ventral nervous system defective



Transcription starts early during cellularization in the region that gives rise to the ventral nervous system (CNS), and part of the procephalic region [Image]. Transcription is also apparent in part of the posterior hindgut anlagen and part of the posterior midgut anlagen. vnd expression is first observed in the blastoderm as two longitudinal stripes that flank the prospective mesoderm. There is a transient alternating pair-rule pattern at stage 6, but soon after gastrulation [Images] the continuity of the stripes is restored and the two longitudinal stripes join at the ventral midline, following mesoderm invagination. The stripes of medial neuroectodermal cells that synthesize VND are converted into clusters of neuroectodermal cells. These cells have very well defined identities, constituting a subset of neuroblasts that proceed to develop into ganglion mother cells and neurons. All continue to express vnd. By late stage 12, neurons expressing the vnd gene integrate into the ladder-like structure that is the ventral nerve cord (known as the CNS). Transcripts are found in the anterior and posterior midgut, starting after 12 hours of development. vnd is also expressed in the CNS of the adult (Mellerick, 1995 and Jimenez, 1995).

Initial expression of vnd in the NE occurs at the blastoderm stage in two longitudinal stripes flanking the presumptive ventral midline, preceding the formation of the S1 proneural clusters. The position of these clusters, defined by the expression of AS-C genes, subdivides the NE into three columns: ventral, intermediate, and dorsal. The DV extent of the vnd stripe appears to match that of the ventral column. After S1 Nb delamination, the ventral-most NE continues expressing vnd. Only ventral Nbs appear to transcribe vnd after S2 Nb delamination. Neuroectodermal expression of vnd is narrowed progressively and, by completion of Nb formation, it is restricted to the ventral-most cell row. At this stage, vnd transcripts are detected in all ventral Nbs and in the three intermediate S2 Nbs located in the posterior compartment of the hemisegment (Nbs 1-2, 6-2, and 7-2, defined by engrailed expression), even though these intermediate Nbs did not express vnd initially. Strong vnd transcription is also found at stage 11 in the neural progeny underlying vnd-positive Nbs (Chu, 1998).

Vnd protein is first detected at the blastoderm stage in bilateral stripes corresponding to the future ventral column of the CNS. Following gastrulation, stage 8 embryos have Vnd protein in the nuclei of the ventral midline cells, as well as in the bilateral ventral column neuroectoderm and neuroblasts. By stage 9, there is little or no Vnd protein in the ventral midline cells, but Vnd levels remain high in the ventral column neuroectoderm and neuroblasts. During these stages, the Vnd protein pattern is identical to the vnd RNA pattern, except that there is a slight lag between the time of transcription and translation. At stage 11 and later, Vnd protein is detected in a complex pattern of neurons including some, but not all, of the neuronal progeny of the ventral column neuroblasts. For example, Vnd protein is detected transiently in the U neurons derived from the ventral column neuroblast, NB 7-1. In contrast, Vnd protein is detected in the pCC interneuron, but not its sibling aCC motoneuron, derived from the ventral column neuroblast, NB 1-1. Vnd is detected in additional neurons derived from ventral column neuroblasts and is also detected in dorsally located neurons that may derive from intermediate or dorsal column neuroblasts that do not express vnd (McDonald, 1998).

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

Expression of the DV patterning genes in the brain

The insect brain is traditionally subdivided into the trito-, deuto- and protocerebrum. However, both the neuromeric status and the course of the borders between these regions are unclear. The Drosophila embryonic brain develops from the procephalic neurogenic region of the ectoderm, which gives rise to a bilaterally symmetrical array of about 100 neuronal precursor cells, called neuroblasts. Based on a detailed description of the spatiotemporal development of the entire population of embryonic brain neuroblasts, a comprehensive analysis was carried out of the expression of segment polarity genes (engrailed, wingless, hedgehog, gooseberry distal, mirror) and DV patterning genes (muscle segment homeobox, intermediate neuroblast defective, ventral nervous system defective) in the procephalic neuroectoderm and the neuroblast layer (until stage 11, when all neuroblasts are formed). The data provide new insight into the segmental organization of the procephalic neuroectodem and evolving brain. The expression patterns allow the drawing of clear demarcations between trito-, deuto- and protocerebrum at the level of identified neuroblasts. Furthermore, evidence is provided indicating that the protocerebrum (most anterior part of the brain) is composed of two neuromeres that belong to the ocular and labral segment, respectively. These protocerebral neuromeres are much more derived compared with the trito- and deuto-cerebrum. The labral neuromere is confined to the posterior segmental compartment. Finally, similarities in the expression of DV patterning genes between the Drosophila and vertebrate brains are discussed (Urbach, 2003).

In addition to the segment polarity genes, the dorsoventral patterning genes ventral nervous system defective (vnd), intermediate neuroblast defective (ind) and muscle segment homeobox (msh) have been shown to confer positional information to the truncal neuroectoderm, which also contributes to the specification of NBs. For the head and brain, a detailed analysis of the expression of these genes has not yet been undertaken. In order to elucidate their putative role in patterning the head and brain, the expression of vnd, ind and msh was analyzed in the procephalic ectoderm and NBs in the early embryo (until stage 11). Although the data are consistent with their role in dorsoventral patterning being principally conserved in the procephalon, significant differences are found in their patterns of expression compared with the trunk (Urbach, 2003).

At the blastodermal stage, Ventral nervous system defective protein (Vnd) is expressed in bilateral longitudinal stripes corresponding to the most ventral neuroectodermal column, and is by stage 11 detected in all ventral and two intermediate NBs of the ventral nerve cord. Interestingly, the latter co-express en and are located in the posterior compartment of each truncal neuromere. At gastrulation the ventral longitudinal vnd domain reaches anteriorly across the cephalic furrow into the procephalic neuroectoderm. By stage 9, vnd maps in the ventral neuroectoderm of the prospective intercalary, antennal and ocular segment and is observed in ventral NBs of the antennal (Dv2, Dv3, Dv6) and ocular neuromere (Pcv1, Pcv3, Pcv6, Ppv2). It appears as if the dorsal part of the Vnd-positive antennal neuroectoderm partly co-expresses ind at that stage, but the NB Dd1, which emerges from this ectodermal region expresses only ind and not vnd. This is possibly due to the transient expression of vnd in most parts of both the ventral antennal ectoderm and corresponding NBs: by stage 10 Vnd is detected in the ventral Dv2, Dv4 and Dd5, but is already downregulated in Dv3 and Dv6, and by stage 11 it is confined to Dd5 and the new Dv8. As a consequence of the downregulation of vnd, some ventral deutocerebral NBs, which delaminate between stage 9 and 11 from this domain were not observed to express vnd (e.g. Dv1, Dv5, Dv7). By stage 11 Vnd is seen in four tritocerebral NBs (Tv2, Tv3, Tv4, Tv5), in two deutocerebral NBs (Dd5, Dv8), and in a cluster of about 13 protocerebral NBs. Interestingly, vnd expression expands along the posterior border of the en intercalary stripe (en is), and is also significantly extended dorsally into the en antennal stripe; the NBs delaminating from there. The fact that vnd and en are co-expressed in Tv5 and in Dd5, Dv8 is in agreement with findings in the ventral nerve cord, where these genes are co-expressed in two intermediate NBs. This indicates that vnd demarcates the ventral part of the posterior border in trunk as well as in brain neuromeres. Furthermore, the posterior border of the ocular vnd domain (including the NBs Pcv1, Pcv2, Pcv3, Ppv1, Ppv2, Ppv3) abuts dorsally the En-positive NBs Ppd5 and Ppd8 (deriving from the en head spot), supporting the view that these NBs demarcate the posterior border of the ocular neuromere (Urbach, 2003).

intermediate neuroblast defective (ind) is expressed in the blastoderm in a bilateral longitudinal column (intermediate column neuroectoderm) just dorsal to the vnd domains. In the trunk, at stage 9 (when ind mRNA is no longer present in the neuroectoderm), it is expressed in all intermediate NBs and finally, at stage 11, it is confined to the NB 6-2. In the head, at stage 9, ind is detected in an intermediate longitudinal ectodermal domain in the intercalary segment, and weakly in an intermediate ectodermal patch in the antennal segment as well as in the deutocerebral NB Dd1 which develops from this patch. At the same stage, a further signal is observed in a dorsal ectodermal patch of the ocular region. The ectodermal ind patches in the intercalary, antennal and ocular segments are both separate from each other and from the ind domain in the trunk. Interestingly, ind mRNA is significantly present longer in the ectoderm of the intercalary and mandibular segment, when compared with the antennal segment and the trunk ectoderm. This presumably mirrors the delayed onset of neurogenesis in both segments. Until stage 10, five NBs derive from the three ind patches: Td1, Td2, Td3, from the intercalary, Dd1 from the antennal and Ppd13 from the ocular ind patch. Subsequently, the ocular ind patch enlarges but never reaches the ocular vnd domain, and by stage 11 about four additional Ind expressing NBs (Pcd7, Pcd13, Ppd6, Ppd9) are identifiable (Urbach, 2003).

muscle segment homeobox (msh) expression is first detected at the blastoderm stage in discontinuous patches in the dorsolateral part of the neuroectoderm that later extend and form a bilateral longitudinal stripe; this domain gives rise to the lateral NBs of the ventral nerve cord. At stage 7 msh expression is detected anterior to the cephalic furrow, which expands until stage 9 to cover, as a broad domain, the dorsal ectoderm of the intercalary and the antennal segment. As evidenced by Msh/Inv double labelling during stage 9 and stage 11, the anterior border of the msh domain coincides with the posterior border of the en hs. This suggests that msh expression in the pregnathal region is restricted to the intercalary and antennal segments, and matches the border between the antennal and ocular segment. This is further supported by Msh/hh-lacZ double labelling in stage 11 embryos, using hh as a marker for the posterior border of the ocular segment. All identified brain NBs delaminating from the dorsal intercalary and antennal neuroectoderm express msh. This suggests that during early neurogenesis, msh controls dorsal identities of the procephalic neuroectoderm and brain NBs, as was shown for the ventral nerve cord. In the ventral nerve cord, most glial precursor cells (glioblasts and neuroglioblasts) derive from the dorsal neuroectoderm, and express msh. In the intercalary segment of the early brain, two glial precursors (Td4 and Td7) were identified. Interestingly, both precursors are also located dorsally and express msh. At least until stage 11 no msh expression is found in the preantennal segments (Urbach, 2003).

In Drosophila the DV patterning genes subdivide the trunk neuroectoderm into longitudinal columns; vnd is required for the specification of the ventral neuroectodermal column and NBs; ind and msh have analogous functions in the intermediate and dorsal neuroectodermal columns and NBs, respectively. Remarkably, homologous genes are found to be expressed in the vertebrate neural plate and subsequently in the neural tube. In the neural tube the order of expression along the DV axis is analogous to that of Drosophila: like vnd, the vertebrate homologs of the Nkx family are expressed in the ventral region; the ind homologs, Gsh-1/2, are expressed in the intermediate region; and the msh homologs, Msx-1/2/3, are expressed in the dorsal region of the neural tube (Urbach, 2003).

Thus, in the brain msh is confined to more posterior regions, and vnd expression extends into anterior regions of the brain. Moreover, the expression border of msh and vnd coincide with neuromeric borders. A comparison of the anteroposterior sequence of DV patterning gene expression in the early brain of Drosophila, with that published for the early mouse brain, reveals striking similarities. Msx3, which presumably represents the ancestral msh/Msx gene, becomes restricted to the dorsal neural tube during later development (in contrast to Msx1/2). The anterior border of the Msx3 domain is positioned within the rostral region of the dorsal rhombencephalon, thus showing the shortest rostral extension of all vertebrate DV patterning genes. This displays analogy to msh, the expression domain of which coincides with the anterior border of the dorsal deutocerebrum, thus representing the shortest anterior extension of DV patterning genes in Drosophila. Mouse Nkx2.2 extends ventrally into the most rostral areas of the forebrain. vnd is expressed ventrally in anterior parts of the ocular and labral protocerebrum. Thus, the expression of the respective homologs in both species displays the most anterior extension among DV patterning genes. Moreover, Nkx2.2 expression in the mouse forebrain suggests that Nkx2.2 may be involved in specifying diencephalic neuromeric boundaries. Similarly, in Drosophila, dorsal expansions of the vnd domain appear to correspond to the tritocerebral and deutocerebral neuromeric boundaries (Urbach, 2003).

Furthermore, Drosophila ind and its mouse homologue Gsh1 show similarities in their expression in the early brain. In the posterior parts of the Drosophila brain, ind is expressed in intermediate positions between vnd and msh. Likewise, in the posterior part of the mouse brain, Gsh1 appears to be expressed in intermediate positions, dorsally to Nkx2.2, and in the hindbrain ventrally to Msx3. Gsh1 has been shown to be expressed in discrete domains within the mouse hindbrain, midbrain (mesencephalon) and the most anterior domain in the posterior forebrain (diencephalon). Correspondingly, in Drosophila ind expression in restricted domains within the gnathocerebrum, the tritocerebrum, deutocerebrum and ocular part of the protocerebrum, demonstrating that the anteriormost extension of ind (and Gsh1) expression lies between that of msh and vnd (Urbach, 2003).

Taken together, considering these similarities, it is suggested that in the Drosophila and vertebrate early brain the expression of DV patterning genes is to some extent conserved, both along the DV axis (as suggested for the truncal parts of the Drosophila and mouse CNS) and along the AP axis. Furthermore, in Drosophila large parts of the anterodorsal procephalic neuroectoderm and NBs (more than 50% of all identified brain NBs) lack DV patterning gene expression. Likewise, in the vertebrate neural tube, gaps between the expression domains of DV patterning genes have been described, raising the possibility that other genes might fill in these gaps. How DV fate is specified in the anterior and dorsal part of the Drosophila procephalic neuroectoderm, and if other genes are involved, remains to be clarified (Urbach, 2003).

Segment-specific requirements for dorsoventral patterning genes during early brain development in Drosophila

An initial step in the development of the Drosophila central nervous system is the delamination of a stereotype population of neural stem cells (neuroblasts, NBs) from the neuroectoderm. Expression of the columnar genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh) subdivides the truncal neuroectoderm (primordium of the ventral nerve cord) into a ventral, intermediate and dorsal longitudinal domain, and has been shown to play a key role in the formation and/or specification of corresponding NBs. In the procephalic neuroectoderm (pNE, primordium of the brain), expression of columnar genes is highly complex and dynamic, and their functions during brain development are still unknown. These genes (with special emphasis on the Nkx2-type homeobox gene vnd) have been investigated in early embryonic development of the brain. At the level of individually identified cells it is shown that vnd controls the formation of ventral brain NBs and is required, and to some extent sufficient, for the specification of ventral and intermediate pNE and deriving NBs. However, significant differences were uncovered in the expression of and regulatory interactions between vnd, ind and msh among brain segments, and in comparison to the ventral nerve cord. Whereas in the trunk Vnd negatively regulates ind, Vnd does not repress ind (but does repress msh) in the ventral pNE and NBs. Instead, in the deutocerebral region, Vnd is required for the expression of ind. In the anterior brain (protocerebrum), normal production of early glial cells is independent from msh and vnd, in contrast to the posterior brain (deuto- and tritocerebrum) and to the ventral nerve cord (Urbach, 2006).

Expression of Vnd protein is first detectable in the blastoderm in bilateral longitudinal columns along the ventral neuroectoderm of the trunk and the ventral procephalic neuroectoderm (pNE), covering the prospective ventral parts of the trito-(TC), deuto-(DC) and protocerebrum (PC). Although in the trunk Vnd expression is maintained within a continuous ventral neuroectodermal column during subsequent stages, it becomes more complex and diverse among head segments. By stage 8 (before first brain neuroblasts have developed0, Vnd becomes downregulated in parts of the procephalic domain. Until stage 10, Vnd has largely vanished in the anterior pNE and NBs of the DC, but its level remains high in the ventral pNE and delaminating NBs of the TC, posterior DC and PC. During stages 10/11, Vnd becomes downregulated at the ventral border between TC and DC. Accordingly, by late stage 11, Vnd expression is restricted to separate domains at the posterior border of the TC, DC and PC, respectively. Whereas the number of Vnd-positive NBs in the domains of the TC and DC is rather small, a large population of about 13 NBs is found in the PC. Thus, in contrast to the situation in the trunk, Vnd expression in the early brain is highly dynamic and becomes progressively confined to three separate ventral domains, encompassing different numbers of NBs and their progeny in the posterior compartments of the TC, DC and PC (Urbach, 2006).

In vnd mutant embryos, it was found at embryonic stages 9 and 11 that ventral NBs in the TC, DC and PC are largely absent, although at different frequencies. Analogously, in the primordium of the VNC of vnd embryos a significant loss of ventral NBs has been reported. In the absence of Vnd, an increase in cell death, which contributes to the loss of ventral brain NBs, was found. Apoptosis acts at the level of both pNE progenitor cells and NBs. It is not yet clear whether the reduction of ventral NBs is solely due to cell death, or whether it also involves activity of proneural genes. In the truncal neuroectoderm, proneural genes of the AS-C complex promote NB formation. There is evidence that vnd interacts with proneural genes, but also that it has additional function in promoting NB formation apart from activating proneural genes. The latter assumption is supported by the finding that, in vnd embryos, lethal of scute (l'sc) can still be expressed in the ventral proneural clusters of, for example, NB5-2, although the respective NB is missing. In the pNE, genes of the AS-C complex are expressed in large proneural domains, of which those of achaete, but especially of l'sc, seem to overlap with the vnd expression domain, suggesting a possible genetic interaction. However, in vnd embryos, no substantial differences were observed in the expression pattern of l'sc transcript compared with the wild type. Thus, similar to the situation in the trunk, Vnd does not appear to exert proneural function through activation of l'sc. However, the data propose a possible interaction between vnd and the proneural gene atonal. In vnd mutants, expression of atonal is often missing in proneural clusters of the sensory organ precursors of the hypopharyngeal-latero-hypopharyngeal organ. Clearly, further investigations are required to clarify in how far interactions between vnd and proneural genes play a role in the formation of ventral brain NBs (Urbach, 2006).

In vnd mutants, not only ventral, but also intermediate brain NBs in the TC and DC show defects in their formation or specification, comparable with the situation in the trunk. As intermediate brain NBs do not express vnd (but ind), these defects appear to be non-cell-autonomous. Another, more likely explanation is that determination occurs at the blastodermal stage, when Vnd is transiently expressed in a much larger population of cells in the pNE, which presumably include progenitors of intermediate NBs. A similar proposal was made for intermediate NBs in the trunk. Furthermore, early commitment of ventral neuroectodermal cells and cell-autonomous expression of ventral and intermediate NB fates has been demonstrated by heterotopic transplantations of neuroectodermal cells from ventral to dorsal sites at the early gastrula stage (Urbach, 2006).

In the trunk, a segmentally reiterated combinatorial code of genes expressed within each particular proneural cluster specifies the individual identity of the NB it gives rise to. These include DV patterning genes and segment polarity genes, which provide positional information in the neuroectoderm, as well as a number of other factors. Most of these genes are also expressed in specific domains of the pNE before NBs delaminate, although in a segment-specific manner. The present data show that Vnd influences the expression of such site-specific marker genes ('NB identity genes') already in the pNE, before NBs are formed. In vnd embryos, a derepression of dorsal-specific genes occurs in the ventral pNE (e.g. of msh and ems in the intercalary and antennal segment, and dac in the ocular segment) and in the descending NBs, and conversely, a loss of ventral-specific gene expression (e.g. lbe in the PC). The altered expression of 'NB identity genes' in vnd mutants reflects a ventral-to-dorsal transformation of ventral pNE and residual NBs. Further evidence for such a transformation is the production of (ectopic) glial cells by these ventral NBs, which normally is a trait specific to dorsal NBs. By contrast, in the trunk, absence of Vnd results in a ventral-to-intermediate transformation, owing to the derepression of ind (instead of msh in pNE), which induces specification of intermediate NB fates (Urbach, 2006).

Together, these data in the vnd loss- and gain-of-function backgrounds indicate that vnd is required, and is at least partially sufficient, for the induction of ventral fate in brain NBs through the activation of genes specific for the ventral pNE, and through the repression of genes specific for dorsal pNE (Urbach, 2006).

This analysis revealed differences in the regulation of DV patterning genes among the intercalary (IC), antennal (AN) and ocular (OC) head segments, giving rise to the TC, DC and PC, respectively. Overexpression of vnd leads to repression of ind within the IC, but loss of vnd-function does not seem to cause ventral expansion of the ind intercalary spot. Unexpectedly, ind is completely absent in the AN of vnd mutants, suggesting that in this segment vnd is necessary for activation and/or maintenance of ind (rather than repression). This is supported by the finding that the ind antennal spot transiently co-expresses Vnd, which is unique in the neuroectoderm, and by the present finding that in vnd gain-of-function background the ind antennal spot is almost unaffected. In the OC, however, ind expression is partially repressed upon Vnd overexpression, and ventrally expanded in the absence of Vnd, similar to the situation in truncal segments. However, because, in wild type the ind ocular spot does not adjoin the ocular vnd domain, its expansion in vnd embryos cannot be due to a cell-autonomous effect (Urbach, 2006).

Overexpression of vnd abolishes Msh almost completely in the neuroectoderm of all body segments. Yet, absence of Vnd reveals segment-specific differences in the regulation of msh. Owing to insulated ind expression in the IC and lack of ind in the AN of vnd mutants, Msh (instead of ind) is found in the ventral pNE of these segments, which is unique in the CNS anlagen, except for the mandibular segment, which exhibits equivalent expression (Urbach, 2006).

Among the pregnathal segments, the degree of conservation with regard to the expression and interactions of DV patterning genes seems to be highest in the posterior IC (TC) [ind and msh being repressed by (ectopic) vnd, and msh by ind]. In the anterior head, endogenous Msh expression in the dorsal pNE reaches the segmental border between AN (DC) and OC (PC), but does not cross it. Ectopic Msh in vnd mutants does also not cross this border, which suggests interference with regulatory factors acting in AP axis (Urbach, 2006).

Significant differences between the anterior head segments and the trunk have also been reported for the initial mode of activation and cross-regulatory interactions of segment-polarity genes (Urbach, 2006).

In the pNE, vnd is necessary for the formation and specification of brain NB. It remains to be shown whether ind and msh exert analogous functions. However, more than 50% of the identified brain NBs do not express any of the three DV patterning genes. Most of these NBs derive from pNE of the preantennal head, which implies that further factors are involved in DV patterning of the anterior pNE and brain. Several other genes have been reported to be crucial for DV patterning in the truncal neuroectoderm, such as the EGF-receptor homolog Egfr, the Sox genes SoxNeuro and Dicheate, and Nk6. For most of them it has been shown that they are involved in formation and/or specification of truncal NBs. Egfr, both Sox genes and Nk6 are also expressed in the pNE, before and during the phase of NB formation. However, in Egfr mutant embryos the number and pattern of brain NBs is unaffected. How far the Sox genes and Nk6 contribute to the formation and/or specification of brain NBs awaits further investigation (Urbach, 2006).

Most of the glial cells in the VNC derive from dorsal NBs (neuroglioblasts or glioblasts), which depend on msh for proper specification. Accordingly, glial cells deriving from these progenitors are missing or improperly differentiated in msh mutants, as well as in sca-vnd embryos. Likewise, in the TC and DC, first glial cells are closely associated with dorsal NBs that descend from Msh-expressing pNE. In the TC, some dorsal NBs have been identified as glial progenitors, e.g. the neuroglioblast Td4 and the glioblast Td7, which are putative serial homologs of the truncal neuroglioblast NB5-6 and the glioblast LGB, respectively. In absence of Vnd, the number of glial cells in the TC, and especially in the DC, were found to be to be increased. This is most probably due to the segment-specific early derepression of Msh in the ventral pNE and NBs of the TC and DC. In the truncal segments, however, ind instead of Msh is derepressed in the ventral NE, and the number of glial cells is not significantly affected in the VNC of vnd mutants. Furthermore, in msh mutants, glial development in the TC and DC is almost completely abolished, which parallels the phenotype observed upon vnd overexpression (leading to repression of msh in the dorsal pNE and NBs). Thus, comparable with the situation in the VNC, Msh promotes glial fate in the TC and DC. However, in the PC, glial development must be regulated differently (at least in its early phase). Until stage 12 no Msh was detected in this part of the brain, and in msh mutants the number of glial cells in the PC is normal. Glial cell fate in the PC is also not affected by loss of vnd, although it remains repressable by ectopic Vnd. Therefore, as opposed to the TC and DC, and to the VNC, normal production of early glial cells in the PC does not depend on msh, nor indirectly on vnd or ind (Urbach, 2006).

There are striking similarities in the spatial order of expression of vnd, ind and msh in the Drosophila neuroectoderm and homologous genes in the neural plate and neural tube of vertebrates: vnd homologs of the Nkx2 family are expressed in ventral regions; the ind homologs Gsh1 and Gsh2 are expressed in the intermediate regions; the msh homologs Msx1, Msx2 and Msx3 are expressed in the dorsal region of the neural tube. This dorsoventral order of expression is conserved not only in the anlagen of the truncal CNS but also in those that form the posterior part of the brain (in Drosophila, TC and DC; in vertebrates, hindbrain). Moreover, the anterior borders of the expression domains of these columnar genes correspond in the early brains of Drosophila and mouse: expression of vnd/Nkx2 extends most rostrally (mouse ventral forebrain), followed by ind/Gsh1 and, finally, msh/Msx3 expression. Thus, the expression of columnar genes in the brain is, to some extent, evolutionarily conserved both along the DV axis and along the AP axis (Urbach, 2006).

This study has presented evidence that in Drosophila vnd mutant embryos a large fraction of ventral brain NBs is missing, and that ventral pNE and residual ventral NBs show significant traits of a ventral-to-dorsal transformation owing to derepression of msh (as opposed to ind in the VNC). Again, this displays obvious similarities to findings made in mice carrying a deletion of Nkx2.1. Consistent with the pattern of expression in wild type, in the mutant embryonic brain a substantial loss of ventral (especially forebrain) structures has been observed. Moreover, ind/Gsh2 expression is not expanded in Nkx2.1 mutants, and residual basal (ventral) pallidal structures become transformed into dorsal striatal structures. Thus, in both Drosophila and mouse, loss of vnd/Nkx2 in the brain leads to a transformation of ventral into dorsal structures, rather than into intermediate structures, which has been shown to be the case in the truncal CNS of both species. Therefore, in the developing brains of Drosophila and vertebrates, vnd/Nkx2 is crucial for the formation and specification of ventral brain structures, and interacts with other dorsoventral patterning genes in a region-specific manner (Urbach, 2006).

Identification of Ind transcription activation and repression domains required for dorsoventral patterning of the CNS

Specification of cell fates across the dorsoventral axis of the central nervous system in Drosophila involves the subdivision of the neuroectoderm into three domains that give rise to three columns of neural precursor cells called neuroblasts. Ventral nervous system defective (Vnd), intermediate neuroblasts defective (Ind) and muscle segment homeobox (Msh) are expressed in the three columns from ventral to dorsal, respectively. The products of these genes play multiple important roles in formation and specification of the embryonic nervous system. Ind, for example, is known to play roles in two important processes. First, Ind is essential for formation of neuroblasts in conjunction with SoxB class transcription factors. Sox class transcription factors are known to specify neural stem cells in vertebrates. Second, Ind plays an important role in patterning the CNS in conjunction with, vnd and msh, which is also similar to how vertebrates pattern their neural tube. This work focuses two important aspects of Ind function. First, multiple approaches were used to identify and characterize specific domains within the protein that confer repressor or activator ability. Currently, little is known about the presence of activation or repression domains within Ind. This study shows that transcriptional repression by Ind requires multiple conserved domains within the protein, and that Ind has a transcriptional activation domain. Specifically, a novel domain, the Pst domain, was identified that has transcriptional repression ability and appears to act independent of interaction with the co-repressor Groucho. This domain is highly conserved among insect species, but is not found in vertebrate Gsh class homeodomain proteins. Second, it was shown that Ind can and does repress vnd expression, but does so in a stage specific manner. It is concluded from this that the function of Ind in regulating vnd expression is one of refinement and maintenance of the dorsal border (Von Ohlen, 2009).

The function of Ind in development of the embryonic nervous system is multifold. Initially, Ind serves to define the intermediate column of the neuroectoderm, this subsequently leads to formation of the corresponding neuroblasts. This study shows that transcriptional repression activity by Ind involves at least two transcriptional repression domains, suggesting that Ind represses transcription via Groucho-dependent and Groucho-independent mechanisms. There are two highly conserved domains in the N-terminal region of the Ind protein. Both appear to be essential for maximal repression activity of Ind. In addition, a third domain was identified that is capable of conferring transcriptional activation ability on a heterologous DNA-binding domain. Also, data is presented demonstrating that Ind functions to define and maintain this domain via transcriptional repression of other columnar genes vnd and msh. Suggesting that, depending on which enhancer it is bound to and possibly association with co-factors, Ind can act as either a transcriptional repressor on as activator. Finally, an Ind protein lacking the Eh1 domain but retaining the Pst domain fails to physically interact with purified Groucho protein. Furthermore, the Gal4-IndδEh1 protein was still a strong repressor of transcription in cultured cells. These results strongly support the hypothesis that the Pst domain confers repressor activity independent of Groucho interaction. However, the possibility that the Pst domain also plays a role in stabilizing the interaction with Groucho or association with other co-factors in vivo cannot be ruled out (Von Ohlen, 2009).

It is not surprising that Ind has incorporated additional repressor activities that are independent of Groucho activity. Formation of the intermediate column of neuroblasts is also dependent on the activity of the Egfr signaling pathway. Specifically, in egfr mutant embryos the intermediate column of neuroblasts fails to form because Ind is not expressed. The readout for activation of the Egfr pathway is the presence of the activated form of Map kinase (dpErk). DpErk is detected in the ventral and intermediate columns of the neuroectoderm at the early stages of development. Interestingly, the activation of DpErk appears to correlate with down-regulation of Groucho activity. Specifically, Map kinase directly phosphorylates Groucho and this phosphorylation of Groucho results in reduced co-repressor activity (Cinnamon, 2008). Since Groucho activity is down-regulated in the region where Ind is expressed and Ind is a Groucho-dependent transcriptional repressor, additional repression activity may be necessary to overcome the effects of Egfr signaling on Groucho activity (Von Ohlen, 2009).

Formation of the proper complement of neuroblasts in the embryonic nervous system of Drosophila and other insects is essential for the proper development of the organism. Initially the neuroblasts form in three columns that correspond to the domains of vnd, ind and msh expression. Therefore, formation of the stripes of homeobox gene expression is essential for the ultimate formation of the CNS. While there is an apparent ventral dominance mechanism in place to initiate the expression of these genes, there is also a cross repressive relationship that is essential for maintaining the boundaries between the domains of gene expression. Ind represses vnd only at stages 9 and 10 of embryonic development and not earlier. This coincides with differences in the ability of Ind to repress vnd reporter gene expression. Thus, Ind can repress transcription from enhancer elements located upstream that regulate expression in neuroblasts. However, Ind is unable to repress transcription of lacZ message from reporter constructs that include the vnd NEE, which is essential for initiation of vnd expression. In conclusion, the temporal differences in the ability of Ind to repress transcription of vnd reflect a role for Ind maintaining the boundary between Vnd and Ind domains. However, Ind was not required for establishing the dorsal border of vnd expression at the earliest stages of embryogenesis (Von Ohlen, 2009).

Effects of Mutation or Deletion

Genetic and developmental analysis of an X-linked vital locus vnd was undertaken. Embryos hemizygous for the original vnd allele do not hatch and exhibit a disorganized ventral nervous system (VNS). The mutation maps in the region 1B6-7 to 1B9-10, a subregion of an area previously shown to be essential to normal neural development. Five new alleles at the locus vnd have been isolated. Genetic complementation analysis of all mutations at the vnd locus, with lethal alleles at adjacent loci, indicates that all lesions at the locus vnd affect only one vital gene function in the region. Four of the five alleles are embryonic lethal; one allele is subvital and behaves like a hypomorphic mutation. Hemizygous embryos for three of the four embryonic lethal alleles were inspected in histological sections; all exhibited a disorganized VNS, similar to the original allele. The developmental analysis in gynandromorphic genetic mosaics shows that (1) vnd+ gene function is not essential in most imaginal-disc cell derivatives; (2) only about 30% of the mosaic zygotes survive as adults; (3) mosaic zygotes with mutant tissue close to the head cuticle are least likely to survive, and (4) mutant tissue in the thoracic ganglion in the adult is not necessarily lethal. The mosaic data are consistent with the vnd+ gene function being necessary in neural cells derived from the anterioventral region of the blastoderm (White, 1983).

Mutations at vnd eliminate a fraction of specific embryonic neuroblasts (Skeath, 1994).

The formation of S1 and S2 Nbs in vnd mutant embryos was analyzed. Morphological features such as size and position, as well as anti-Hunchback (Hb) antibodies (a general Nb marker), were used to assess the presence of Nbs as described. Additionally, resolution of mapping was improved by the use of two lacZ enhancer trap lines in genes svp and hkb, each being expressed in a different subset of Nbs in a precise temporal sequence. These markers greatly aid mapping of lacZ-expressing Nbs as well as those surrounding them, despite possible displacements of their position after delamination. In these mutant embryos, formation of all ventral Nbs is affected dramatically, but Nbs of the intermediate and dorsal columns form normally. However, a few residual Nbs are still observed in the ventral column. To test if the possible maternal expression of vnd is responsible for the residual ventral Nbs, homozygous vnd germ-line clones were generated, and mutant embryos devoid of any maternal vnd product were analyzed. Residual S1 + S2 ventral Nbs are still observed at a similar frequency. Although not studied in detail, formation of S3-S5 Nbs seems to be only partially affected. Thus, instead of the three ventral Nbs (MP2, 3-1, and 4-1) present in rows 3/4 at the S3 stage in the wild type, only one Nb at most can be found at the same position in vnd embryos, and this happens in only 33% of cases. On the contrary, the S3 Nb 6-1 (identified by normal expression of ming-lacZ and Gsb-d) is detected in >90% of cases (Chu, 1998).

In the vnd mutant embryos some early Nbs were still observed in the ventral column; this led to an examination of their developmental potential. Virtual absence of several progeny of S1 ventral Nbs, determined by using different specific markers, suggests that the residual Nbs do not possess ventral identities. These absences include (1) Eve- and FasII-positive aCC/pCC neurons and Repo-positive SPG glial cells, progeny of Nb 1-1; (2) 22C10-positive dMP2/vMP2 neurons, progeny of MP2, and (3) Eve-positive CQ neurons and the Eve- and Fas II-positive U motoneurons, progeny of Nb 7-1. Collectively, these data indicate that early ventral Nb lineages are affected in the vnd embryos. Absence of specific lineages could mean that the Nb identities are modified, or that the residual Nbs do not develop further, or that vnd is also required for the formation of the neurons in question. Several observations indicate that residual early ventral Nbs have altered identities in vnd embryos (Chu, 1998).

Loss of AS-C expression in the proneural clusters of S1 ventral Nbs MP2 and 7-1 occurs in vnd mutant embryos. Because vnd expression in the ventral NE precedes Nb formation, an investigation was carried out to see if loss of vnd also influences other distinctive identity features of the ventral proneural clusters. Indeed, proneural clusters of Nb 1-1, Nb 2-2, and Nb 5-2 fail to express Odd, hkb-lacZ, and svp-lacZ, respectively, in vnd embryos. The neuroectodermal expression of Gsb-d in the ventral column is also reduced strongly. Therefore, vnd seems to control Nb fates already at the proneural cluster stage. msh, a homeobox gene implicated in the control of dorsal Nb fate, is not expressed in the ventral NE of the wild type. Initially, msh expression is restricted to the dorsal NE; at stage 10, it is also expressed in the putative proneural clusters of some intermediate Nbs. In stage 10 vnd embryos, the msh expression domain, determined with the enhancer trap insertion rH96, is expanded into part of the ventral NE (Chu, 1998).

Proneural gene expression is insufficient to promote Nb formation in the absence of vnd. In vnd mutant embryos AS-C gene expression in proneural clusters corresponding to ventral Nbs 1-1 and 5-2 is unaffected. Nevertheless, these two Nbs fail to delaminate frequently, suggesting an additional requirement for vnd in Nb formation, which is distinct from its capacity to control AS-C expression. Because Kr-GAL4-driven vnd expression restores Nb formation, Kr-GAL4 was to drive AS-C expression in vnd embryos. Kr-GAL4-driven expression of either UAS-l'sc or UAS-ac+ UAS-sc is insufficient to induce formation of S1 ventral Nbs above the levels found in vnd embryos alone. On the contrary, the Kr-GAL4/UAS-ac combination can rescue formation of S1 ventral Nbs fully in Df(1)sc19 (ac-sc-l'sc) embryos. Likewise, the rho-GAL4/UAS-ac combination, which rescues MP2 formation in Df(1)y3PLsc8R (ac-sc-) embryos very efficiently, is unable to do so in vnd embryos. These results confirm that the role of vnd in Nb formation is not solely through the activation of proneural AS-C gene expression (Chu, 1998).

To assess if Vnd expression can modify the fate of intermediate and dorsal Nbs, UAS-vnd/Kr-GAL4 or sca-GAL4 embryos were analyzed. Cell-specific staining data collected from these embryos at the earliest stages indicate that in the gain-of-function situation, intermediate and dorsal Nbs exhibit two kinds of changes: (1) acquisition of specific features of ventral Nbs, and (2) suppression of some of their normal characteristics. Furthermore, intermediate and dorsal NE exhibit certain ventral-like features. As a rule, the penetrance of the transformations is not complete, affecting the intermediate Nbs more frequently than the dorsal ones. Also, it is unlikely that the transformations are perfect, although in this analysis transformations of intermediate Nbs in every row were observed and the transformed intermediate Nb progeny exhibited all identity markers tested. In addition to numerous examples that illustrate the acquisition of ventral-like fates and the suppression of normal fates, when Vnd is expressed in the intermediate and dorsal NE, situations that defy simple interpretation were also observed. This is not unexpected, as the positional informational matrix depends on the interplay of complex regulatory networks in which both the concentration and timing of the ectopic expression will influence the final outcome (Chu, 1998).

This study describes secondary structures, DNA binding properties, and thermal denaturation behavior of six site-directed mutant homeodomains encoded by the vnd/NK-2 gene from Drosophila melanogaster. Three single site H52R, Y54M, and T56W mutations, two double site H52R/T56W and Y54M/T56W mutations, and one triple site H52R/Y54M/T56W mutation were investigated. These positions were chosen based on their variability across homeodomains displaying differences in secondary structure and DNA binding specificity. Multidimensional NMR, electrophoretic mobility shift assays, and circular dichroism spectropolarimetry studies were carried out on recombinant 80-amino acid residue proteins containing the homeodomain. Position 56 on its own, and even more significantly, position 56 in combination with position 52, play an important role in determining the length of the recognition helix. The H52R mutation alone does not affect the length of this helix but does increase the thermal stability. Introduction of site mutations at positions 52 and 56 in vnd/NK-2 does not modify their high affinity binding to the 18-base pair DNA fragment containing the vnd/NK-2 consensus binding sequence, CAAGTG. Site mutations involving position 54 (Y54M, Y54M/T56W, and H52R/Y54M/T56W) all show a decrease of 1 order of magnitude in their binding affinity. The roles of individual atom-atom interactions in structure and sequence specificity are described (Weiler, 1998).

This paper describes the structural and DNA binding behavior for an analog of the vnd/NK-2 homeodomain, which contains a single amino acid residue alanine to threonine replacement in position 35 of the homeodomain. Multidimensional nuclear magnetic resonance, circular dichroism, and electrophoretic gel retardation assays were carried out on recombinant 80-aa residue proteins that encompass the wild-type and mutant homeodomains. The mutant A35T vnd/NK-2 homeodomain is unable to adopt a folded conformation when free in solution at temperatures down to -5 degrees C, in contrast to the behavior of the corresponding wild-type vnd/NK-2 homeodomain, which is folded into a functional three-dimensional structure below 25 degrees C. The A35T vnd/NK-2 binds specifically to the vnd/NK-2 target DNA sequence, but with an affinity that is 50-fold lower than that of the wild-type homeodomain. Although the three-dimensional structure of the mutant A35T vnd/NK-2 in the DNA bound state shows characteristic helix-turn-helix behavior similar to that of the wild-type homeodomain, a notable structural deviation in the mutant A35T analog is observed for the amide proton of leucine-40. The wild-type homeodomain forms an unusual i,i-5 hydrogen bond with the backbone amide oxygen of residue 35. In the A35T mutant this amide proton resonance is shifted upfield by 1.27 ppm relative to the resonance frequency for the wild-type analog, thereby indicating a significant alteration of this i,i-5 hydrogen bond (Xiang, 1998).

The Sox-domain containing gene Dichaete/fish-hook acts in concert with vnd and ind to regulate cell fate in the Drosophila neuroectoderm

The Sox-domain-containing gene Dichaete/fish-hook plays a crucial role in patterning the neuroectoderm along the DV axis. Dichaete is expressed in the medial and intermediate columns of the neuroectoderm, and mutant analysis indicates that Dichaete regulates cell fate and neuroblast formation in these domains. Molecular epistasis tests, double mutant analysis and dosage-sensitive interactions demonstrate that during these processes, Dichaete functions in parallel with ventral nerve cord defective and intermediate neuroblasts defective, and downstream of EGF receptor signaling to mediate its effect on development. These results identify Dichaete as an important regulator of dorsoventral pattern in the neuroectoderm, and indicate that Dichaete acts in concert with ventral nerve cord defective and intermediate neuroblasts defective to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

vnd, ind and Egfr are key factors that regulate pattern and cell fate along the DV axis of the neuroectoderm. To ask if Egfr pathway activity depends on vnd or ind, MAPK activity was assayed in homozygous vnd or ind single mutant embryos. In both backgrounds, the initial activation of Egfr signaling in the medial and intermediate columns is normal. Thus, in the early neuroectoderm, Egfr acts either upstream or in parallel to vnd and ind. To investigate whether Egfr acts upstream of vnd or ind, vnd and ind expression was assayed in embryos homozygous mutant for the Egfr null allele flbIK35 (referred to as Egfr mutant embryos). ind expression is absent in Egfr mutant embryos, indicating that Egfr activates ind expression in the intermediate column. By contrast, vnd expression in Egfr mutant embryos appears normal through the onset of stage 8. However, during stage 8, vnd expression begins to dissipate in medial column cells, and by early stage 10 these cells no longer express vnd. Conversely, medial column NBs that form in Egfr mutant embryos express vnd normally and retain vnd expression throughout embryogenesis. Thus, Egfr functions to maintain vnd expression in the neuroectoderm but is dispensable for vnd expression in NBs. These data indicate that Egfr resides atop the genetic hierarchy known to subdivide the neuroectoderm along the DV axis (Zhao, 2002).

These results suggest that Egfr patterns the neuroectoderm, at least in part, through its regulation of vnd and ind. To determine if additional genes act downstream of Egfr in this process, the phenotypes of embryos singly mutant for Egfr and ind were compared. It was reasoned that if Egfr patterns the intermediate column solely through regulation of ind, then Egfr and ind mutant embryos should exhibit identical intermediate column phenotypes. To compare the early CNS phenotypes of Egfr and ind, a precise analysis of msh expression and the NB pattern was carried out. In both cases, Egfr exhibits a more severe phenotype than ind. msh expression expands more medially in Egfr mutant embryos than in ind mutant embryos. In addition, lateral NBs are most often separated from medial NBs by a gap in ind mutant embryos, while lateral NBs develop immediately adjacent to medial NBs in Egfr mutant embryos. These data indicate a greater disruption to the intermediate column in Egfr mutant embryos than in ind mutant embryos. These phenotypic differences are consistent with the presence of additional genes acting downstream of Egfr and in parallel to ind to control cell fate in the intermediate column. However, Egfr maintains vnd expression in the neuroectoderm; thus, these data do not exclude the possibility that the differences in phenotype between Egfr and ind arise due to the late regulation of vnd expression by Egfr (Zhao, 2002).

To test whether the phenotypic differences between ind and Egfr mutant embryos are an indirect result of the regulation of vnd expression by Egfr, it was asked whether these differences are equalized in double mutants where vnd function is also removed. In vnd;ind mutant embryos, msh is expressed throughout the neuroectoderm, although its expression is higher in the lateral column relative to the medial column. By contrast, msh is expressed at uniformly strong levels throughout the neuroectoderm in vnd;Egfr mutant embryos. Thus, removal of vnd and Egfr causes a stronger derepression of msh in the neuroectoderm than loss of vnd and ind. These results suggest that additional gene(s) act downstream of Egfr and in parallel to vnd and ind to regulate DV pattern in the neuroectoderm. They also suggest that in the absence of vnd and Egfr function, the entire neuroectoderm acquires a lateral column fate (Zhao, 2002).

ind normally represses ac expression in the intermediate column, because in ind mutant embryos, ac expression is completely derepressed within rows 3 and 7 of the intermediate column. The Dichaete and ind phenotypes demonstrate that both genes are necessary for intermediate column fates. To determine if Dichaete and ind function in a linear pathway to regulate intermediate cell fates, ind expression was followed in Dichaete mutant embryos and Dichaete expression in ind mutant embryos. ind expression is normal in Dichaete mutant embryos and Dichaete expression is normal in ind mutant embryos. Thus, ind and Dichaete are regulated independently of each other (Zhao, 2002).

Double labeling Dichaete mutant embryos for ac and ind, and double labeling ind mutant embryos for ac and Dichaete reveals an interdependent relationship between Dichaete and ind. In Dichaete mutant embryos, a significant number of row 3 and 7 intermediate column cells and NBs co-express ac and ind -- an occurrence never observed in wild-type embryos. Thus, the ability of ind to repress ac in the intermediate column requires Dichaete activity. Reciprocally, in ind mutant embryos, all row 3 and 7 intermediate column cells co-express ac and Dichaete. Thus, the ability of Dichaete to repress ac in the intermediate column requires ind activity (Zhao, 2002).

These loss of function analyses have identified Dichaete as a regulator of DV pattern and cell fate in the neuroectoderm. To place Dichaete within the known genetic regulatory hierarchy that governs DV pattern in the neuroectoderm, systematic molecular epistasis tests were performed for Dichaete, ind, vnd and Egfr. Initially, vnd and ind expression, as well as Egfr activity was assayed in Dichaete mutant embryos. Dichaete mutant embryos exhibit no obvious defects to the expression of vnd or ind, or the activity of Egfr. Thus, Egfr, vnd and ind function upstream or in parallel to Dichaete (Zhao, 2002).

To investigate whether Egfr, vnd or ind regulate Dichaete, Dichaete expression was assayed in embryos mutant for each gene. No alterations were observed to the initial pattern of Dichaete expression in vnd or ind mutants, or in embryos doubly mutant for vnd and ind. Dichaete expression remains normal in ind mutant embryos throughout embryogenesis. However, by stage 11 in vnd and vnd; ind mutant embryos, Dichaete expression narrows inappropriately to an irregularly patterned stripe two-to-four cells wide immediately adjacent to the ventral midline. These results show that Dichaete is regulated independently of ind and is activated independently of vnd, but that vnd helps maintain Dichaete expression in the neuroectoderm (Zhao, 2002).

In contrast to vnd and ind, the initial pattern of Dichaete in Egfr mutant embryos is greatly reduced in the intermediate column and moderately reduced in the medial column during early neurogenesis. By stage 11, Dichaete expression narrows inappropriately to a thin and irregular stripe zero-to-three cells wide immediately adjacent to the ventral midline; Dichaete expression in the ventral midline is normal. These data identify Egfr as a key positive regulator of Dichaete in the neuroectoderm, and indicate that at least one other gene acts with Egfr to activate Dichaete expression in the medial column (Zhao, 2002).

To investigate whether vnd acts with Egfr to promote Dichaete expression in the medial column, Dichaete expression was followed in vnd; Egfr mutant embryos. The initial pattern of Dichaete in these embryos is the same as that observed in Egfr mutant embryos. However, by stage 11, Dichaete expression is completely absent from the neuroectoderm, although Dichaete expression is normal in the ventral midline. These results indicate that vnd and Egfr collaborate to maintain Dichaete expression in the neuroectoderm (Zhao, 2002).

To determine if Egfr activity is sufficient to activate Dichaete expression, the GAL4/UAS system system was used to activate Egfr signaling throughout the early Drosophila embryo. Ubiquitous Egfr signaling activates Dichaete expression throughout the neuroectoderm but not in the dorsal ectoderm. Thus, Egfr is necessary and sufficient to activate Dichaete in the neuroectoderm. However, in the dorsal ectoderm, either factors exist that inhibit the ability of Egfr to activate Dichaete or this domain lacks co-factors required for Egfr to activate Dichaete. Molecular epistasis tests place Egfr upstream of Dichaete and indicate that vnd, ind and Dichaete function largely in parallel to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

The parallel genetic activities of Dichaete, vnd and ind, the co-expression of Dichaete with vnd and ind, and the similarity of the early Dichaete CNS phenotype to those of vnd and ind led to a test of whether Dichaete interacts genetically with vnd and ind. To ascertain whether Dichaete interacts with vnd, the double mutant vnd;Dichaete was made and the formation of medial column SIII NBs 4-1 and 6-1 was assayed. In Dichaete mutant embryos, NBs 4-1 and 6-1 formed in 69.1% and in 92.1% of hemisegments, respectively. In vnd mutant embryos it was found that NBs 4-1 and 6-1 formed in 39.3% and 35.5 of hemisegments, respectively. In vnd; Dichaete mutant embryos NBs 4-1 and 6-1 formed in 10.8% and 9.1% of hemisegments, respectively. The increased defects in NB formation in vnd; Dichaete mutant embryos relative to either single mutant confirms that Dichaete and vnd do not act in a linear pathway to regulate NB formation -- rather, they demonstrate that Dichaete and vnd function in parallel to control NB formation in the medial column (Zhao, 2002).

Defects in NB formation in vnd; Dichaete mutant embryos are more severe than would be expected if these genes functioned independently. For example, if two genes act independently to promote NB formation, then the frequency of NB formation in the double mutant would be the product of the individual probabilities that the indicated NB will form in each single mutant. Thus, if vnd and Dichaete function independently, it would be expected that NB 4-1 would form 27.2% of the time (0.393 x 0.691=0.272) and NB 6-1 to form 32.7% of the time (0.355 x 0.921=0.327) in vnd; Dichaete mutant embryos. However, NBs 4-1 and 6-1 form ~10% of the time in vnd; Dichaete mutant embryos -- roughly threefold more severe than predicted for independently acting genes. These results reveal a genetic interaction between Dichaete and vnd. Furthermore, these results are interpreted to suggest that the activities of vnd and Dichaete are more convergent than parallel with respect to NB formation (Zhao, 2002).

Next, genetic interactions between Dichaete and ind were tested. The partial derepression of ac expression and the incomplete loss of an Eve-positive RP2 neuron are the most sensitive assays for Dichaete function in the intermediate column. However, strong alleles of ind cause a complete derepression of ac expression, and a complete loss of RP2 neurons in this domain. Thus, an analysis of Dichaete ind double mutant embryos using these markers would be uninformative. To circumvent this problem, a test was performed to see whether ind dominantly enhances the Dichaete intermediate column ac and RP2 phenotypes. Embryos heterozygous for ind exhibit wild-type ac expression and RP2 formation. However, Dichaete ind/Dichaete + mutant embryos exhibit enhanced derepression of ac expression and an approximately threefold enhancement of the RP2 loss phenotype relative to Dichaete mutant embryos. The dominant enhancement of the Dichaete phenotype by ind reveals a genetic interaction between Dichaete and ind (Zhao, 2002).

Initial interest in Dichaete arose from the observation that vnd; Egfr mutant embryos exhibit a more severe neuroectodermal phenotype than vnd; ind mutant embryos. This suggests that at least one other gene acts downstream of Egfr, and in parallel to vnd and ind to pattern the early neuroectoderm: this led to the analysis of Dichaete. To determine if the continued function of Dichaete in vnd; ind mutant embryos can explain the phenotypic differences between vnd; ind and vnd; Egfr mutant embryos, msh expression was followed in vnd;Dichaete;ind triple mutant embryos. In this background, a complete and uniform derepression of msh expression was observed throughout the neuroectoderm. The msh phenotype of vnd; Dichaete; ind embryos is essentially identical to that of vnd; Egfr embryos, and more severe than that of vnd; ind embryos. Thus, with respect to msh expression the difference between the vnd; ind and vnd; Egfr mutant phenotypes appears to result from the persistent function of Dichaete in vnd; ind mutant embryos (Zhao, 2002).

The results in this paper indicate that Dichaete is a key regulator of DV pattern in the neuroectoderm. Dichaete is expressed in the medial and intermediate columns and regulates cell fate and NB formation in these domains. Within the neuroectoderm, Dichaete acts downstream of Egfr and in parallel to vnd and ind. Together with biochemical research on Sox-domain-containing genes in vertebrates this work supports a model in which Dichaete protein physically associates with Vnd and Ind to regulate target gene expression and NB formation in distinct neuroectodermal columns (Zhao, 2002).

Interest in Dichaete arose owing to the observation that removal of vnd and Egfr function caused a stronger derepression of msh expression in the neuroectoderm than removal of vnd and ind function. These results contrast slightly with previous research that did not identify a phenotypic difference between vnd; ind and vnd; Egfr mutant embryos. This work analyzed msh expression in the neuroectoderm at a later stage (late stage 9) than the current work. At late stage 9, identical alterations to msh expression were also observed in vnd; ind mutant embryos relative to vnd; Egfr mutant embryos. However, the msh expression pattern is dynamic -- rapidly changing from uniform expression in the lateral column during stage 8 to a segmentally modulated pattern of cell clusters located within the lateral half of the neuroectoderm by stage 10. The differences in these observations are attributed to the different stages used to assay the effects of vnd, ind and Egfr on neuroectodermal development in the two studies (Zhao, 2002 and references therein).

How might Dichaete exhibit region specific effects on putative target genes? Work from vertebrate systems suggests that individual Sox-domain-containing proteins exhibit a widespread ability to partner with different transcription factors. Thus, Dichaete protein could exhibit column-specific functions via its association with different transcription factors in different domains. The formation of distinct protein complexes containing Fish could alter the output of Fish activity in at least two ways. Different protein complexes that contain Fish could exhibit different effects on transcription: repression versus activation. Alternatively, different Fish-containing protein complexes could exhibit distinct DNA-binding properties and therefore bind distinct recognition sites. These two possibilities are not mutually exclusive, and different Fish-containing protein complexes may both bind different recognition sites and exert different transcriptional effects on target genes (Zhao, 2002).

Examples of both forms of regulation are known. In the early Drosophila embryo, the transcription factor Dorsal activates one set of target genes ventrally and represses a distinct set dorsally. On its own, Dorsal functions as a transcriptional activator. However, in the dorsal region of the embryo, the interaction of Dorsal with a co-factor that binds to adjacent sites on target promoters converts Dorsal to a repressor. Although less well-defined mechanistically, the vertebrate Sox2 protein appears capable of activating or repressing target gene expression depending on cell-type and the target promoter (Botquin, 1998). In addition, work on vertebrate Sox domain proteins indicates that the composition of Sox-protein containing complexes modulates the DNA-binding specificity of these complexes. For example, in lens cells, Sox2 interacts with the DNA-binding factor deltaEF3 and binds to a bipartite recognition site on the delta-crystallin enhancer (Kamachi, 1998; Kamachi, 1999). In embryonic stem cells, Sox2 interacts with Oct3/4 and binds to a different recognition site in the Fgf4 minimal enhancer (Ambrosetti, 1997). In both enhancers, Sox2 binds to the same individual sequence. However, the specificity for the entire recognition site in one enhancer over the other arises as a consequence of the interaction of Sox2 with different transcription factors in different cell types and the distinct DNA-binding preferences of the entire complex (Zhao, 2002).

Based on these data, Dichaete is expected to associate with different transcription factors in the medial and intermediate columns to carry out its column-specific effects on target genes. The results in this paper identify Vnd and Ind as excellent candidates to be column-specific factors that associate with Dichaete and enable Dichaete to regulate transcription in a region specific manner: (1) Dichaete is co-expressed with Vnd in the medial column and Ind in the intermediate column; (2) the neuroectodermal Dichaete mutant phenotype is similar to those of vnd and ind; (3) Dichaete functions in parallel to vnd and ind in the neuroectoderm; (4) Dichaete exhibits dose-sensitive interactions with ind and genetic interactions with vnd, consistent with these proteins interacting physically. Based on these data, it is speculated that physical interactions between Dichaete and Vnd in the medial column and Dichaete and Ind in the intermediate column mediate the ability of distinct Dichaete protein complexes to bind to and either activate or repress distinct target genes. Validation of this model awaits the determination of whether Dichaete associates with Vnd or Ind, and how these proteins regulate target gene activity. However, recent results provide precedence for the model since genetic interactions between Dichaete, single-minded and drifter during midline development in the Drosophila CNS have led to experiments that show Dichaete physically associates with the Single-minded and Drifter proteins (Zhao, 2002).

These results place Dichaete within the known genetic regulatory hierarchy that controls pattern and cell fate along the DV extent of the neuroectoderm. In the future, it is expected that many additional genes will be joined into this pathway. For example, the Sox-domain-containing gene sox-neuro is expressed throughout the entire neuroectoderm and it may exhibit region-specific effects in the neuroectoderm in a manner similar to that proposed for Dichaete. In addition, the Ras-pathway antagonist yan is expressed in the lateral half of the neuroectoderm during early neurogenesis and may help regulate pattern and cell fate in this domain. A complete understanding of the genetic and molecular mechanisms that pattern the neuroectoderm requires the identification of all such genes and the elucidation of how these genes interact to regulate cell fate along the DV axis of the neuroectoderm (Zhao, 2002).

ventral veins lacking is required for specification of the tritocerebrum in embryonic brain development of Drosophila

The homeotic or Hox genes encode a network of conserved transcription factors which provide axial positional information and control segment morphology in development and evolution. During embryonic brain development of Drosophila, the Hox gene labial (lab) is essential for tritocerebral neuromere specification; lab loss of function results in tritocerebral cells that fail to adopt a neuronal identity, causing axonal pathfinding defects. Evidence is presented that the POU-homeodomain DNA-binding protein ventral veins lacking (vvl) acts genetically downstream of lab in the specification of the tritocerebral neuromere. In the embryonic brain, vvl expression is seen in all brain neuromeres, including the tritocerebral lab domain. Lab mutant analysis shows that vvl expression in the tritocerebrum is dependent on lab activity. Loss-of-function analysis focussed on the tritocerebrum reveals that inactivation of vvl results in patterning defects which are comparable to the brain phenotype caused by null mutation of lab. In the absence of vvl, mutant tritocerebral cells are generated and positioned correctly, but these cells fail to express neuronal markers indicating defects in neuronal differentiation. Moreover, longitudinal axon pathways in the tritocerebrum are severely reduced or absent and the tritocerebral commissure is missing in the vvl mutant brain. Genetic rescue experiments show that vvl is able to partially replace lab in the specification of the tritocerebral neuromere. These results indicate that vvl acts downstream of the Hox gene lab and regulates specific aspects of neuronal differentiation within the tritocerebral neuromere during embryonic brain development of Drosophila (Meier, 2006).

vvl is expressed in the embryonic brain from the extended germ band stage onwards. For an analysis of the protein distribution pattern of vvl in the embryonic brain, immunocytochemical experiments with a polyclonal antibody against the Vvl protein were carried out in combination with an anti-HRP antibody; anti-HRP immunoreactivity reveals the entire neural lineage of the developing CNS excluding the glial lineage. At late stage 11, vvl expression is first detected in few neuroblasts of the developing brain anlage, and by stage 13 became abundant in neuroblasts and their progeny within each brain neuromere. By stage 15, when neural progeny are generated and axonal projections are formed, Vvl protein is observed in specific cell clusters within all brain neuromeres (Meier, 2006).

Expression of vvl in the tritocerebrum suggests a possible overlap with the expression of the Hox gene lab. To investigate this, double-immunocytochemical experiments were carried out either on transgenic flies expressing a lab-lacZ reporter construct in which antibodies against Vvl were used together with anti-βgal antibodies, or on wildtype embryos in which antibodies against Vvl were used together with anti-Lab antibodies. These experiments revealed that the majority of cells expressing vvl in the tritocerebrum are located within the lab expression domain. Together with the observation that vvl appears to be differentially regulated by lab, the co-expression of lab and vvl in the tritocerebrum suggests that vvl activity might be lab-dependent in this neuromere. To study this further, whether mutational inactivation of lab affects vvl expression in the tritocerebrum was investigated (Meier, 2006).

Mutational inactivation of lab results in regionalized axonal patterning defects which are due to both cell-autonomous and cell-nonautonomous effects. Thus, in the absence of lab, mutant cells are generated and positioned correctly in the brain, but these cells do not extend axons. Additionally, extending axons of neighboring wildtype neurons stop at the mutant domains or project ectopically, resulting in the disruption of the longitudinal connectives and a lack of the tritocerebral commissure. To characterize vvl expression in a lab-/- background, double-immunocytochemical experiments were carried out on homozygous lab null mutant embryos using antibodies against Vvl and HRP. These experiments revealed that vvl immunoreactivity is lacking in the tritocerebral lab mutant domain, in addition to the expected lack of anti-HRP immunoreactivity despite the persistence of cells in this region. This suggests that vvl expression in the posterior tritocerebrum is affected by loss of lab function during late stages of embryonic brain development, indicating that vvl expression in the tritocerebrum is lab-dependent (Meier, 2006).

To assess the functional role of vvl in tritocerebral neuromere formation, vvl null mutants were analyzed using immunocytochemical markers including anti-HRP, anti-ELAV, anti-REPO, and anti FASII, which label general neuronal (or glial) domains and tracts in the developing embryonic brain. In vvl loss-of-function mutants, a pronounced brain phenotype is observed in the late stage embryonic brain. Immunolabelling with neuron-specific anti-HRP and anti-FASII antibodies identified a gap separating the deutocerebral brain region from the neuromeres of the more posterior subesophageal ganglion. This dramatic phenotype is associated with severe axonal patterning defects in the embryonic brain. The longitudinal connectives that normally run from the deutocerebral and tritocerebral neuromeres to the subesophageal ganglion are severely reduced or missing and the tritocerebral commissure, which interconnects the brain hemispheres at the level of the tritocerebrum, is completely absent. Analysis of FASII immunoreactivity revealed that descending and ascending axons, which in the wildtype normally project through the tritocerebrum in well formed fascicles, fail to project through this domain in vvl mutants. Moreover, a loss of anti-ELAV immunolabelling was observed in the tritocerebral domain, whereas glia-specific anti-REPO immunoreactivity revealed that glial cells are present in the vvl mutant but fail to be correctly localized in the affected region. In addition to the observed defects in the tritocerebral brain region, marked axonal patterning defects in the protocerebrum are also seen in vvl mutant embryos. Moreover the organization of the subesophageal ganglion and the VNC is affected in the vvl mutant (Meier, 2006).

At the gross histological level, the vvl mutant brain phenotype is, in part, reminiscent of the mutant brain phenotype observed for lab. Since lab and vvl show overlapping expression in the tritocerebral neuromere, and vvl expression is lacking in lab mutants, these findings suggested that either lab expression itself or lab expressing tritocerebral cells are affected in vvl mutant embryos. To investigate this anti-Lab immunolabelling was carried out in late vvl loss-of-function mutant brains. Surprisingly, despite the expected lack of expression of neuronal differentiation markers, anti-LAB immunolabelling was detected in a wildtype-like pattern in the vvl mutant tritocerebral domain. This suggests that the expression of lab os not affected in the absence of vvl during late stages of embryonic brain development. Moreover, the lab expressing cells in the vvl mutant generally have the same relative position in the brain as does the normal lab expressing cells in the wildtype. Thus, despite the severe axonal patterning defects observed in this domain, mutant cells are generated and appear to be properly positioned in the developing tritocerebrum of the vvl null mutant. This, in turn, suggests that the pattern of proliferation in the tritocerebrum is initiated correctly in the absence of the vvl gene product, but that the cells that normally express vvl might become incorrectly specified in the vvl mutant leading to axogenesis defects. Moreover, the lack of anti-ELAV and anti-HRP immunolabelling together with the observed severe fasciculation defects in the vvl mutant tritocerebrum strongly suggest that mutational inactivation of vvl affects neuronal differentiation in the developing tritocerebrum (Meier, 2006).

These data imply that vvl might be involved in the specification of tritocerebral neuronal identity—either by acting directly or indirectly downstream of tritocerebral lab activity. To further assess a possible lab-dependent vvl activity in the developing tritocerebrum, the potential of vvl to rescue the lab mutant brain phenotype was determined using the Gal4-UAS system. For this, a transgenic fly line carrying a Gal4 transcriptional activator under the control of the lab promoter together with CNS-specific upstream enhancer elements of the lab gene was used. By crossing this lab::Gal4 line to different UAS-responders it was possible to express the responder constructs in a pattern that corresponded to that of the endogenous lab gene. Using this approach, it was shown that the lab mutant brain phenotype can be rescued by transgenic expression of the Lab protein in a lab null mutant background. To determine whether vvl might also be able, at least in part, to rescue the lab mutant brain defects, a transgenic UAS::vvl line was used in which the vvl coding sequence was placed under UAS control (Meier, 2006).

As a control, it was first determined whether lab::Gal4 driven misexpression of vvl in a lab+background has any effects on the development and specification of the tritocerebral Lab domain. In none of these experiments were morphological abnormalities detected in the tritocerebrum or in any other part of the embryonic brain. The UAS::vvl responder was then expressed under the control of the lab::Gal4 driver in the lab mutant domain. Remarkably, the Vvl protein is able to rescue specific lab mutant brain defects. Thus, the longitudinal pathways are restored and cells in the mutant domain show wildtype-like anti-HRP immunolabelling. Moreover, FASII immunoreactivity revealed that descending and ascending axons from other parts of the brain again project through the tritocerebral lab mutant domain. The vvl responder achieved a rescue efficiency (97.3%) which is comparable to the rescue efficiency of Lab, which was taken as 100%. In contrast, lab::Gal4-specific expression of vvl in the lab mutant domain does not rescue tritocerebral commissure formation, nor correct axonal projection of the frontal connective. This suggests that lab::Gal4 driven misexpression of vvl is sufficient to restore both neuronal marker gene expression like HRP and correct axonal patterning of longitudinal connectives in the lab mutant tritocerebrum. These findings together with the vvl mutant brain phenotype indicate that vvl acts genetically downstream of lab in the specification of the triocerebral neuromere (Meier, 2006).

Taken together, these findings demonstrate that vvl function is required for the specification of the developing tritocerebrum. The vvl gene is important for correct axon guidance and fasciculation of longitudinal connectives in the tritocerebral neuromere. In the absence of vvl, longitudinal and commissural axon pathways are severely affected. Comparable findings have been reported for the role of vvl in VNC development, where vvl mutant embryos exhibit aberrantly localized midline glia and axonal defects in that commissures are often fused and the longitudinal connectives are severly reduced or even disrupted. These findings suggest that vvl acts genetically downstream of the Hox gene lab in the control of regionalized neuronal identity and tritocerebral brain neuromere specification. In accordance with this notion is the successive timing of lab and vvl expression in the tritocerebral neuromere. From stage nine onwards, lab expression commences in the intercalary segment and by early stage 11, lab is detected in all neuroblasts of the developing tritocerebrum. Accordingly, by late stage 11, vvl expression is first seen in the tritocerebral neuromere and thus succeeds initial lab expression. Interestingly, this time of initial vvl expression exactly coincides with the temporal requirement of lab for tritocerebral neuronal fate specification. Moreover, the results demonstrate that tritocerebral vvl expression is lab-dependent. In addition, in vvl mutants, lab is expressed normally in the tritocerebrum and yet cells in the affected tritocerebral domain phenocopy the lab mutant brain and do not express molecular markers characteristic of neuronal cells. Furthermore, the vvl gene can mediate neuronal specification and longitudinal connective formation in the absence of the Hox gene lab if expressed under appropriate spatiotemporal control. This indicates that vvl is required for the specification of neuronal identity in the tritocerebral lab domain and sufficient to provide a permissive substrate for the migration of axons originating from outside this region. However, vvl cannot rescue tritocerebral commissure formation in the lab mutant brain. This indicates that lab exerts at least some of its effects on tritocerebral development through other subordinate genes than vvl (Meier, 2006).

The columnar gene vnd is required for tritocerebral neuromere formation during embryonic brain development of Drosophila

In Drosophila, evolutionarily conserved transcription factors are required for the specification of neural lineages along the anteroposterior and dorsoventral axes, such as Hox genes for anteroposterior and columnar genes for dorsoventral patterning. This report analyses the role of the columnar patterning gene ventral nervous system defective (vnd) in embryonic brain development. Expression of vnd is observed in specific subsets of cells in all brain neuromeres. Loss-of-function analysis focussed on the tritocerebrum shows that inactivation of vnd results in regionalized axonal patterning defects, which are comparable with the brain phenotype caused by mutation of the Hox gene labial (lab). However, in contrast to lab activity in specifying tritocerebral neuronal identity, vnd is required for the formation and specification of tritocerebral neural lineages. Thus, in early vnd mutant embryos, the Tv1-Tv5 neuroblasts, which normally express lab, do not form. Later in embryogenesis, vnd mutants show an extensive loss of lab-expressing cells because of increased apoptotic activity, resulting in a gap-like brain phenotype that is characterized by an almost complete absence of the tritocerebral neuromere. Correspondingly, genetic block of apoptosis in vnd mutant embryos partially restores tritocerebral cells as well as axon tracts. Taken together, these results indicate that vnd is required for the genesis and proper identity specification of tritocerebral neural lineages during embryonic brain development of Drosophila (Sprecher, 2006; full text of article).

During an initial phase of embryonic neurogenesis, vnd expression is seen in the neurectoderm and delaminating neuroblasts in the ventral domains of the protocerebral, deutcerebral and tritocerebral brain neuromeres. During later stages of embryogenesis, vnd expression is also seen in specific cell clusters within these brain neuromeres. Thus, at late stage 12, a large expression domain is seen in the protocerebral neuromere and two smaller expression domains are observed in the deutocerebrum and in the tritocerebrum. Although the tritocerebral and deuterocerebral vnd expression clusters are in close proximity to each other, they do not overlap. Towards the end of embryogenesis, at embryonic stage 15, expression of vnd is still visible in these three neuromeric domains. Throughout embryonic neurogenesis, vnd expression is found in neuroblasts, ganglion mother cells and neurons as judged by immunolabelling with anti-PROS and neuron-specific anti-ELAV antibodies. Immunolabelling with glia-specific anti-REPO antibody indicates that none of the glia cells of the embryonic brain express vnd. vnd-expressing cells are also seen in the neuromeres of the s ganglion and the VNC, as well as in peripheral sense organs (Sprecher, 2006).

Mutational inactivation of vnd results in a pronounced brain phenotype in the late stage embryonic brain. Immunolabelling with neuron-specific anti-HRP and anti-ELAV antibodies identifies a large gap separating the anterior deutocerebral brain region from the neuromeres of the posterior s ganglion. Evaluation of the penetrance of the vnd-null mutant phenotype reveals that in 36% of the mutant embryos this gap is completely devoid of Elav- and HRP-immunoreactive cells, while in the majority of vnd mutant embryos (63%) a thin strand of ELAV- and HRP-immunoreactive cells remains and interconnects the protocerebrum and the s ganglion. This cell loss is associated with axonal patterning defects in the embryonic brain. The longitudinal connectives that normally run from the protocerebrum to the s ganglion are missing or strongly reduced and the tritocerebral commissure is completely absent. Glia-specific anti-REPO immunoreactivity reveals that glial cells are present in the mutant but fail to be correctly localized in the affected region, most probably owing to the absence of neuronal tissue (Sprecher, 2006).

To delineate the region affected in vnd mutants in more detail, the expression of engrailed (en), which in the wild-type embryonic brain is located in several small clusters of cells that demarcate the posterior boundary of the brain neuromeres, was studied. The b1 en-stripe (or en head spot) delimits the posterior protocerebrum (several en cells are also seen more anteriorly in the protocerebrum as the secondary head spot), the b2 en-stripe (or en antennal stripe) delimits the posterior deutocerebrum, and the b3 en-stripe (or en intercalary stripe) delimits the posterior tritocerebrum. In late vnd mutant brain (embryonic stage 13 onwards), only the b1 en-stripe and the secondary head spot are visible; neither the b2 en-stripe nor the b3 en-stripe can be identified. This supports the observation that major parts of the embryonic tritocerebrum and parts of the deutocerebrum are lacking in the vnd mutant. In addition to the cell loss defect in the tritocerebral/deutocerebral brain region, a less marked reduction in overall size of the protocerebrum is also seen in vnd mutant embryos. Moreover the organization of the s ganglion and the VNC is affected in the vnd mutant. These latter two phenomena were not studied further (Sprecher, 2006).

At the gross histological level, the vnd mutant brain phenotype described above is, in part, reminiscent of the mutant brain phenotype observed for the Hox gene labial (lab). In lab-null mutants, tritocerebral cells are generated and positioned correctly; however, these cells fail to differentiate into neurons and marked axogenesis defects occur, including the disruption of longitudinal connectives and lack of the tritocerebral commissure. As lab and vnd also show overlapping expression in a subset of tritocerebral neuroblasts, these findings suggest that lab-expressing tritocerebral neuroblasts are affected in vnd mutant embryos. To investigate this, focus was placed on the developing tritocerebrum, and specifically on the lab expression domain of this neuromere, and whether loss of vnd function affects formation of lab-expressing neuroblasts was determined (Sprecher, 2006).

During the early phase of brain neurogenesis, the lab-expressing neuroectodermal domain gives rise to 15 neuroblasts, which include all of the tritocerebral neuroblasts and two deutocerebral neuroblasts. By stage 11, all of these neuroblasts are present and express lab; they include a ventral group of tritocerebral neuroblasts, Tv1-Tv5, a more dorsal group of tritocerebral neuroblasts, Td1-Td8, and two deutocerebral neuroblasts, Dv2 and Dv4. In the wild type, the most ventral part of the neuroectodermal domain, from which the tritocerebral neuroblasts Tv1-Tv5 and the two deutocerebral neuroblasts originate, dynamically co-expresses lab and vnd between stages 8 and 11 (Sprecher, 2006).

In vnd mutants this ventral-most part of the lab-expressing domain appears to be reduced in size and accordingly, the number of lab-expressing neuroblasts that derive from this brain region is diminished. Generally only four to six large rounded cells are observed that co-express lab and the neuroblast-specific marker Deadpan (this may be a slight underestimate as a few enlarged rounded cells in sub-ectodermal position lacking Deadpan expression are sometimes observed in this region). Based on the expression of molecular markers indicative of dorsal neuroblasts [e.g. ladybird early, empty spiracles, wingless, this reduction in lab-expressing neuroblasts appears to affect preferentially ventral neuroblasts of the tritocerebrum and adjacent part of the deutocerebrum. These data imply that vnd is required for the formation of a ventral subset of lab-expressing neuroblasts in the developing tritocerebrum (Sprecher, 2006).

Although the reduction in tritocerebral neuroblast number seen in vnd mutants can account for some of the tritocerebral defects, this mechanism alone is unlikely to be the exclusive cause for the massive cell loss phenotype observed in the late embryonic vnd mutant brain. This is because a dorsal subset of the lab-expressing tritocerebral neuroblasts, as well as large number of lab-expressing neural progeny are generated in the tritocerebrum of stage 11 vnd mutant brains. Hence, in addition to defective neuroblast formation, other phenomena must be responsible for the gap-like phenotype observed in vnd mutant brains, implying that vnd is required also later in embryogenesis - either by acting directly on lab expression in tritocerebral cells or through a lab-independent requirement (Sprecher, 2006).

To investigate this, whether vnd and lab show overlapping expression during later stages of tritocerebral neuromere formation was determined. Immunocytochemical analysis indicates that a partial overlap of vnd and lab expression persists in the differentiating tritocerebrum throughout embryogenesis and is prominent in the ventral region (according to neuraxis) of this neuromere. lab expression was analyzed in late vnd loss-of-function mutant brains. Owing to extensive cell loss in the vnd mutant tritocerebrum, this analysis was limited to the remaining strand of cells that interconnects the protocerebrum and the remaining part of the deutocerebrum with the s ganglion. Despite the extensive cell loss seen in vnd mutant brains, remaining cells of the interconnecting strand do show lab expression (Sprecher, 2006).

Whether expression of vnd occurs in the lab mutant tritocerebrum was investigated by studying lab loss-of-function mutants. For this, advantage was taken of the fact that in lab-null mutants, cells in the tritocerebral mutant domain are generated and can be visualized by a 7.31 lab-lacZ reporter construct. Surprisingly, despite the lack of expression of neuronal differentiation markers in cells of the lab mutant domain, vnd is expressed normally and shows partial overlap with tritocerebral lab mutant cells, as visualized by the lab-specific reporter construct. This indicates that expression of vnd is not affected by the absence of lab during late stages of embryonic brain development (Sprecher, 2006).

These results indicate that the homeotic gene lab, which is part of the anteroposterior patterning system, and the columnar gene vnd, which is involved in dorsoventral patterning, act in an integrated manner but independently in the formation and specification of the tritocerebral neuromere. Although vnd and lab show overlapping expression in tritocerebral neuroblasts and subsequently in neural cells of the posterior tritocerebrum, expression of vnd appears unaffected in lab mutant cells. Conversely, vnd does not act on lab expression; the complete absence of lab expression in vnd mutants (with the exception of a rare thin strand of neuronal cells) reflects a secondary defect because of the absence of cells that normally express lab. This independent genetic activity of vnd and lab is further supported by the fact that blocking apoptosis restores tritocerebral lab expression in vnd-null mutant embryos (Sprecher, 2006).

Thus, although the lab and vnd mutant brain phenotypes result in comparable axonal patterning defects (loss of the tritocerebral commissure and perturbation of the longitudinal connectives that normally run through this neuromere), their mode of action within the developing tritocerebrum is discriminable. The results suggest that vnd is required for the specification of neural lineages within the developing tritocerebral neuromere, whereas the Hox gene lab appears to be independently required for the specification of neuronal identity within the same territory during later stages. This indicates that the activity of the columnar gene vnd is integrated into pattern formation along the anteroposterior neuraxis by ensuring proper formation and development of tritocerebral neural lineages that subsequently become further specified by the activity of the Hox gene lab (Sprecher, 2006).

The Drosophila columnar gene vnd belongs to the highly conserved Nkx2 class of transcription factors that have been found in various animals, including mammals. Notably, the vnd/Nkx2 family of genes is exceptionally well conserved, both in terms of expression and function. Thus, the vertebrate homologues of vnd are expressed in the neural plate, or tube, in topologically similar positions as is vnd in the Drosophila ventral neuroectoderm and in the absence of vnd/Nkx2 genes, ventral-most cells in the spinal cord and the Drosophila VNC are missing or transformed. Moreover, this evolutionary conservation in expression and function of vnd/Nkx2 genes appears to apply to some extent to brain development. A comparison of the anteroposterior order of vnd/Nkx2 gene expression in the early embryonic brains of Drosophila and mouse reveals remarkable similarities. In terms of function, genetic knockouts in mice have shown that Nkx2 genes appear to play a crucial role in patterning and neuronal specification during embryonic development of the telencephalon and hindbrain. Nkx2.1 mutant mice display numerous brain patterning defects: the entire pituitary is missing; the number of cortical interneurons is halved; there is a complete absence of TrkA-expressing cells in the developing telencephalon; and the ventral-most aspect of the telencephalon (the medial ganglionic eminence) becomes trans-fated to that of the adjacent more dorsolateral ganglionic eminence. Thus, comparable with the role of vnd during Drosophila brain development, Nkx2.1 is involved in pattern formation and in cell fate determination during embryonic brain development in mice (Sprecher, 2006).

In addition, recent studies have shown that Nkx2.2 is involved in neural lineage specification in the developing hindbrain. In particular, the sequential generation of visceral motoneurons and serotonergic neurons from a common pool of neural progenitors located in the ventral hindbrain crucially depend on the integrated activities of Nkx2.2- and Hox1/2-class homeodomain proteins. An important function of these proteins is to coordinate the spatial and temporal activation of the homeodomain protein Phox2b, which in turn acts as a binary switch in the selection of motor neuron or serotonergic neuronal fate. De-repressive activity of Nkx2.2 at or in vicinity of Pbx/Hox-binding sites proximal to the Phox2b enhancer enhances transcriptional activation of Phox2b by Hox1 and Pbx factors. These data suggest that comparable with the integrated activity of vnd and lab in Drosophila brain neuromere specification, integrated activity of the Nkx2.2 and Hox1/2 proteins is involved in the specification of segmental neural lineages. Thus, integration of anteroposterior and dorsoventral patterning systems by homeodomain transcription factors of the Hox and vnd/Nkx2 genes might represent an ancestral feature of insect and mammalian brain development (Sprecher, 2006).

Intrinsic dorsoventral patterning and extrinsic EGFR signaling genes control glial cell development in the Drosophila nervous system

Dorsoventral patterning and EGFR signaling genes are essential for determining neural identity and differentiation of the Drosophila nervous system. Their role in glial cell development in the Drosophila nervous system is not clearly established. This study demonstrates that the dorsoventral patterning genes, vnd, ind, and msh, are intrinsically essential for the proper expression of a master glial cell regulator, gcm, and a differentiation gene, repo, in the lateral glia. In addition, it was shown that esg is particularly required for their expression in the peripheral glia. These results indicate that the dorsoventral patterning and EGFR signaling genes are essential for identity determination and differentiation of the lateral glia by regulating proper expression of gcm and repo in the lateral glia from the early glial development. In contrast, overexpression of vnd, msh, spi, and Egfr genes repress the expression of Repo in the ventral neuroectoderm, indicating that maintenance of correct columnar identity along the dorsoventral axis by proper expression of these genes is essential for restrictive formation of glial precursor cells in the lateral neuroectoderm. Therefore, the dorsoventral patterning and EGFR signaling genes play essential roles in correct identity determination and differentiation of lateral glia in the Drosophila nervous system (Kim, 2015).

This study demonstrates that the DV patterning genes, ind, msh, and esg, are required for expression of the glial cell identity marker, gcm, and of the glial cell differentiation marker, Repo, in the proper region of the LTG in the Drosophila VNE. msh and esg acts locally in the formation and differentiation of the LG from the lateral column of the VNE, and esg strongly influences the formation and differentiation of the PG. ind is also locally involved in the initial formation and differentiation of the SG from the VNE. Considering that DV patterning genes, such as ind and msh, are required for the identity determination and formation of NBs in the intermediate and lateral columns along the DV axis, it is plausible that these two genes play essential roles in the proper development of the LTG in the corresponding columns. Interestingly, the zinc finger transcription factor, Esg, plays an important role in the formation and differentiation of the PG that originate from the lateral column, where esg is expressed. Although esg, together with snail and worniu, is required for the asymmetric division of NBs, the precise role of esg in embryonic CNS development has not been clearly determined. Thus, experimental results obtained in this study on esg's role in glial cell formation and differentiation is the first of its kind to analyze the role of esg in gliogenesis during embryonic CNS development (Kim, 2015).

Unexpectedly, vnd, which is essential for identity determination of the medial column NBs, showed the strongest influence on the proper formation and differentiation of all glia, including the LG, SG, and even PG in the VNE. Since the region of msh expression is ventrally expanded in the vnd mutant, disruption of the expression of gcm and Repo in the lateral column may have caused a decrease in the number of LG, LTG, and PG that originate from this region. In addition, the overexpression of vnd also repressed the expressions of Repo and MAPK in the Kr domain, presumably by promoting identity determination of the medial column in the intermediate and lateral columns. Original reports on the role of the vnd in formation and identity determination of the medial column NBs using the vnd target gene, NK6, showed that intermediate and lateral column identity markers are repressed by overexpression of vnd in the Kr-expression domain. One of the reasons for the wider influence of vnd in DV patterning than other DV patterning genes may be that vnd is expressed earliest among these genes, repressing expression of other DV patterning genes such as ind and msh in the medial column, in a process termed 'ventral dominance' (Kim, 2015).

The data revealed that the EGFR signaling receptor and ligand, Egfr and spi, play more global roles in glial cell development than do the DV patterning genes. Egfr and spi are required for initial glial cell formation as shown by reduced expression of gcm and Repo in the LGBs of the VNE. In addition, Repo expression in the differentiated glia was markedly reduced, especially in Egfr embryos, and in spi embryos, to a lesser degree. Interestingly, Repo expression is almost absent in the SG and remains only in the LGs of spi as well as of ind embryos. Since ind expression is activated by the EGFR signaling ligand, Spi, in the VNE to establish the identity of the intermediate column, it is plausible that glial phenotypes in spi and ind mutants are similar to each other. This result indicated that once the intermediate column identity is determined by ind-mediated repression of msh expression in the lateral column, EGFR signaling provides a consolidating extrinsic cue to make ind a repressor of some of the target genes in the intermediate column via MAPK-mediated phosphorylation. This interpretation is compatible with the results obtained by overexpression of Spi and Vn through Kr- and sca-Gal4 drivers, which show repressed Repo expression in the VNE due to the repressor activity of Ind, which in turn is activated by EGFR signaling. Thus, the results indicated that EGFR signaling globally activates many types of glial cell lineages in the VNE and delimits the area where glial cells originate by repressor activity that is chemically modified by EGFR signal transduction (Kim, 2015).

Establishment of proper identity along the DV axis by expression of the DV patterning and EGFR signaling genes is essential for correct formation and differentiation of glia from the VNE This study revealed that the DV patterning and EGFR signaling genes play important roles in the initial formation and differentiation of various types of glia in the Drosophila CNS. The DV patterning genes and EGFR signaling genes are locally and globally required, respectively, for glial cell formation and differentiation using loss-of function mutants of the genes. Unexpectedly, overexpression of the DV patterning and EGFR signaling genes also repressed the initial formation and differentiation of glia. Overexpression of vnd showed stronger repressor activity than msh on the Repo expression in most types of glial cells including the LG, whereas msh showed mild reduction in the Repo expression mainly in the SG, but not in the LG. The repressor activity of vnd started from the initial formation of the LGBs and continued until the glial cells differentiated into mature glia (Kim, 2015).

There are several possible explanations for the repressive effect in both loss-of-function and gain-of function mutants. First, vnd and EGFR signaling genes together play important roles in establishing identities of the medial and intermediate columns in DV patterning of the VNE. Therefore, overexpression of these genes also promote identities of the medial and intermediate in the lateral columns, where many glial cells, including the LG, PG, and some of the SG originate after neurons are formed. This identity change may block glial cell formation and differentiation from the lateral neuroectoderm. Second, overexpression of these genes may also promote neurogenesis over gliogenesis during developmental stages when overexpression was driven by Kr- and sca-Gal4. In addition, repressor activity appears to play a more dominant role than activator activity upon overexpression of vnd, considering that the DV patterning genes, vnd, int, and msh, act as successive repressors to establish and maintain their identity in the VNE. The results obtained using the loss-of-function and overexpression mutants demonstrate that the expression of a proper level of the DV patterning genes promote identity determination of neurons, while their overexpression represses formation of the glia in the VNE by default. In addition, repressor activity of the DV patterning genes appears to play a dominant role in the establishment of the three columnar divisions along the DV axis (Kim, 2015).

Similarly, overexpression of the EGFR signaling ligands, Spi and Vn, and the activated form of EGFR signaling receptor, EgfrAC, repressed Repo expression in all types of glial cells in the VNE. This may be due to the repressor activity of int, since activation of EGFR signaling induces phosphorylation of int and vnd to consolidate their repressor activity. In addition, since Egfr overexpression can cause expansion of vnd expression from the medial column to the lateral area, the intermediate and lateral columns may have acquired the medial identity, such that the LG and various types of other glia originating from the VNE are not generated after overexpression of Spi in the VNE (Kim, 2015).

These studies on the glial cell development in the Drosophila VNE revealed that the DV patterning and EGFR signaling genes play prominent roles in promoting neural identity, rather than glial identity during the early stages of CNS development, since their overexpression did not activate glial identity, but rather repressed it. Later, expression of the glial master gene, gcm, is required to promote glial cell identity in the VNE. It appears that the two-step mode of CNS development first ensures generation of a neural circuit and then provides supporting glial cells in the CNS. The results indicated that the DV patterning genes act locally to promote glial cell formation in their expression domains, but EGFR signaling genes act broadly throughout the VNE. Among the DV patterning genes, vnd, appears to influence glial cell formation and differentiation globally, since it represses int and msh to establish and maintain medial identity from the earliest developmental stage. It remains to be investigated how the DV patterning and EGFR signaling genes control the spatial and temporal regulation of glial cell formation and how they interact to promote glial identity in the CNS (Kim, 2015).

ventral nervous system defective: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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