prospero

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 Yan^ACT 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 Yan^ACT 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 Yan^ACT. 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 lz^gal4 reporter system was used to show that hyperstable Yan^ACT 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-yan^ACT 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-yan^ACT 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 Yan^ACT. 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).

N^ICD 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).

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 gcm^N7-4 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 dlg^v55 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 dlg^v55 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-1^null 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 pros^null alleles tested (Kuzin, 2005).

To determine if the axon guidance phenotype observed in pros^null (pros^I13) 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-1^null embryos. Although both pros^null and nerfin-1^null 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 pros^null embryos was considerably wider when compared to nerfin-1 mutants or wild-type embryos. The axon scaffolding phenotype observed in nerfin-1^null; pros^I13 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 pros^null 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-1^null 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, CycE^AR95-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 CycE^AR95 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 CycE^AR95-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/p27^Cip/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 pros^C7 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, pros^C7 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 svp^e22, pros^C7 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).

Splicing of prospero

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 b