more severe than that of either single mutant (Kuzin, 2005).

Analysis of two other transcription factor genes that are widely expressed in the developing CNS, lola and fru, has revealed that they too are required for proper axon guidance and both are required for proper longitudinal axon fasciculation. However, unlike the severe axon guidance phenotype observed in prosnull embryos, the disorganization of the CNS axon scaffolding is not as extensive in lola or fru loss-of-function mutants. To determine the epistatic relationship between nerfin-1 and lola or fru, the expression of each gene in each other's loss-of-function background was analyzed. In contrast to the marked reduction of nerfin-1 expression in pros mutants, no such reduction in nerfin-1 expression was found in lola or fru mutants, nor was the expression of lola or fru altered in nerfin-1null embryos (Kuzin, 2005).

A critical role for cyclin E in cell fate determination in the central nervous system of Drosophila; CycE functions upstream of pros and gcm

This study examined the process by which cell diversity is generated in neuroblast (NB) lineages in the central nervous system of Drosophila. Thoracic NB6-4 (NB6-4t) generates both neurons and glial cells, whereas NB6-4a generates only glial cells in abdominal segments. This is attributed to an asymmetric first division of NB6-4t, localizing prospero (pros) and glial cell missing (gcm) only to the glial precursor cell, and a symmetric division of NB6-4a, where both daughter cells express pros and gcm. This study shows that the NB6-4t lineage represents the ground state, which does not require the input of any homeotic gene, whereas the NB6-4a lineage is specified by the homeotic genes abd-A and Abd-B. They specify the NB6-4a lineage by down-regulating levels of the G1 cyclin, DmCycE (CycE). CycE, which is asymmetrically expressed after the first division of NB6-4t, functions upstream of pros and gcm to specify the neuronal sublineage. Loss of CycE function causes homeotic transformation of NB6-4t to NB6-4a, whereas ectopic CycE induces reverse transformations. However, other components of the cell cycle seem to have a minor role in this process, suggesting a critical role for CycE in regulating cell fate in segment-specific neural lineages (Berger, 2005).

In Drosophila, individual neuroblasts deriving from corresponding neuroectodermal positions among thoracic and abdominal segments generally acquire similar fates. However, some of these serially homologous neuroblasts produce lineages with segment-specific differences that contribute to structural and functional diversity within the CNS. The NB6-4 lineage was selected as a model to determine how this diversity evolves from a basic developmental ground state. As an experimental system, NB6-4 has an additional advantage, since Eagle (Eg) is expressed in all the cells of both thoracic and abdominal lineages and can thus be used as a lineage marker (Berger, 2005).

First the expression patterns of different homeotic genes were examined in thoracic and abdominal lineages of NB6-4. Antennapedia (Antp) is expressed in NB6-4t lineages of thoracic segments T1-T3. Abdominal A (Abd-A) is expressed in the NB6-4a lineage of abdominal segments A1-A6, whereas Abdominal B (Abd-B) is expressed in the NB6-4a lineage of segments A7-A8. Whereas loss of Antp function does not affect the NB6-4t lineage in any of the thoracic segments, loss-of-function mutations in abd-A and Abd-B cause NB6-4a-to-NB6-4t homeotic transformations in their corresponding segments. Interestingly, Ultrabithorax (Ubx), which is expressed in most of the cells of T3, is specifically absent in the NB6-4t lineage of that segment, and its loss-of-function alleles do not show any thoracic phenotypes. However, overexpression of Ubx as well as abd-A causes NB6-4t-to-NB6-4a transformations. Thus, it seems that the NB6-4t fate is the ground state and the NB6-4a state is imposed by the function of homeotic genes of the bithorax-complex (BX-C). This is consistent with previous reports that the T2 state is the ground state (for epidermis, including adult appendages) and other segmental identities are conferred by the function of homeotic genes (Berger, 2005).

The mechanism was examined by which abd-A or Abd-B specify the NB6-4a lineage compared with the NB6-4t lineage. As the mode and number of mitoses is the most obvious characteristic by which the NB6-4a lineage differs from NB6-4t, it was wondered whether factors regulating the cell cycle might be involved in controlling NB6-4 cell fate. One major factor that regulates the cell cycle is the G1 Cyclin CycE, which is needed for various aspects of the G1-to-S-phase transition (Berger, 2005).

To examine possible effects on cell fate decisions in the NB6-4t lineage, CycEAR95-mutant embryos were stained for gcm transcripts and Pros and Repo proteins. In wild-type embryos, gcm is initially distributed to both daughter cells during the first division of NB6-4t, but subsequently gets rapidly removed in the cell that functions as a neuronal precursor. Pros is transferred asymmetrically into only one cell, where it is needed to maintain and enhance the expression of gcm, thereby promoting glial cell fate. In CycEAR95 embryos, even at late stages (up to stage 14), gcm mRNA is strongly expressed in both daughter cells after the first division of NB6-4t. Even distribution of Pros was observed in both daughter cells, which could be the cause of continued expression of gcm. Furthermore, the glial marker Repo revealed that both cells differentiate as glial cells. The NB6-4a lineage is not affected in CycEAR95-mutant embryos, suggesting that the requirement for zygotic CycE is specific to NB6-4t (Berger, 2005).

Whether ectopic expression of CycE in abdominal lineages causes the opposite effect was tested. The sca-GAL4 line was used to drive UAS-CycE to achieve early expression in the neuroectoderm. An asymmetric distribution of Pros to one of the two progeny cells was observed just after the first division of NB6-4a. At later stages an increase was observed in the number of cells in the NB6-4a lineage (up to 5 cells). Some of these cells migrated medially, as NB6-4 glial cells normally do, maintaining Pros expression at a lower level. They also expressed Repo, which confirmed their glial identity. Other cells stayed in a dorso-lateral position and did not stain for Repo, suggesting neuronal identity (Berger, 2005).

To further investigate if ectopic CycE had indeed induced a neuronal sublineage in NB6-4a, and to test whether CycE can function cell-autonomously, a cell transplantation technique was employed. Single progenitor cells (stage 7) from the abdominal neuroectoderm of horseradish peroxidase (HRP)-labelled donor embryos overexpressing CycE were transplanted into the abdominal neuroectoderm of unlabelled wild-type hosts (at the same stage). The lineages produced by the transplanted cells were identified by morphological criteria. In all six cases, where cell clones were derived from NB6-4a, they were composed of both glial cells and neurons exhibiting their respective characteristic structures and positions. Because the clones are located in a wild-type abdominal environment, this experiment provides evidence that ectopic expression of CycE causes asymmetric division of NB6-4a and confers neuronal identity to one part of the lineage in a cell-autonomous manner. In these single-cell transplantation experiments, similar observations were made for NB1-1 and NB5-4, which also generate segment-specific lineages. Thus, CycE seems to have a general role in establishing segment-specific differences in neuroblast lineages (Berger, 2005).

Next whether the requirement for CycE to specify the neuronal lineage in NB6-4t is due to altered cell-cycle phases was examined. In string mutants, NB6-4t (whose proliferation is blocked before its first division) expresses gcm mRNA, as well as Pros and hunchback protein, although it does not differentiate as a glial cell. The composition of NB6-4t and NB6-4a lineages were further analysed in embryos mutant for other factors that interact with CycE in cell-cycle regulation. dacapo (dap) is the Drosophila homologue of members of the p21/p27Cip/Kip inhibitor family, which specifically block CycE-cdk complexes. Interestingly, in dap-null-mutant embryos an additional glial cell was observed in the NB6-4a lineage, but the appearance of any neuron-like cells was not observed. Consistent with these results, overexpression of Dap resulted in a reduction in the number of neurons in the NB6-4t lineage (from the normal number of 6 to 2-4), but homeotic transformation of the lineage did not occur. The number of glial cells was never affected, neither in the thorax nor in the abdomen. Ectopic expression of p21, the human homologue of the Drosophila dacapo gene, generated similar phenotypes (Berger, 2005).

The influence of the transcription factor dE2F, which mediates the activation of several genes needed for the initiation of S phase, was tested. In dE2F-mutant embryos, unlike in CycE mutants, no homeotic transformation of NB6-4t to NB6-4a was observed, although the number of neurons was reduced from 5-6 to 2-4. Ectopic expression of dE2F resulted in an increase in cell number in some abdominal hemisegments. In only a small percentage of those embryos, cells at lateral positions in abdominal segments did not show expression of gcm or Repo, suggesting their neuronal identity. Thus, although dE2F activation in the CNS depends on CycE, ectopic expression of dE2F cannot fully bypass the requirement for CycE in a NB6-4a-to-NB6-4t transformation. Similarly, ectopic expression of Rbf, a potent inhibitor of E2F target genes, did not cause any changes in the segregation of gcm mRNA, Pros or Repo in the NB6-4t lineage (Berger, 2005).

Whether interfering with another checkpoint of the cell cycle, the transition from G2 to M phase, affects NB6-4 cell fate was tested. Previous studies show that loss of CycA function prevents further mitosis after the first division of NB6-4t. However, the first division of NB6-4t follows the normal pattern; it gives rise to one glial and one neuronal cell. Similar effects were observed in CycA mutants. The cyclin-dependent kinase cdc2 heterodimerizes with CycA and CycB, and high levels of cdc2 expression have been shown to be required for maintaining the asymmetry of neuroblast divisions. In a cdc2 loss-of-function background, NB6-4t generated a normal lineage consisting of two glial cells and five to six dorso-lateral neurons. These observations show that NB6-4 cell fates do not change after manipulation of the transition from G2 to M phase (Berger, 2005).

These results suggest a critical role for CycE per se in regulating the NB6-4t lineage. Therefore whether CycE itself is differentially expressed between thoracic and abdominal NB6-4 lineages was tested. In situ hybridization with CycE RNA on wild-type embryos revealed that CycE is expressed just before the first division in NB6-4t. After the first division, CycE mRNA was detected in the neuronal precursor only and not in the glial precursor. In abdominal segments, no CycE expression was detected in NB6-4a before or after the division. Consistent with the role of CycE in specifying the NB6-4t lineage, notable levels of CycE transcripts were detected in the homeotically transformed NB6-4a lineages in abd-A-mutant embryos. Conversely, overexpression of abd-A caused down-regulation of CycE levels in thoracic segments and homeotic transformation of NB6-4t to NB6-4a. The importance of CycE in generating neuronal cells in the NB6-4t lineage was confirmed in an epistasis experiment involving abd-A and CycE mutants. As described above, loss of abd-A leads to transformation of NB6-4a to NB6-4t. Such homeotic transformation was suppressed by mutations in CycE, suggesting an absolute requirement for CycE in specifying the NB6-4t lineage. Finally, nine potential AbdA-binding sites (five of which are evolutionarily conserved in Drosophila pseudoobscura) were identified in a 5.0-kb enhancer fragment of CycE that is known to harbour cis-acting sequences for driving CycE expression in the CNS (Berger, 2005).

It is concluded that, in addition to its role in cell proliferation, CycE is necessary and sufficient for the specification of cell fate in the NB6-4 lineage. These results suggest that the function of CycE in regulating cell fate in NB6-4 lineages is independent, albeit partially, of its role in cell proliferation. The absence of any cell fate changes in the loss-of-function mutants of string, dap, cdc2 or CycA and in the Dap or Rbf gain-of-function genetic background may be attributed to the presence of CycE, which is strongly expressed in the NB6-4t, but not the NB6-4a, lineage. However, CycE may still function by controlling cell-cycle progression. NB6-4a, which does not express CycE, divides once followed by a cell-cycle arrest, presumably in G1. After the first division in the thorax, one daughter cell expresses high levels of CycE and divides roughly three times to generate neuronal cells. Therefore, this daughter cell presumably progresses through S phase. Chromatin reorganization during S phase might allow cell fate regulators to access their target genes, driving neuronal differentiation. Contrary to this interpretation, the other daughter cell of NB6-4t, which does not express CycE, divides twice but still generates glial cells. Thus, it remains to be investigated whether the role of CycE in neuronal cell fate determination is entirely independent of its role in cell proliferation. The results on the role of CycE in specifying neuronal compared with glial cell fate in the CNS are consistent with data from Xenopus on the role of cyclin-cdk complexes in specifying neuronal cell fate, inhibition of which promotes glial cell fate. In addition, this study shows that homeotic genes contribute to regional diversification of cell types in the CNS through the regulation of CycE levels (Berger, 2005).

Timing of identity: spatiotemporal regulation of hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero

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).

Neural stem cell transcriptional networks highlight genes essential for nervous system development

Neural stem cells must strike a balance between self-renewal and multipotency, and differentiation. Identification of the transcriptional networks regulating stem cell division is an essential step in understanding how this balance is achieved. It has been shown that the homeodomain transcription factor Prospero acts to repress self-renewal and promote differentiation. Among its targets are three neural stem cell transcription factors, Asense, Deadpan and Snail, of which Asense and Deadpan are repressed by Prospero. This study identifies the targets of these three factors throughout the genome. A large overlap in their target genes was found, and indeed with the targets of Prospero, with 245 genomic loci bound by all factors. Many of the genes have been implicated in vertebrate stem cell self-renewal, suggesting that this core set of genes is crucial in the switch between self-renewal and differentiation. It was also found that multiply bound loci are enriched for genes previously linked to nervous system phenotypes, thereby providing a shortcut to identifying genes important for nervous system development (Southall, 2009).

Recent work on Drosophila neural stem cells (or neuroblasts) has provided important insights into stem cell biology and tumour formation. Neuroblasts divide in an asymmetric, self-renewing manner producing another neuroblast and a daughter cell that divides only once to give post-mitotic neurons or glial cells. During these asymmetric divisions the atypical homeodomain transcription factor, Prospero, is asymmetrically segregated to the smaller daughter cell, the ganglion mother cell (GMC), where it can enter the nucleus and regulate transcription. Neuroblasts lacking Prospero form tumours in both the embryonic nervous system and the larval brain. Using the chromatin profiling technique DamID, together with expression profiling, it has been showm that Prospero represses neuroblast genes and is required to activate neuronal differentiation genes. Therefore, Prospero acts as a binary switch to repress the genetic programs driving self-renewal (by directly repressing neuroblast transcription factors) and to promote differentiation. It was found that Prospero represses the neuroblast transcription factors, Asense, Deadpan and Snail, suggesting that these transcription factors may control genes involved in neural stem cell self-renewal and multipotency (Southall, 2009).

To identify the transcriptional networks promoting neural stem cell fate the binding sites of Asense, Deadpan and Snail were profiled, on a whole genome scale. These three proteins are members of a small group of transcription factors that are expressed in all embryonic neuroblast. The first, Asense, is a basic-helix-loop-helix protein, a member of the achaete-scute complex, and a homologue of the vertebrate neural stem cell factor, Ascl1 (Mash1). Unlike the other members of the achaete-scute complex, Asense is not expressed in proneural clusters in the embryo. Asense expression is initiated in the neuroblast and is maintained in at least a subset of GMC daughter cells. Asense is also expressed in most larval brain neuroblasts but is markedly absent from the DM/PAN neuroblast (Bello, 2008; Bowman, 2008). In these lineages, Asense expression is delayed and the daughter cells (secondary neuroblasts) of the Asense-negative DM/PAN neuroblasts undergo multiple cell divisions, expanding the stem cell pool before producing GMCs (Bello, 2008; Boone, 2008; Bowman, 2008). Ectopic expression of Asense limits the division potential of DM/PAN neuroblast progeny (Bowman, 2008). A study in the optic lobe showed that Asense expression coincides with the upregulation of dacapo and cell-cycle exit. Perhaps in combination, these results suggest that Asense may also have a pro-differentiation role (Southall, 2009).

The second transcription factor, Deadpan, is a basic-helix-loop-helix protein related to the vertebrate Hes family of transcription factors. Deadpan is expressed in all neuroblasts and has been shown to promote the proliferation of optic lobe neural stem cells. Unlike Asense, Deadpan is also expressed in the DM/PAN neuroblasts of the larval brain (Southall, 2009).

The third factor, Snail, is a zinc-finger transcription factor whose vertebrate homologues have roles in the epithelial to mesenchymal transition and in cancer metastasis. The Snail family members (Snail, Worniu and Escargot) are known to regulate neuroblast spindle orientation and cell-cycle progression (Southall, 2009).

To further understand the role of these pan neural stem cell transcription factors, their targets were mapped throughout the genome. This, combined with expression profiling, allows building of the gene regulatory networks governing neural stem cell self-renewal, and enhancement of knowledge of the function and mode of action of these transcription factors in neural stem cells (Southall, 2009).

To identify the genes regulated by Asense, Deadpan and Snail in the embryo, their binding sites in vivo were mapped by DamID, as has been done previously for Prospero (Choksi, 2006). In brief, DamID involves tagging a DNA or chromatin-associated protein with a Escherichia coli DNA adenine methyltransferase (Dam). Wherever the fusion protein binds, surrounding DNA sequences are methylated. Methylated DNA fragments can then be isolated, labelled and hybridised on a microarray. This study expressed Dam fusion proteins in vivo, in transgenic Drosophila embryos. Methylated DNA fragments from transgenic embryos expressing Dam alone serve as a reference. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation, by mapping to polytene chromosomes and by 3D microscopy data (Southall, 2009).

In comparing the results for Asense, Deadpan, Snail and Prospero, a high degree of overlap was seen between their targets. The average overlap for the four factors in pairwise comparisons is 40%, with the highest overlap between Deadpan and Snail (66%). The similarity in binding is illustrated by the binding of all four factors to the intronic regions of the cell-cycle regulation gene CycE. 245 genes are bound by all four proteins, including genes involved in neuroblast cell fate determination, cell-cycle control and differentiation. These loci are unlikely to represent regions of chromatin accessible to all transcription factors; only 17/245 (7%) were also bound by another neural transcription factor, Pdm1. The large overlap in the targets of Asense, Deadpan, Snail and Prospero implies that these may be a core set of genes involved in neuroblast self-renewal and differentiation (Southall, 2009).

Genome-wide analysis of Asense DamID peaks shows that Asense binding is associated with increased levels of DNA conservation (determined by the alignment of eight insect species. A representation of Asense binding around a generic gene shows an enrichment of ~2 kb upstream of the transcriptional start site, binding within intronic regions (32%) and also downstream of the gene (20%). This distribution is consistent with transcription factor-binding analysis and regulatory sequence studies in mice and humans (Southall, 2009).

The resolution of DamID is ~1 kb and there are currently no motif discovery tools available that can analyse the large amount of sequence data generated by full genome DamID. Therefore, a motif discovery tool, called MICRA (Motif Identification using Conservation and Relative Abundance) was developed to identify overrepresented motifs in low-resolution data. In brief, 1 kb of sequence from each binding site is extracted and filtered for conserved sequences. The relative frequency of each 6-10 mer is then calculated and compared with background frequency. Using MICRA the E-box, CAGCTG, was identified as the most overrepresented 6 mer in the regions of Asense binding (131% overrepresented using a conservation threshold of 0.6. In support of the in vivo binding data, in vitro studies had previously shown that Asense binds to CAGCTG, which is also the binding site of the vertebrate Asense homologue Ascl1 (Mash1) (Southall, 2009).

A GO annotation analysis of the genes bound by Asense shows a highly significant overrepresentation of genes involved in nervous system development and cell fate determination. Similar analyses were performed for Deadpan and Snail and for both transcription factors; DNA conservation was enriched surrounding their binding sites. Deadpan and Snail targets fall broadly into the same gene ontology classes as Asense and Prospero and the binding peaks show a similar distribution relative to gene structure as for Asense. Motif discovery using MICRA identifies sites consistent with previously published in vitro studies for Deadpan (CACGCG and CACGTG) and Snail (CAGGTA). These analyses provide unbiased support for the Deadpan and Snail DamID experiments (Southall, 2009).

When comparing the data sets for Asense, Deadpan, Snail and Prospero genomic loci in which multiple transcription factors bind were found. This phenomenon has been described previously in a Drosophila cell line and, more recently, in mouse embryonic stem (ES) cells in which these loci are termed 'multiple transcription factor-binding loci' (MTL). The ES cell MTLs are associated with ES-cell-specific gene expression and are thought to identify genes important for stem cell self-renewal. The data provide an independent and direct, in vivo demonstration of the phenomenon described in these two earlier studies. Analysis of neural MTLs (as determined by binding of Asense, Deadpan, Prospero and Snail within a 2 kb window) shows increased sequence constraint, correlating with the number of transcription factors bound. The increase in conservation is higher than expected solely based on the combined binding sites of the factors studied. This suggests that further factors may bind to these loci. The loci associated with MTLs are enriched for genes required for proper neural development and for viability (Southall, 2009).

To investigate further the relationship between the number of transcription factors bound at a locus and the importance of the associated target gene in neural development, a database ( was assembled comprising DamID data, expression profiling of neural transcription factors, and data on Drosophila nervous system development collated from genetic screens, expression screens, gene homology and text mining screens. Using a random permutation algorithm and training sets of known nervous system development genes weighted scores were assiged to each screen. A total score is calculated for each gene, providing an indication of the gene's involvement in nervous system development. Multiple gene lists can be searched in the database, which is a useful method to pinpoint key genes in user generated gene lists (e.g. expression array results) (Southall, 2009).

Using the data collected for the database, a correlation was consistently found between gene sets bound by increasing numbers of transcription factors and genes in Drosophila genetic screens for defects in nervous system development, eye development and cell-cycle progression or in text mining screens (occurrence of the gene or its homologue with neural or stem cell terms; r=0.98) (Southall, 2009).

This study has shown that Asense, Deadpan, Prospero and Snail bind to genes essential for neural development. This finding enables highlighting of novel genes that may be involved in neural development. The neuroBLAST database ranks genes based on the number of transcription factors bound, together with their appearance in external screens. In this way it identifies known key players in neural development such as prospero, brain tumour, miranda, seven up and glial cells missing. The majority of these genes are identified by multiple binding information (DamID data), independent of external screens and weighted scores (Southall, 2009).

Interestingly, there are many high scoring genes that have not previously been characterised for a role in Drosophila neural development. These include CG32158, an adenylate cyclase known to be expressed in the CNS, two putative transcription factors (CG2052 and CG33291), an NADH dehydrogenase (CG2014) and an F-box protein (CG9772). There is also cenG1A, an ARF GTPase activator, is bound by all four transcription factors and is expressed in neuroblasts. CG9650 is bound by Prospero and Deadpan, and is a homologue of the BCL11b oncogene, which is essential for proper corticospinal neuron development in vertebrates. Another high scoring gene identified by this method is canoe (bound by all four transcription factors, neuroBLAST score of 33.7), which has recently been shown to regulate neuroblast asymmetric divisions (Southall, 2009).

Using the binding data for these four transcription factors as a foundation, attempts were made to construct the transcriptional networks governing neural stem cell self-renewal and differentiation. Although DamID reports protein-binding sites, it cannot show how individual target genes are regulated in response to binding. Expression profiling of neuroblasts and GMCs from wild type and mutant embryos can provide this information, and provide greater insight into the biological function of each of the transcriptional regulators (Southall, 2009).

Expression profiling of asense mutants was performed on 50-100 neuroblasts and GMCs microdissected from the ventral nerve cord of stage 11 wild type and mutant embryos. Genes that are bound by Asense exhibited a significant change in expression level in asense mutant neuroblasts and GMCs. In many cases, neuronal differentiation and Notch pathway genes (enhancer of split complex [E(spl)-C] and bearded complex) are upregulated in the mutant, suggesting that Asense normally represses them, whereas neuroblast genes are downregulated, suggesting they require Asense for expression. This contrasts with the data for Prospero, which represses neuroblast genes and is required for the activation of differentiation genes. Combined with the fact that Prospero represses expression of Asense, these data support an antagonistic relationship between Prospero and Asense. For example, the neuroblast genes miranda and grainy head are activated by Asense and repressed by Prospero, whereas transcription of the differentiation gene Fasciclin I is promoted by Prospero but inhibited by Asense. Interestingly, however, there are also examples of differentiation and cell-cycle exit genes activated by Asense, such as commissureless, hikaru genki and dacapo. Furthermore, when the full expression array data from prospero mutants and asense mutants are compared by cluster analysis two clusters were found in which genes are regulated antagonistically, but also two clusters in which genes are similarily regulated. These data suggest a dual role for Asense: activating the expression of neuroblast genes and repressing differentiation genes in the neuroblast, whereas promoting differentiation when present in the GMC (Southall, 2009).

This study has combined in vivo chromatin profiling and cell-specific expression profiling to identify the gene regulatory networks directing neural stem cell fate and promoting differentiation in the Drosophila embryo. Asense, Deadpan, Snail and Prospero were found to bind to many of the same target genes. The targets of Asense, Deadpan and Snail include neuroblast genes but also many differentiation genes. The binding of these neural stem cell factors to differentiation genes is not entirely unexpected. In vertebrates, stem cell transcription factors bind to and repress differentiation genes to maintain the stem cell state. Additionally, it is becoming apparent that transcription factors can have roles in both activation and repression, in Drosophila and in vertebrate stem cell transcriptional networks. The ability to either repress or activate is likely to be due to interaction with co-factors, and the ability to recruit chromatin remodelling complexes to specific loci (Southall, 2009).

It was shown previously that Prospero represses the expression of Asense and Deadpan in GMCs, supporting a model whereby a core set of genes involved in neuroblast self-renewal and multipotency is activated by the neuroblast transcription factors and repressed by Prospero. This study has shown that, in part, Asense acts oppositely to Prospero, promoting the expression of neuroblast genes and repressing certain differentiation genes. However, the data also indicate that Asense can promote the expression of some genes required for differentiation, including the cell-cycle inhibitor dacapo, which is a member of the p21/p27 family of cdk inhibitors. dacapo expression inititates in the GMC; a reduction was observed in levels of dacapo mRNA in the asense mutant neuroblasts and GMCs, similar to what has been reported in the developing optic lobe. asense mRNA is known to be expressed in at least a subset of GMCs, and Asense protein is present in larval GMCs. This suggests that Asense has a secondary role, to promote GMC cell-cycle exit and differentiation. Asense is absent in larval PAN neuroblasts whose progeny, unlike GMCs, divide in a stem cell-like manner. Ectopic expression of Asense prevents formation of these daughter cells, which can undergo extra divisions (Bowman, 2008), possibly by the upregulation of dacapo, and other differentiation genes (Southall, 2009).

The expression pattern, function and binding site specificity of Asense all correlate strongly with its vertebrate counterpart, Ascl1 (Mash1). Mash1 is expressed in neural precursors in vertebrates, is known to regulate genes involved in Notch signalling (Delta, Jag2, Lfng and Magi1), cell-cycle control (Cdc25b) and neuronal differentiation (Insm1)and recognises the E-box sequence, CAGCTG. Furthermore, Mash1 is consistently found to promote neuronal differentiation, consistent with a pro-differentiation role for Asense. Conversely, it was shown that Asense activates the expression of certain neuroblast genes, such as miranda, which is expressed in all neuroblasts and repressed by Prospero. Deadpan and Snail bind to many neuroblast genes. Given that the expression of deadpan and snail is restricted to pan-neural neuroblasts, it is likely that they can also activate the expression of neuroblast genes. However, confirmation of this awaits expression profiling of deadpan and snail mutant neuroblasts and GMCs (Southall, 2009).

Finally, this study has shown that multiple transcription factor binding is associated with genes that have critical functions in neural development. This relationship can be used to identify novel genes involved in neural development, including those with vertebrate counterparts. A similar gene network and data mining study, using two pair-rule genes in Drosophila, has recently been used to identify a new marker for kidney cancer. Therefore, large-scale analysis of gene regulatory networks, as used here, provides a powerful approach to identifying key genes involved in development and disease (Southall, 2009).

Drosophila homologs of mammalian TNF/TNFR-related molecules regulate segregation of Miranda/Prospero in neuroblasts

During neuroblast (NB) divisions, cell fate determinants Prospero (Pros) and Numb, together with their adaptor proteins Miranda (Mira) and Partner of Numb, localize to the basal cell cortex at metaphase and segregate exclusively to the future ganglion mother cells (GMCs) at telophase. In inscuteable mutant NBs, these basal proteins are mislocalized during metaphase. However, during anaphase/telophase, these mutant NBs can partially correct these earlier localization defects and redistribute cell fate determinants as crescents to the region where the future GMC 'buds' off. This compensatory mechanism has been referred to as 'telophase rescue'. The Drosophila homolog of the mammalian tumor-necrosis factor (TNF) receptor-associated factor (TRAF1) and Eiger (Egr), the homolog of the mammalian TNF, are required for telophase rescue of Mira/Pros. TRAF1 localizes as an apical crescent in metaphase NBs and this apical localization requires Bazooka (Baz) and Egr. The Mira/Pros telophase rescue seen in inscuteable mutant NBs requires TRAF1. These data suggest that TRAF1 binds to Baz and acts downstream of Egr in the Mira/Pros telophase rescue pathway (Wang, 2006).

In telophase NBs, segregation of cell fate determinants, such as Pros, into future GMCs, is critical for their proper development. Telophase rescue appears to be one of the safeguard mechanisms that acts to ensure that GMCs inherit the cell fate determinants and adopt the correct cell identity when the mechanisms, which normally operate during NB divisions, fail (e.g., in insc mutant). Telophase rescue is a phenomenon for which the underlying mechanism involved remains largely unknown. The current data demonstrate that TRAF1 and Egr are two members of the Insc-independent telophase rescue pathway specific for Mira/Pros (Wang, 2006).

Although it is apically enriched in mitotic NBs and can directly interact with Baz in vitro, TRAF1 does not seem to be involved with the functions normally associated with the apical complex proteins. One distinct feature of TRAF1 differs from the other known apical proteins is its localization pattern; it is cytoplasmic in interphase and the apical crescent is prominent only at metaphase. In contrast, proteins of the apical complex are largely undetectable during interphase and form distinct apical crescents, starting from late interphase or early prophase. The protein localization difference between TRAF1 and other apical proteins suggests that TRAF1 and apical proteins are not always colocalized during mitosis. If TRAF1 is a bona fide member of the apical complex, the localization defects of other apical proteins are expected to be observed in TRAF1 mutant, as well as mislocalization of basal proteins, which was not detect. In addition, no spindle orientation or geometry defects were observed in the absence of TRAF1. Based on these observations, it is concluded that TRAF1 is not involved with the functions normally associated with the apical complex proteins (Wang, 2006).

The in vitro GST fusion protein pull-down assay suggests that TRAF1 may physically bind to Baz. This result is consistent with genetic data, indicating that TRAF1 acts downstream of baz and that its apical localization requires baz. These observations are consistent with the view that TRAF1 is recruited to the apical cortex by apical Baz in mitotic NBs. Baz, even at very low levels, can recruit TRAF1 to the apical cortex of the mitotic NBs. For example, in insc mutant NBs, TRAF1 remains apical probably owing to the low levels of Baz that remain localized to the apical cortex. This speculation is supported by Mira/Pros telophase rescue data, which clearly demonstrate that the telophase rescue seen in insc mutant NBs is severely damaged in baz mutant, suggesting that the Baz function required for Mira/Pros (and Pon/Numb) telophase rescue is intact in insc mutant NBs (Wang, 2006).

It has been shown that Pins/Gαi asymmetric cortical localization can be induced at metaphase by the combination of astral microtubules, kinesin Khc-73 and Dlg in the absence of Insc; this coincides with the observation that TRAF1 also forms tight crescent only at metaphase in both WT and insc mutant NBs. Does TRAF1 apical crescent formation also require the functions of astral microtubules, kinesin Khc-73 and Dlg? The data do not favor this hypothesis based on the following observations. (1) In TE35BC-3, a small deficiency uncovering sna family genes insc is not expressed but Pins and Gαi are asymmetrically localized, indicating that the astral microtubules, kinesin Khc-73 and Dlg pathway remain functional. TRAF1 is delocalized and is uniformly cortical in this deficiency line. (2) Similarly, in egr insc NBs, TRAF1 is cytoplasmic whereas the functions of astral microtubules, kinesin Khc-73 and Dlg are intact. (3) In egr NBs TRAF1 is cytoplasmic, whereas the apical complex is normal and astral microtubules, kinesin Khc-73 and Dlg are present. (4) TRAF1 apical localization remains unchanged in dlg mutant NBs. Based on these observations, it is concluded that TRAF1 apical localization is unlikely to share similar mechanism with Pins and Gαi and is likely to be independent of astral microtubules, kinesin Khc-73 and Dlg. TRAF1 apical localization appears to specifically require Egr and Baz (Wang, 2006).

In TRAF1 insc double-mutant embryos, the complete segregation of Mir/Pros into future GMCs occurs only in about 12% of the total population, and in the remaining NBs, only a fraction of Mira/Pros segregate into future GMCs as indicated by the Mira 'tail' extending into the future NBs at telophase. As it is difficult to address the global effect of this partial segregation of Mira/Pros on GMC specification in TRAF1 insc double mutant, focus was place on a well-defined GMC, GMC4-2a in NB4-2 lineage, to evaluate this issue. It is assumed that as long as the RP2 neuron (progeny of GMC4-2a, Even-skipped (Eve)-positive) was identified in a particular hemisegment, the GMC cell fate of GMC4-2a in that hemisegment should have been correctly specified. In insc mutants, almost all hemisegments contain RP2s, indicating that GMC4-2a has adopted the correct GMC cell fate in 99% of the total hemisegments. When TRAF1 insc double-mutant embryos were stained with anti-Eve, it was found the frequency of loss of Eve-positive RP2 neuron increased (to 8%) in late embryos, suggesting that about 8% of the GMCs in TRAF1 insc double mutant did not inherit sufficient Pros to specify the GMC fate in these embryos. The relatively low frequency (8%) of mis-specification of GMCs suggests that the threshold amount of Pros protein needed is sufficiently low such that just a partial inheritance of Pros, even when telophase rescue is compromised, is sufficient for most GMCs to be correctly specified (Wang, 2006).

Although Mira/Pros and Pon/Numb share similar basal localization patterns in insc NBs, further removal of either TRAF1 or Egr compromised telophase rescue only for Mira/Pros, but not for Pon/Numb. This difference between Mira/Pros and Pon/Numb indicates that the detailed mechanisms of basal localization and segregation of Mira/Pros differ from those of Pon/Numb, which is consistent with the observations that the dynamics of Mira/Pros and Pon/Numb localization early in mitosis are different and the basal localization for Mira/Pros and Pon/Numb requires different regions of the Insc coding sequence (Wang, 2006).

Dlg/Lgl/Scrib are required for correct basal localization of Mira/Pros and Pon/Numb in mitotic NBs. Dlg has been shown to be involved in the Mira telophase rescue. In dlg insc double-mutant NBs, not only was spindle geometry symmetric but Mira telophase rescue was also affected. It would be interesting to know if Dlg belongs to the same pathway as TRAF1 and Egr and if Dlg is also involved in Pon/Numb telophase rescue (Wang, 2006).

Two other members of the TRAF family have also been identified in Drosophila: DTRAF2 (DTRAF6) and DTRAF3. In contrast to the specific and strong expression of TRAF1 in the embryonic NBs, only low levels of ubiquitous signals similar to the control background were seen in the NBs with DTRAF2 and DTRAF3 probes. It is likely that DTRAF2 and DTRAF3 are not expressed in NBs and do not play an important role in Mira/Pros telophase rescue pathway as the Mira/Pros telophase rescue is dramatically compromised in TRAF1 insc and egr insc NBs (Wang, 2006).

In mammals, the TNF pathway works as a typical receptor-mediated signal transduction pathway. TNFR is a key player in transducing external signal to the cytoplasm. In the Drosophila compound eyes, ectopic Egr, Wgn and TRAF1 seem to work in a similar receptor-mediated signal pathway to induce apoptosis through the activation of the JNK pathway. Does the same Egr, Wgn and TRAF1 receptor-mediated signal pathway play a role in Mira/Pros telophase rescue? If it does, the coexpression of Egr, Wgn and TRAF1 might be expected to be seen in dividing NBs, along with the potential interaction between TRAF1 and the cytoplasmic domain of Wgn. Three observations argue against this hypothesis: (1) wgn is not expressed in embryonic NBs but in the mesoderm. (2) The domain analysis suggests that the Drosophila Wgn cytoplasmic domain is unique with no sequence homology to any mammalian TNFR family members and has neither a TRAF-binding domain nor a death domain, which is required for the interaction between TNFR and TRAF in mammals. (3) More informatively, Wgn knockdown by a UAS head-to-head inverted repeat construct of wgn (UAS-wgn-IR) driven by a strong maternal driver, mata-gal4 V32A, in WT embryos did not affect TRAF1 apical localization. These observations are consistent with the view that the receptor Wgn may not be involved in Mira/Pros telophase rescue or is redundant in this pathway. If this is the case, then how do TRAF1 and Egr function in Mira/Pros telophase rescue? It has been reported that TRAFs associate with numerous receptors other than the TNFR superfamily in mammals. It is speculated that Egr and TRAF1 may adopt an alternative receptor in NBs for Mira/Pros telophase rescue. However, until an anti-Wgn antibody and wgn mutant alleles are available, the possibility that Wgn is involved in Mira/Pros telophase rescue cannot be ruled out (Wang, 2006).

Hindsight modulates Delta expression during Drosophila cone cell induction: Hindsight regulates Delta to high enought levels to induce the cone-cell determinants Prospero and D-Pax2 in neighboring cells

The induction of cone cells in the Drosophila larval eye disc by the determined R1/R6 photoreceptor precursor cells requires integration of the Delta-Notch and EGF receptor signaling pathways with the activity of the Lozenge transcription factor. This study demonstrates that the zinc-finger transcription factor Hindsight (Hnt) is required for normal cone-cell induction. R-cells in which hindsight levels are knocked down using RNAi show normal subtype specification, but these cells have lower levels of the Notch ligand Delta. HNT functions in the determined R1/R6 precursor cells to allow Delta transcription to reach high enough levels at the right time to induce the cone-cell determinants Prospero and D-Pax2 in neighboring cells. The Delta signal emanating from the R1/R6 precursor cells is also required to specify the R7 precursor cell by repressing seven-up. As hindsight mutants have normal R7 cell-fate determination, it is inferred that there is a lower threshold of Delta required for R7 specification than for cone-cell induction (Pickup, 2009).

This study shows that Hnt function is necessary to elevate the Dl ligand in the R1/R6 precursor cells to a level high enough to achieve cone-cell induction. Notably, Hnt is not an on/off switch for Dl expression; rather it potentiates the level of Dl transcription in the R1/R6 precursor cells. The data suggest that this modulation is likely to be independent of Chn, which is itself a transcriptional repressor of Dl. Although this paper does not show that this Hnt effect is due to direct action, the exact sequence for two Hnt binding sites was found in the upstream and intronic sequences of the Delta transcription unit (Pickup, 2009).

Earlier reports describing Hnt function in the ovary show that Hnt expression is regulated by the Notch signaling pathway and controls follicle cell proliferation and differentiation. This paper reports that Hnt acts upstream of Notch activation by regulating Dl ligand expression levels. These two modes of regulation are not necessarily mutually exclusive, but it is not thought that Notch activates the hnt gene in the eye. (1) Hnt is expressed in all the R-cell precursors in the eye, whereas the Notch pathway is activated at high levels only in a subset of these precursors, as well as in the accessory cone and pigment cell precursors, where Hnt is not expressed at all. (2) When Notch activity is attenuated by using the Nts mutant, Hnt expression in the furrow expands to all cells that now acquire a neuronal fate. This result cannot be interpreted as a simple repression of Hnt expression by Notch activation in non-neuronal cells, as Hnt expression is not complementary to Notch activation in the eye disc. (3) Notch activation cannot be sufficient to induce Hnt expression in the eye disc, since no expansion of Hnt expression into adjacent, non-determined cells is seen when Dl is ectopically expressed early in the cone-cell precursors (with the lz-Gal4 driver). (4) It was shown that the expression of Dl in the R-cell precursors is partly dependent on Hnt function. Others have clearly demonstrated that this late Dl expression does not require Notch activity, since it is unaffected in a Nts1 mutant (Pickup, 2009).

The two-signal model of R7 fate hypothesizes that R7 determination requires a strong RTK signal (achieved by the additive effects of Sevenless and EGFR activation) together with Notch activation. These signals are necessary to activate pros and repress svp expression, respectively. Since the cone-cell precursor cells do not contact the determined R8 cell at the appropriate time, they will not 'see' the SEV ligand BOSS. Cone cell precursors, then, will not ordinarily activate their Sev receptors. In this model, different fates have been reinforced in the R7/cone equivalence group by adding a second, activating ligand for EGFR (Pickup, 2009).

This paper suggests a further level of complexity. It was shown, by manipulating the level of Dl in the R1/R6 signaling cells, that activation of the key players in cone-cell determination requires high levels of the Notch activation in the cone-cell precursor cell. Several lines of evidence support the idea that the level of the Dl ligand is translated into cell-fate differences in a responding R precursor cell. Since there is low Dl expression in the R7 precursor cell and only late expression of Dl in the cone-cell precursor cell, the adjacent R1/R6 precursor cells never activate their Notch receptors. Both the R7 precursor and the cone-cell precursor cells receive their ligand signal from the R1/R6 precursor cells. In this hypothesis, the R7 precursor cell requires only a low level of ligand signal to activate the R7-like program: turning on pros and off svp (Pickup, 2009).

It is suggested that the cone-cell precursor requires a high level of ligand signal to activate the cone-cell program. Expressing a dominant-negative form of Dl in the R1/R6 signaling cells prevents cone-cell, but not R7-cell, determination. Since both the cone and R7 precursor cells receive their Dl input from the same R1/R6 cells, it is possible that an intrinsic feature of the R7 precursor cell - possibly the high RTK activation - antagonizes N signaling, so that D-Pax2 transcription does not occur in that cell. The transcriptional repressor, Lola, may also be involved in this distinction, since it is known to bias precursor cells towards R7-over cone-cell fate (Pickup, 2009).

Although a role for Notch signaling in cone-cell induction has been shown to be necessary for D-Pax2 expression, it has not been directly demonstrated as necessary for pros regulation in cone cells. The experiments presented in this study suggest that high levels of Notch signaling may indirectly or directly be required for Pros expression in the cone-precursor cells. This requirement is independent of the role of SU(H) in inducing D-Pax2, since there are normal levels of Pros in the cone-cell precursors of a D-Pax2 null mutant. Ectopically activating the Notch pathway in the R1/R6 precursor cells occasionally induces ectopic Pros (but eliminates ELAV) in these cells. Although this effect on Pros expression may be a secondary result of a cell-fate transformation, it could also be interpreted as a more direct effect of Notch signaling on pros transcription. In a different context, Pros expression has been shown to be affected by Dl-activated Notch signaling in a subset of glial cells in the embryonic CNS (Pickup, 2009).

Why would there be two Dl thresholds for different cell fates? There is some preliminary work that suggests different mechanisms for Notch-activated transcriptional readout in the responding cell, depending on the level of signal received. In the cone-cell equivalence group, the cone-cell determination pathway requires that D-PAX2 and Pros be expressed. It is hypothesized that D-Pax2 may require a higher level of Notch activation than Pros, which is also required for R7 determination. These experiments indicate that there may be coordinated regulation of both D-Pax2 and Pros expression in the cone cells. It is postulated that the mechanism of Pros-gene induction in the cone cells is different from pros regulation in R7. By potentiating the level of Dl gene expression in the R1/R6 signaling cells, it is possible to overlay the cone-cell fate over the transcriptional module necessary for R7-cell fate. This simple change has, thus, allowed for the elaboration of very different cell fates from the same equivalence group (Pickup, 2009).

Expression profiling of prospero in the Drosophila larval chemosensory organ: Between growth and outgrowth

The antenno-maxilary complex (AMC) forms the chemosensory system of the Drosophila larva and is involved in gustatory and olfactory perception. It has been shown that a mutant allele of the homeodomain transcription factor Prospero (prosVoila1, V1), presents several developmental defects including abnormal growth and altered taste responses. In addition, many neural tracts connecting the AMC to the central nervous system (CNS) were affected. These earlier reports on larval AMC did not argue in favour of a role of pros in cell fate decision, but strongly suggested that pros could be involved in the control of other aspect of neuronal development. In order to identify these functions, microarray analysis was performed of larval AMC and CNS tissue isolated from the wild type, and three other previously characterised prospero alleles, including the V1 mutant, considered as a null allele for the AMC. A total of 17 samples were first analysed with hierarchical clustering. To determine those genes affected by loss of pros function, a discriminating score was calculated reflecting the differential expression between V1 mutant and other pros alleles. A total of 64 genes were identified in the AMC. Additional manual annotation using all the computed information on the attributed role of these genes in the Drosophila larvae nervous system, enabled identification of one functional category of potential Prospero target genes known to be involved in neurite outgrowth, synaptic transmission and more specifically in neuronal connectivity remodelling. The second category of genes found to be differentially expressed between the null mutant AMC and the other alleles concerned the development of the sensory organs and more particularly the larval olfactory system. Surprisingly, a third category emerged from this analyses and suggests an association of pros with the genes that regulate autophagy, growth and insulin pathways. Interestingly, EGFR and Notch pathways were represented in all of these three functional categories. It is propose that Pros could perform all of these different functions through the modulation of these two antagonistic and synergic pathways. The current data contribute to the clarification of the prospero function in the larval AMC and show that pros regulates different function in larvae as compared to those controlled by this gene in embryos. In the future, the possible mechanism by which Pros could achieve its function in the AMC will be explored in detail (Guenin, 2010).

In the AMC, Prospero is expressed in a cluster of cells (composed of neuronal and support cells, but not glial cells) that emerge during embryonic life and are maintained until the end of the larval stages. In embryos, Pros was reported to be involved in cell fate decision and in cell-cycle control. By contrast, earlier data from the larval AMC rather suggested that pros could assume more restricted functions, such as the control of neuron-specific functions. The present study confirms this hypothesis and shows that in the chemosensory organs dedicated to larval olfactory and gustatory sensing, prospero could regulate genes involved in neurite outgrowth and synaptic transmissionz (Guenin, 2010).

Since pros was clearly shown to control axonal and dendritic outgrowth, the possibility cannot be excluded that the connection of pros with several genes that drive synaptic activity could be the indirect consequence of its involvement in neurite outgrowth control. In this respect, it is interesting to mention that a recent study showed that axon targeting of the R7 Drosophila photoreceptor cells to their synaptic partner requires R7-specific transcription factor Prospero. That study proposed that Pros could promote cell-type-specific expression of sensory receptors and cell-surface proteins regulating synaptic target specificity (Guenin, 2010).

As previously mentioned, some of the genes identified in this functional class are also involved in neural connectivity remodelling. How can this be achieved if the AMC is completely histolysed? In fact, in Drosophila, not all sensory neurons degenerate; Some larval neurons persist and remodel to take on a new role in the adult system. During the metamorphosis larval arbors of these neurons are pruned back and new adult-specific arbors are generated through a subsequent period of outgrowth. It seems that the neurites of these persistent larval neurons are used to partly guide axons of adult sensory neurons towards and within the CNS. Therefore, histolysis and remodelling are two processes that are achieved during metamorphosis and could concern distinct neurons (Guenin, 2010).

Does Pros play any role in AMC neuronal remodelling? The question cannot actually be answered. However, it has been previously reported that the insulin and epidermal growth factor signalling pathways, as well as ubiquitin-specific proteases are all required for the regulation of Drosophila neuronal remodelling. Interestingly, all of these components emerge clearly from the current analysis (Guenin, 2010).

Actually, no work was done on the Drosophila larvae anterior sense organ in order to check whether some of the sensory neurons (which have also an embryonic origin) persist and remodel to take place in the adult olfactory or gustatory system. Therefore, the question is left open. At least the answers will provide important insights into the mechanisms that govern developmental plasticity in insect nervous systems (Guenin, 2010).

In summary, these data collected from larval AMC and the previous genome wide expression profiling done on embryos confirms that pros is associated with the regulation of neuronal specific genes. In this respect, it is essential to note that except for a few genes (126), most of the Pros target genes identified (~1000) were not represented the current microarrays. For this reason, and because the current experiments were performed on isolated individual larval tissues, it is not possible to determine whether the genes identified by these authors are specifically expressed in embryos and/or in tissues other than AMC (Guenin, 2010).

In Drosophila, the insulin/TOR signalling pathway is divided into two branches. The insulin and its downstream effectors P13 and FOXO (forkhead box) represent one branch of this pathway, while the other branch acts through the TOR family of Serine-Threonin kinases. It has been shown that the insulin/TOR signalling pathway inhibits autophagy and controls growth by regulating ribosome biogenesis and protein biosynthetic capacity. It has been demonstrated that the TOR pathway is a nutritional checkpoint that participates in the systemic control of larval growth emanating from the Fat body (Guenin, 2010).

Microarray analysis has revealed a group of highly correlated pros candidate genes (correlation index: 0.9) that are either controlled by the insulin/TOR signalling pathway or are directly involved in the signalling cascade. This is the case for Ash2 which was found to be regulated by TOR signalling. Similarly, FK506-bp1 affects autophagy through the modulation of FOXO and Lk6 was reported to be a direct FOXO Target. Therefore it seems that in the larval AMC, Pros could be associated with growth, autophagy and nutrient sensing through the regulation of genes that are directly or indirectly linked to the insulin/TOR pathway. Interestingly, TOR was found to be differentially expressed in the V1 pros mutant in the CNS (Guenin, 2010).

As described previously, loss of pros function in the AMC induced several alterations including axon pathfinding defects and abnormal growth and taste responses. This is consistent with microarray results showing that in the larval AMC, Pros expression is associated with the regulation of genes involved in the control of neurite outgrowth, mediation of growth and autophagy and in the organisation and function of the olfactory system. The mechanism by which all of these functions are achieved by pros in the AMC is presently not known but EGFR and/or Notch pathways could play a central role. Several lines of evidence are in favour of this hypothesis (Guenin, 2010).

Four ligands are known to bind EGFR receptor: Keren, Gurken, Spitz, and Vein. Two of these were identified as potential targets of Prospero: Keren in both larval AMC and CNS and Gurken (Grk) in the larval CNS only. Moreover, Notch and EGFR were identified as putative Pros target genes in both embryos and larval AMC, indicating that they could play a central role (Guenin, 2010).

It has been reported that EGFR signalling is required for the development of some of the neurons and cuticular structures present in the AMC. In this respect, it is interesting to point out that EGFR involvement has been reported during the development of mouse gustatory epithelia in the palate and tongue (Guenin, 2010).

The expression of Notch, EGFR and Pros have been shown to be tightly linked. It has been demonstrated that normal levels of Pros expression in photoreceptor R7 cells in the Drosophila eye require EGFR signalling as well as Notch activation. In addition, a recent analysis has shown that in R7 cells, Notch and EGFR cooperate in a complex way to promote pros transcription (Guenin, 2010).

Although these data suggest that Notch and EGFR could play a central role in the mechanism by which Prospero carries out its function in the larval AMC, this hypothesis has still to be validated. In the future, it will be of great interest to explore in detail the mechanism by which all of these functions are accomplished by the homeodomain transcription factor Prospero (Guenin, 2010).

The bHLH repressor Deadpan regulates the self-renewal and specification of Drosophila larval neural stem cells independently of Notch: Dpn is a potential repressor of Pros

Neural stem cells (NSCs) are able to self-renew while giving rise to neurons and glia that comprise a functional nervous system. However, how NSC self-renewal is maintained is not well understood. Using Drosophila larval neuroblasts as a model, this study demonstrates that the Hairy and Enhancer-of-Split (Hes) family protein Deadpan (Dpn) plays important roles in NB self-renewal and specification. The loss of Dpn leads to the premature loss of NBs and truncated NB lineages, a process likely mediated by the homeobox protein Prospero (Pros). Conversely, ectopic/over-expression of Dpn promotes ectopic self-renewing divisions and maintains NB self-renewal into adulthood. In type II NBs, which generate transit amplifying intermediate neural progenitors (INPs) like mammalian NSCs, the loss of Dpn results in ectopic expression of type I NB markers Asense (Ase) and Pros before these type II NBs are lost at early larval stages. These results also show that knockdown of Notch leads to ectopic Ase expression in type II NBs and the premature loss of type II NBs. Significantly, dpn expression is unchanged in these transformed NBs. Furthermore, the loss of Dpn does not inhibit the over-proliferation of type II NBs and immature INPs caused by over-expression of activated Notch. These data suggest that Dpn plays important roles in maintaining NB self-renewal and specification of type II NBs in larval brains and that Dpn and Notch function independently in regulating type II NB proliferation and specification (Zhu, 2012).

Dpn was initially identified as a pan-neural protein and has been widely used as a NB marker. However, the function of Dpn in NBs has been elusive. This study provides evidence that Dpn plays an important role in maintaining NB self-renewal. In type II NBs, in addition to maintaining the self-renewal, Dpn is also required to suppress Ase expression when these NB exit quiescence. Furthermore, Notch and Dpn may function independently in larval NBs. While both Dpn and Notch are required for maintaining the identity and self-renewal of type II NBs, knockdown of Notch does not affect the expression of Dpn in type II NBs (Zhu, 2012).

In a developing nervous system, NSCs must be maintained when they divide in order to generate the complete array of neurons and glia that form a functional neuronal circuit. Current studies are focused on determining how NSC self-renewal is maintained, as well as mechanisms governing NSC terminal differentiation. The findings that dpn mutant MB NBs as well as other type I NBs are prematurely, progressively, lost demonstrate that Dpn functions cell-autonomously to maintain the self-renewal of larval NBs. Interestingly, in dpn mutant larvae, the premature loss of type I NBs mainly occurred within 48 hours after larval hatching (with the exception of the MB NB). Zacharioudaki (2012) has reported recently that Dpn and E(spl) proteins function redundantly to maintain NB self-renewal but have different temporal expression patterns. Dpn expression in NBs is activated at the newly hatched larval stage, whereas E(spl)mγ expression becomes obvious only when NBs start to divide at the 2nd instar larval stage. The difference in temporal expression patterns between Dpn and E(spl) proteins probably explains why loss of type I NBs occurred mainly within 48 hours after larval hatching in dpn mutants. Interestingly, despite the redundant function of Dpn and E(spl) proteins in maintaining NB self-renewal, loss of Dpn alone resulted in the premature loss of MB NBs at late larval/early pupal stages, indicating that E(spl) proteins may not be involved in maintaining MB NBs at late larval/early pupal stages. The current findings as well as those of Zachariousdaki suggest that Dpn is required for maintaining NB self-renewal rather than NB formation or specification as was proposed by San-Juan and Baonza (2011). It is likely that differences in identifying and quantifying NBs at different developmental stages accounts for this discrepancy (Zhu, 2012).

The role of Dpn in NB self-renewal is also supported by the observation that ectopic/over-expression of Dpn promoted non-dividing immature intermediate INPs and terminally dividing GMCs to enter self-renewing divisions, and prolonged the self-renewal of both types of NBs. These ectopic self-renewing GMCs and immature INPs, which normally do not express Dpn, may de-differentiate to acquire a NB-like fate and contribute to the increased number of NBs, similar to what has been observed in brat, numband klumpfuss mutant type II NB lineages. However, type II NB lineages show more severe over-proliferation phenotypes than type I NB lineages in response to ectopic/over-expression of Dpn. The difference in degree of over-proliferation between the type I and type II NB lineages is likely related to intrinsic differences between the type I and type II NB daughters, rather than a difference in how Dpn itself is acting. Type I NBs produce GMCs that express genes such as pros and ase that limit proliferation, counteracting the pro-self-renewal function of Dpn. In contrast, type II NBs and immature INPs express the ETS family protein Pointed but do not express Ase or Pros, making them particularly susceptible to the ectopic expression of genes, such as dpn, that promote self-renewal. It is proposed that the significantly enhanced proliferation of type II NB progeny in response to ectopic/increased Dpn expression is most likely due to a disparity in the inherent self-renewal potential of the type I and type II NB daughters (Zhu, 2012).

The function of Dpn in maintaining NB self-renewal is consistent with mammalian Hes family proteins' function in maintaining NSCs. In the developing mammalian nervous system, the loss of Hes1, Hes3, and Hes5 leads to accelerated neurogenesis and premature depletion of neuroepithelial cells and radial glial cells, whereas forced expression of Hes proteins maintains NSCs (Zhu, 2012).

While Dpn is expressed in both type I and type II NBs, the data showed that the loss of Dpn not only resulted in the premature loss of type II NBs at early larval stages, but also led to the ectopic expression of type I NB markers Ase and Pros in type II NBs when they exited quiescence, making type II NBs appear as type I-like NBs. This indicates Dpn has two roles in type II NBs: Dpn maintains NB self-renewal just as it does in type I NBs, and Dpn is also required to maintain type II NB identity. Moreover, it appears that Dpn's role in maintaining type II NB identity is temporally restricted. Results from this study as well as others showed that dpn mutant embryonic brains contained a comparable number of Dpn+ Ase- NBs as wild type embryonic brains. In dpn mutant clones, our results showed that type II NBs did not ectopically express Ase even 4 days after clone induction when Dpn is no longer detectable. Therefore, it seems that Dpn's function to suppress Ase expression is limited to a narrow temporal window during the reactivation of type II NBs at the 1st instar larval stage. How might Dpn act to maintain type II NB identity? In mammals, Hes family proteins are well known for their roles in antagonizing the expression and/or activity of proneural genes (Ase is a member of the achaete-scute family of proneural genes). Negative interactions between dpn and the achaete-scute complex (AS-C) genes occur during Drosophila sex determination as well as neurogenesis. One potential model could be that a proneural protein(s) might be expressed in quiescent type II NBs and that Dpn is required to antagonize its expression and/or activity in order to promote type II NB fate when NBs exit quiescence. Since Dpn is expressed in both type I and type II NBs, it is postulated that its role in maintaining type II NB fate is associated with the differential expression and/or activity of another, currently unidentified gene (Zhu, 2012).

This work suggests that the premature loss of dpn mutant type I NBs could be mediated by Pros. This is supported by the findings that nuclear Pros precociously accumulates in dpn mutant type I NBs and that dpn mutant type I NBs are maintained even in adult brains in the absence of Pros. It has been shown that over-expressing Pros in embryonic and larval NBs is sufficient to induce ectopic nuclear Pros localization and terminal division. Therefore, one possibility is that Dpn negatively regulates pros expression. In the absence of Dpn, Pros expression increases, leading to the nuclear accumulation of Pros and thus premature terminal division. In type I NBs, dynamic cortical and cytoplasmic localization of Pros makes it difficult to compare the levels of Pros in wild type and dpn mutant type I NBs by immunostaining. However, ectopic Pros expression in dpn mutant type II NBs, which normally do not have Pros, provide evidence that Dpn negatively regulates Pros expression, either directly or indirectly. The existence of putative Dpn binding sites in the pros promoter suggests that Dpn could directly regulate pros expression. Alternatively, Dpn could indirectly regulate pros by inhibiting the expression and/or activity of proteins, such as Ase, that promote pros expression. In support of this notion, it has been shown that mammalian Hes proteins can inhibit the expression of proneural proteins such as Mash1 in the developing cortex, whereas forced expression of the proneural protein Mash1 in neuroepithelial cells is sufficient to promote the expression of Prox1, the mammalian homolog of Pros that plays an anti-proliferative and pro-differentiation role in the developing mammalian hippocampus and retina (Zhu, 2012).

Unlike the majority of mammalian Hes proteins or other members of the fly Hes family, which typically act downstream of Notch, results from this study as well as Zacharioudaki (2012) do not support a model in which Dpn functions as a direct target of Notch signaling in larval NBs as was proposed by San Juan and Baonza (2011). First, although these studies, as well as the work from other investigators, showed that the knockdown of Notch or disruption of Notch signaling led to premature loss of type II NBs and ectopic expression of Ase in type II NBs as was observed in dpn mutant larval brains, knockdown of Notch did not affect the expression of Dpn in type II NBs, which is consistent with previous findings. Second, the data and those of Zacharioudaki showed that removing Dpn did not abolish the over-proliferation of type II or type I NBs caused by over-expression of activated Notch. Nor did reducing Notch expression exacerbate the loss of type II NBs in dpn7 heterozygous animals. These genetic interaction data suggest that Dpn does not function downstream of Notch signaling. Thus, Dpn may be similar to the mammalian Hes2 and Hes3, which are not transcriptionally regulated by Notch.Notch and Dpn likely employ distinct mechanisms to maintain the self-renewal and suppress Ase expression in type II NBs. Zacharioudaki showed that some E(Spl) proteins, particularly E(spl)mγ and m8, depend on Notch signaling for their expression in larval NBs. However, loss of E(Spl) proteins does not result in ectopic expression of Ase in type II NBs. Therefore, Notch must function through molecules, which are yet to be identified, to regulate Ase expression in type II NBs (Zhu, 2012).

Splicing of prospero

An intronic enhancer regulates splicing of the twintron of Drosophila melanogaster prospero pre-mRNA by two different spliceosomes

The second intron of pros pre-mRNA contains two complete sets of splice sites, an arrangement referred to as twintron. Examined here was the alternative splicing of the Drosophila melanogaster prospero twintron, which contains splice sites for both the U2- and U12-type spliceosome and generates two forms of mRNA, pros-L (U2-type product) and pros-S (U12-type product). Twintron splicing is developmentally regulated: pros-L is abundant in early embryogenesis while pros-S displays the opposite pattern. A Kc cell in vitro splicing system has been established that accurately splices a minimal pros substrate containing the twintron, and the sequence requirements for pros twintron splicing were examined. Systematic deletion and mutation analysis of intron sequences establishes that twintron splicing requires a 46-nucleotide purine-rich element located 32 nucleotides downstream of the U2-type 5' splice site. While this element regulates both splicing pathways, its alteration showed the severest effects on the U2-type splicing pathway. Addition of an RNA competitor containing the wild-type purine-rich element to the Kc extract abolishes U2-type splicing and slightly represses U12-type splicing, suggesting that a trans-acting factor(s) binds the enhancer element to stimulate twintron splicing. Thus, an intron region critical for prospero twintron splicing has been established as a first step towards elucidating the molecular mechanism of splicing regulation involving competition between the two kinds of spliceosomes (Scamborova, 2004).

Introns are removed from pre-mRNA by two transesterification steps catalyzed by a large multicomponent complex known as the spliceosome. Five small nuclear RNAs, U1, U2, U4/U6, and U5, and more than 60 polypeptides form the active U2-type spliceosome. Spliceosome assembly proceeds in an ordered fashion that is directed by the recognition of conserved sequence motifs within the pre-mRNA by small nuclear RNAs and protein factors. These sequences allow efficient and accurate removal of introns and are located at the 5' and 3' splice sites as well as the branch point (Scamborova, 2004).

A small subset of introns is removed via a unique and divergent spliceosome (U12-type), whose composition and splice site recognition signals differ. U11, U12, and U4atac/U6atac are the functional analogues of U1, U2, and U4/U6 small nuclear RNPs in the U12-type spliceosome; U5 is the only small nuclear RNP shared by both types of spliceosomes. As in its U2-type counterpart, interactions of conserved sequence elements at the 5' and 3' splice sites and branch point with the components of the U12-type spliceosome direct the correct recognition and removal of introns from the pre-mRNA (Scamborova, 2004).

In addition to the intrinsic quality of the splice sites themselves, splice site selection can depend on other properties of the pre-mRNA, such as exon sequences and relative splice site proximity, RNA secondary structure, exon size, and intronic sequences. For many pre-mRNAs, the splicing reaction produces only a single product from the pre-mRNA transcript. However, other genes undergo alternative splicing, a process in which splice sites in a single primary transcript are differently paired to generate two or more mRNAs encoding multiple protein isoforms with slightly altered or opposing functional properties. It has been estimated that nearly 60% of all human genes undergo at least one alternative splicing event (Scamborova, 2004).

A remarkable variety of alternative splicing patterns has so far been observed, involving differential 5' or 3' splice site selection, alternative exon selection, and intron retention. In many instances, alternative splicing is regulated in a developmental or tissue-specific manner. Such complex patterns have been suggested to derive from numerous distinct mechanisms. For many examples of alternatively spliced genes, exonic or intronic cis-acting RNA sequences that positively or negatively regulate splice site choice have been identified. Binding of specific proteins to these regulatory sequences, in turn, dictates the specificity and efficiency of splicing resulting in promotion or repression of each splicing event (Scamborova, 2004).

The Drosophila prospero pre-mRNA provides a rare and unusual example of alternative splicing. The prospero gene locus encodes a homeodomain-containing neuronal transcription factor, Prospero, which is involved in control of axon outgrowth and in cell fate specification in the developing central nervous system. The second intron of pros pre-mRNA contains two complete sets of splice sites, an arrangement referred to as twintron. One set of splice sites is of the U2-type (GT-AG termini), whose usage leads to a production of the pros-L mRNA isoform. The U2-type splice sites are nested inside an intron defined by a second set of splice sites of the U12 type (with AT-AC termini), whose usage leads to the production of the pros-S mRNA isoform, 87 nucleotides shorter than pros-L. Excision of the larger flanking U12-type intron thus removes 29 amino acids with 24 amino acids upstream and 5 amino acids within the N terminus of the homeodomain, a region demonstrated to influence the interaction of the Prospero protein with Drosophila Deformed and mouse Hoxa-5 proteins (Scamborova, 2004 and references therein).

Fundamental to understanding the alternative splicing of the prospero twintron is an elucidation of the mechanism(s) involved in splice site selection. The existence of two competing splicing pathways within one intron raises the question of whether the two splicing pathways are coregulated or separately controlled through different mechanisms. The temporal splicing profile of the two alternatively spliced pros mRNA isoforms was examined and it was found that pros-L predominates during the first half of Drosophila embryogenesis while pros-S is more abundant at later stages. Possible regulatory elements within the twintron were sought. The splicing of pros minigene constructs containing both sets of splice sites was analyzed both in vitro and in vivo. The results show that twintron splicing depends on a 46-nucleotide intronic sequence, called the purine-rich element, located 32 nucleotides downstream of the U2-type 5' splice site. Its deletion or modification decreases or completely abolishes U2-type splicing. Lastly, it was found that the same element is involved in the control of U12-type splicing, since mutation of the element induces a marked decrease in the splicing efficiency of that pathway (Scamborova, 2004).

The stage-specific profile of prospero pre-mRNA splicing in the Drosophila embryo and early larva was investigated and it was found that pros-L predominates in early embryogenesis while the pros-S predominates in middle to late embryogenesis and early larval development. Similar stage-specific splicing has been reported for another alternatively spliced Drosophila homeotic gene, ultrabithorax (ubx), where the RNA levels have been shown to fluctuate during Drosophila embryonic development with the long ubx isoforms (Ia and Ib) predominating during the first 7.5 h and a short isoform (IV) predominating from 18 to 22.5 h of Drosophila embryogenesis (Scamborova, 2004).

What functional differences might derive from the structural differences between the proteins encoded by the two pros mRNAs? Pros protein has been reported to regulate multiple target genes in different lineages and different stages of neuronal development by repressing or activating their transcription. It is therefore possible that the two pros isoforms interact with different coactivators in different tissues, and at different developmental stages. Recent structural studies on the Pros-L C-terminal domain (encompassing the homeodomain and Prospero domain) suggested three regions of potential Prospero-DNA contacts, among them, the N-terminal arm of the homeodomain appears to contact the minor groove of the DNA. The Pros homeodomain is essential for the sequence-specific DNA binding function of Prospero and its N terminus has been shown to associate with two homeobox proteins, Engrailed and Deformed. Because the Pros-S homeodomain contains a different N terminus, it is tempting to speculate that the two Pros proteins exhibit different DNA-binding capacities that lead to target gene selectivity during neurogenesis in a tissue or stage-specific manner. As a result, production of various amounts of the two protein isoforms would be expected at different stages of development or in different tissues (Scamborova, 2004).

Two additional examples of twintrons in the second intron of the prospero gene are found in D. pseudoobscura and D. virilis. Sequence alignment further reveals a homologous purine-rich element in a similar location in D. pseudoobscura; it is located 40 nucleotides downstream of the 5' U2-type splice site and contains the same number of GA repeats flanked on both sides by purines. The 5' half of the purine-rich region is 100% conserved between the two species (apart from three missing residues, which could be due to sequencing error) while the 3' end is less conserved. D. virilis (which is more diverged from D. melanogaster than D. pseudoobscura also contains a purine-rich region located 28 nucleotides downstream of its U2-type 5' splice site, but this region contains only five GA repeats. These observations support the functional importance of the purine-rich element as a regulatory cis-element in twintron splicing (Scamborova, 2004).

Additional Prospero orthologs, called Prox1, have been identified in Caenorhabditis elegans, chicken, mouse, and human. Their protein expression patterns indicate that they are critical for the development and function of the developing central nervous system, eye, and midgut. While Prox1 is very well conserved in vertebrates, including 102 amino acids at the 3' terminus of the Prox1 homeodomain that are 62% identical to corresponding residues of Drosophila Prospero, the first seven amino acids of the Prox 1 homeodomain do not share homology with Drosophila Prospero. Strikingly, all of the vertebrate prox 1 genes contain a single U12-type intron located near the N terminus of the homeobox in exactly the same position as the U12-type intron in Drosophila prospero. However, no alternatively spliced variants of Prox 1 have been identified, and no hints of U2-type splicing signals have been found in the second intron of mouse or human Prox 1. Furthermore, sequence analysis of human prospero has not revealed any pronounced intronic purine-rich sequences. This suggests that the twintron and its intronic purine-rich element are unique to Drosophila species, where they may play a special role in development (Scamborova, 2004).

To date prospero has provided the only example of a twintron whose splicing can proceed via either the U12- or U2-type pathway. Documentation of temporal specificity in embryos lends further support to the idea that regulatory signals within the pros pre-mRNA act as sensors of developmental and/or tissue-specific cues. A minimal splicing substrate was generated and an in vitro splicing system was established in Kc nuclear extract as a means to map and characterize cis-regulatory elements in a systematic way. Deletion and replacement mutant analysis of the intron led to identification of a 46-nucleotide purine-rich regulatory element, denoted the purine-rich element, that is located 32 nucleotides downstream of the U2-type 5' splice site and is required for twintron splicing both in vitro and in vivo. Complete replacement of the purine-rich element impairs U2-type splicing both in vitro and in vivo. The presence of multiple GA repeats is consistent with a partite, redundant element structure, a common feature of many intronic regulatory sequences. Indeed, in vitro studies indicate that only the first 20 nucleotides of the purine-rich element are required for pros twintron splicing. Furthermore, replacements of the purine-rich element by purine-rich SR protein binding sites did not completely restore splicing, while the deletion mutants in which only the first 8 or 10 nucleotides of the purine-rich element were kept did restore splicing (Scamborova, 2004).

Although in vitro splicing in Kc cell extract yielded high levels of pros-S and low levels of pros-L products, the same transcripts spliced in vivo in the Schneider (S2) cell line showed the opposite effect. The linearity of the transfection data, obtained in S2 cells under the same conditions, excludes the possibility that the observed differences result from artificial saturation of the U12-type splicing machinery by high levels of pre-mRNA expressed from the transfected constructs. Although the basis of reversed levels of pros-S and pros-L in vivo compared to the in vitro system is unknown, reduction in pros-S levels and increase in pros-L levels similar to those in S2 cells are also observed when the pros substrate is spliced in vitro in HeLa nuclear extracts (Scamborova, 2004).

A likely explanation for this discrepancy stems from the origin of the two cell lines. Although both were derived from late-stage Drosophila embryos, Kc cells are of hematopoietic origin, whereas the Schneider cell line is of macrophage origin. Antagonistic splicing factors have been shown to influence alternative splicing in vivo in a concentration-dependent manner. For instance, in vivo variations in hnRNP A1 protein levels have been shown to influence 5' splice site choice. Factors can also vary between cell types, such as levels of ASF/SF2 differing nearly 20-fold between heart and testis and levels of X16/SRp20 protein being high in pre-B cells and thymus but not abundant in other cells. Therefore, the enhancement of pros-L splicing observed in S2 cells could be due to various levels of hnRNP, SR proteins, or other splicing factors (Scamborova, 2004).

The location of the purine-rich element and its enhancer function resemble the situation with several previously characterized intronic enhancer elements. Intronic enhancer sequences are often purine-rich, but are diverse in sequence and distinct from exonic enhancers. For instance, purine-rich regions act as important regulatory signals for neural splicing of mammalian c-src, calcitonin/CGRP, and agrin. In the c-src gene, the intronic enhancer located just downstream of the N1 exon is required for the N1 exon to be positively selected; it assembles a complex of proteins, including hnRNP F, hnRNP H, and KSRP (Scamborova, 2004).

While intronic enhancers generally have a complex architecture, the prospero purine-rich element sequence appears distinct in that it is composed almost exclusively of purines. Furthermore, its deletion or modification had two effects: the almost complete abolition of major-class splicing and the down regulation of minor-class splicing. Specifically, the mutant in which the entire 46-nucleotide of the purine-rich element was replaced with a non-purine-rich sequence yielded no observable major class product, while deletions within purine-rich element decreased major class splicing to a lesser degree. The purine-rich region immediately downstream of the purine-rich element was able to partially replace portions of the purine-rich element in the deletion mutants (perhaps by being brought closer to the 5' splice sites) but not in the replacement mutants. Thus, activating sequences in addition to the purine-rich element may exist in the vicinity of the pros twintron (Scamborova, 2004).

There are at least two possible mechanisms by which the prospero purine-rich element may exert its stimulatory effect: one is that it participates in formation of a secondary structure that improves the accessibility of splicing factors to the splice sites, and another is that the element serves as a target of a trans-acting factor(s). Competition experiments show that the stimulatory effect of the purine-rich element can be titrated by competitor purine-rich element RNA, strongly supporting the latter mechanism. Thus it is proposed that the purine-rich element serves as a target sequence for the formation of a protein enhancer complex. The data further suggest that the purine-rich element functions as a bipartite element in which nucleotides 32 to 37 and 77 to 105 influence the overall splicing efficiency of both splicing pathways, while nucleotides 37 to 77 primarily influence the ratio of pros-S to pros-L. Future research will focus on the factors that bind the purine-rich element and flanking sequences, their binding locations and how they function to influence spliceosome assembly and promote twintron splicing in general (Scamborova, 2004).

midlife crisis encodes a conserved zinc-finger protein required to maintain neuronal differentiation in Drosophila

This study reports the identification of the presumptive splice factor midlife crisis (mdlc; CG4973) as a gene required for the maintenance of neuronal differentiation and for neuroblast proliferation in Drosophila. mdlc encodes a ubiquitously expressed zinc-finger-containing protein with conserved orthologs from yeast to humans that are reported to have a role in RNA splicing. Using clonal analysis, it was demonstrated that mdlc mutant neurons initiate but fail to complete differentiation, as judged by the loss of the pro-differentiation transcription factor Prospero, followed by derepression of the neuroblast factors Deadpan, Asense and Cyclin E. Loss of Mdlc decreases pros transcript levels and results in aberrant pros splicing. Importantly, misexpression of the full-length human ortholog, RNF113A, completely rescues all CNS defects in mdlc mutants. It is concluded that Mdlc plays an essential role in maintaining neuronal differentiation, raising the possibility that RNF113A regulates neuronal differentiation in the human CNS (Carney, 2013).

This study has identified Mdlc as a ubiquitous nuclear protein that is required to maintain neuronal differentiation. Mdlc maintains the expression of Pros in larval postmitotic neurons and inhibits the expression of neuroblast genes, thus maintaining neuronal differentiation. Mdlc is not required for Pros expression in the oldest neurons located most distal from the neuroblast in mdlc mutant clones; there might be enough Mdlc protein or RNA present at the time of mutant clone induction to allow the first neurons born after clone induction to successfully pass through middle-age without dedifferentiation. This suggests that after a certain age, neurons do not require Mdlc to maintain neuronal differentiation. Alternatively, neurons born at early larval stages might not have the same requirement for Mdlc as neurons born at later larval stages (Carney, 2013).

Interestingly, mdlc mutant clones never show EdU incorporation (used to directly measure de novo DNA synthesis) in ectopic Dpn+ cells, raising the question of whether they are true neuroblasts or have a mixed neuroblast/neuronal fate. It was note that loss of Mdlc results in cell cycle delay in parental neuroblasts, so it is perhaps unsurprising that the ectopic Dpn+ cells do not proliferate. Nevertheless, the data show that Mdlc is not required to suppress cell cycle entry in postmitotic neurons. Similarly, the data show that Pros is not required to suppress cell cycle entry in postmitotic neurons, as pros RNAi specifically within postmitotic neurons removes all detectable Pros protein but does not trigger entry into the cell cycle. This is comparable to the situation in wild-type embryonic neurons, which rapidly lose Pros but never re-enter the cell cycle, and contrasts with the role of Pros in GMCs, where it is required to repress neuroblast genes and promote cell cycle exit. The maturation step that is taken by neurons to make them incapable of re-entering the cell cycle in the absence of Pros is not well understood (Carney, 2013).

Pros is known to bind the dpn, ase and CycE loci, and is known to keep the expression of these genes low in embryos. Does Pros directly or indirectly maintain repression of the dpn, ase and CycE neuroblast genes in larval neurons? To attempt to address this question, RNAi was used to ablate either Pros or Mdlc in postmitotic neurons, assaying for derepression of the neuroblast factor Dpn. Driving UAS-pros RNAi with either atonal-Gal4 or acj6-Gal4 specifically eliminated Pros protein in multiple clusters of postmitotic neurons, yet did not lead to the derepression of Dpn. However, RNAi knockdown of mdlc using the same Gal4 lines also did not cause Dpn derepression. Several factors may account for this unexpected result. First, the lineages expressing atonal-Gal4 and acj6-Gal4 might not require Mdlc, consistent with the finding that some mdlcc04701 mutant clones had no Pros loss, Dpn/Ase derepression or EdU incorporation phenotypes. Alternatively, or in addition, the postmitotic Gal4 lines might have eliminated Mdlc and Pros in neurons after they passed through the susceptible middle-aged stage. However, it is noted that acj6-Gal4 appears to drive expression in one lineage in all ages of neurons (only excluding the neuroblast and GMCs). This indicates either that this lineage does not require Pros or Mdlc to maintain Dpn repression or that Mdlc is required at the GMC level in this lineage. Appropriate drivers are lacking to distinguish between these possibilities. Given these caveats, it was not possible to determine whether neuronal loss of Pros alone is sufficient for derepression of neuroblast genes. It is thought likely that Pros does have some role in the neuronal repression of neuroblast genes, however, because derepression of Dpn/Ase is nearly always observed in neurons that have already lost Pros (Carney, 2013).

The CCCH zinc-finger domain, which is implicated in RNA-binding in other proteins, is essential for Mdlc function in the nervous system and for organismal viability. The S. cerevisiae and human orthologs have roles in splicing. In yeast, Cwc24p is reported to be a splicing efficiency factor primarily affecting primary transcripts with atypical branchpoints. For example, splicing of the transcripts snR17A and B, which encode the U3 snoRNAs, was strongly affected, resulting in defects in the processing of pre-rRNA. The observation that loss of Mdlc causes specific splicing defects (both increased and decreased intron retention) in the pros transcript, together with the finding that RNF113A can rescue the mdlc loss-of-function phenotypes, suggest that the fly and human proteins might have a more complex role in regulating splicing than that of the yeast general splicing factor Cwc24p (Carney, 2013).

What might be the CNS splicing targets of Mdlc, in addition to pros? In mammals, alternatively spliced transcripts resulting in protein isoforms that influence stemness or differentiation are known, including within the nervous system. Splice isoforms that are differentially regulated by the neuroblast transcription factor Wor have been identified. The analysis of genome-wide changes in splicing in mdlc RNAi brains is in progress but beyond the scope of this paper (Carney, 2013).

The pros twintron undergoes developmentally regulated alternative splicing (Scamborova, 2004) to generate protein isoforms that differ by 29 amino acids at the homeodomain N-terminus. The two nested pairs of splice sites in the pros twintron are utilized mutually exclusively by two separate spliceosomes: U2 and U12 (Scamborova, 2004). Loss of Mdlc specifically reduces pros U12 splicing, so most of the other introns in Drosophila that are known to utilize the U12 spliceosome were examined. The pros intron was the sole example of differential retention. This indicates that Mdlc does not preferentially affect the U12 spliceosome (Carney, 2013).

The RING-type zinc-finger proteins constitute one of the largest protein families, with over 600 members. Many of these proteins have been shown to function as E3 ubiquitin ligases, and the presence of a RING domain is often sufficient for such an annotation. The Mdlc human ortholog RNF113A is no exception and is thought to function as an E3 ligase; consistent with this assumption, RNF113A was found to physically interact with one of the human E2 proteins, UBE2U (Li, 2008; van Wijk, 2009). Moreover, the Mdlc RING domain is very well conserved from yeast to humans, suggesting its functional importance. It was were therefore surprising to find that the RING domain was completely dispensable not only for CNS function but also for organismal viability, since the ubiquitous misexpression of a version of Mdlc lacking the RING domain was able to substitute for the full-length protein (Carney, 2013).

A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells

Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH) transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da) protein and its mammalian counterparts (E12/E47) act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. This study demonstrates that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. This paradox can be explained by demonstrating that Da induces the differentiation factor Prospero (Pros) whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. These results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis (Yasugi, 2014).

To further characterize the overproliferation caused by da RNAi, da RNAi was induced by insc-Gal4 in all larval NBs. The number of Deadpan (Dpn) expressing NBs increased at the expense of Embryonic lethal abnormal vision (Elav) expressing neurons. Although da was expressed in all NBs of the central brain and in some progenitor cells, no phenotype was found in these lineages when da3 amorphic mutant clones were induced using mosaic analysis with a repressible cell marker (MARCM) technique (Yasugi, 2014).

The visual processing centers of the fly brain consist of the so-called optic lobes. The medial surface of the optic lobes is surrounded by medulla NBs that differentiate from NE cells and generate medulla neurons on the inner side of the brain. In the optic lobe, da is expressed in NE cells and in medulla NBs. To induce daRNAiin the optic lobe, a dpn-Gal4 driver line was used that showed strong Gal4 expression in NE cells and medulla NBs and weak expression in medulla neurons (called dpnOL-Gal4). Expression of daRNAi using dpnOL-Gal4 caused a strong increase of Dpn positive NBs. The da RNAi phenotype was examined with the mitotic marker Phospho-Histone H3 (PH3), the NB marker Miranda (Mira) and the neuronal marker Elav. In the wild type, PH3 positive mitotic cells (NBs and GMCs) were restricted to the periphery of the optic lobe. In da RNAi samples, PH3 positive cells were mislocalized and ectopically found in the inner side of the brain. To confirm this phenotype, da3 mutant clones were induced in the optic lobe. In da3clones, Dpn positive NBs were found in the region that was normally occupied by medulla neurons. Thus, da is required for cell fate determination in medulla NBs (Yasugi, 2014).

To test whether the ectopic NBs in da RNAi brains have unlimited growth potential and can induce malignant tumors, optic lobes expressing GFP under the control of insc-Gal4, were dissected and implanted into the abdomen of wild type adult host flies. Transplanted cells from da RNAi brains proliferated and GFP positive cells were observed in the host flies, while no substantial growth was observed in control samples. PH3 positive mitotically active cells were observed in the tissue from transplanted daRNAi samples, and this tumor tissue consisted of both Dpn-expressing NB-like cells and Elav-expressing neuron-like cells. This suggests that the da tumor cells proliferate and some of the cells keep the stem cell state, but these cells also produce differentiating cells. This is consistent with the result from da3 clones, in which both ectopic NBs and differentiated neurons were observed. From these results, it is concluded that da acts as a tumor suppressor in optic lobe NB lineages (Yasugi, 2014).

Da is an E-box protein that heterodimerizes with other bHLH type transcription factors, such as the proneural proteins of the AS-C. The AS-C is composed of four transcription factors called Achaete (Ac), Scute (Sc), Lethal of Scute (L(1)sc), and Asense (Ase). While Ac is not expressed in the optic lobe, three of four AS-C proteins show specific expression. Sc is expressed in the NE cells and NBs, L(1)sc is transiently expressed in the transition zone between NE cells and NBs, and Ase is expressed in NBs and GMCs in the developing medulla. To test which of the AS-C genes might act with da during cell fate determination in medulla NBs, clones of several deletion lines were induced that uncover the AS-C region. Ectopic NBs were observed in clones of Df(1)260-1 uncovering all AS-C genes or in ase1 that uncovers the ase coding region. On the other hand, no phenotype was observed in clones of Df(1)sc19, which deletes ac, sc, and l(1)sc. Since the phenotype of Df(1)260-1 or ase1 clones was similar to the phenotype of da3 mutant clones and heterodimerization between Ase and da has been shown, it is concluded that da acts together with Ase to regulate cell fates in the optic lobe. It has been reported that da is required for the timely differentiation from NE cells to NBs and L(1)sc is involved in this transition during the optic lobe development. From the expression pattern of AS-C genes and results from the clonal analysis using deficiency lines, a dual function is proposed for Da: As a heterodimer with L(1)sc, da promotes the transition from NE cells to NBs. Later, da acts with Ase in NBs to promote differentiation and prevent tumor formation (Yasugi, 2014).

To identify the downstream targets of da and Ase, the expression was tested of candidate genes. The homeodomain transcription factor Pros acts as a cell fate determinant in embryonic and larval NBs and is regulated by da and Ase in embryos. In the larval optic lobe, Pros is localized to the basal cortex of dividing NBs and nuclear in GMCs and newly born medulla neurons. Whether Pros expression is dependent on da and/or Ase was tested. Pros expression decreased in da3 or ase1 mutant clones suggesting that Pros acts downstream of da and Ase. To test whether pros is required for cell fate determination in the optic lobe, induced pros17 mutant clones were induced. In pros17 mutant clones, ectopic NBs were observed in the medulla neuron layer, which was similar to the phenotype of da3 or ase1mutant clones. Overexpression of Pros, on the other hand, resulted in a decrease of medulla NBs. To test whether Pros acts downstream of Da, Pros was overexpressed in a da RNAi background. A reduced number of medulla NBs were observed in optic lobes overexpressing Pros in a da RNAi background, indicating that pros is epistatic to da. Thus, Pros is a key downstream target of da and Ase in optic lobe NBs (Yasugi, 2014).

Next, it was asked whether Pros expression is regulated by da in the central brain where da is not required for NB self-renewal. Nuclear Pros expression was found in differentiating daughter cells in the wild type. Pros expression remained in da3 mutant clones. Thus, unlike in the optic lobe, da is not essential for Pros expression in the central brain. This explains why the da phenotype is specific to the optic lobe NBs, while pros mutations cause overproliferation in all larval NBs. It is speculated that other factors may act redundantly to regulate Pros expression in the central brain (Yasugi, 2014).

If Pros is induced by da and Ase, then how are their functions turned off after asymmetric division? To test whether Pros can terminate the expression of ase, Ase expression was examined in pros17 clones. While Ase expression was restricted to the periphery of the optic lobe in wild type, Ase expression continued on the inner side of the optic lobe in pros17 clones. Thus, Pros turns off Ase expression and this transcriptional negative feedback loop regulates the proliferation and differentiation of NBs (Yasugi, 2014).

A prevailing view in stem cell biology is that a self-renewal program allows prolonged proliferation in stem cells and is turned off upon differentiation. The current data challenge this view and demonstrate that the ability to differentiate is pre-programmed in neural stem cells. This explains why transcription factors like da and Ase that are thought to be required for NB specification can be required for proper differentiation and act as tumor suppressors. It is proposed that a regulatory transcriptional loop assures cell fate determination and inhibits tumor formation. In a medulla NB, Da and Ase heterodimers induce Pros expression but Pros is excluded from the nucleus and therefore can not terminate Ase expression. After asymmetric cell division, however, Pros enters the nucleus of the GMC where it initiates differentiation and cell cycle exit. In the GMC, Pros terminates Ase expression and therefore triggers an irreversible decision towards differentiation. The data from embryonic NBs suggest that Pros can directly bind to the ase region and regulates its expression. In the absence of this regulation, GMCs maintain the stem cell fate and continue to grow into malignant tumors (Yasugi, 2014).

The role of Da, Ase, and Pros in neural stem cells could be conserved in mammals. Mammalian class I bHLH genes, namely E2A (encoding the E12 and E47 proteins), E2-2, and HEB are expressed in the developing brain. E2A, HEB, or E2A/HEB transheterozygous mutant mice show a brain size defect, suggesting that class I factors also regulate mouse brain development. Mash1 and Prox1, the vertebrate orthologs of Ase and Pros, are expressed in proliferating neural precursor cells of the developing forebrain and spinal cord. Like in Drosophila, Mash1 induces Prox1 and Mash1 promotes an early step of differentiation in neural stem cells. Like in vertebrates, NE cells in the Drosophila optic lobe first proliferate by symmetric cell division and then become asymmetrically dividing NBs. From these molecular and developmental similarities, it is speculated that the transcriptional regulatory mechanism this study identified might be well conserved in mammalian brains (Yasugi, 2014).

The data are of particular relevance in light of the recently postulated role of stem cells in the formation of malignant tumors. Failure to limit self-renewal capacity in stem cells or defects in progenitor cell differentiation can both lead to the formation of cells that continue to proliferate and ultimately form tumors. While genes acting in stem cells are thought to promote self-renewal, genes required in differentiating cells are thought to promote differentiation and limit proliferation and are therefore candidate tumor suppressors. The current data challenge this view and show that the path to differentiation is initiated in the stem cell and therefore even genes specific to stem cells can act as tumor suppressors. It will be interesting to determine whether a similar mechanism acts in mammalian neural stem cells as well. If it does, the expression pattern of a gene can no longer be used as a main criterion for whether it promotes or inhibits self-renewal in stem cell lineages (Yasugi, 2014).

prospero: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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