intermediate neuroblasts defective

DEVELOPMENTAL BIOLOGY

Embryonic

The temporal and spatial pattern of IND mRNA accumulation was determined using RNA blots and whole-mount in situ hybridization. A single 1.3-kb IND transcript appears at 2-4 hr of development with peak accumulation at 4-8 hr of development; no transcripts were detected later in embryogenesis. Whole-mount in situ hybridization first detects ind expression in two parallel, lateral columns in stage 5 cellular blastoderm embryos. At this stage each column is about five cells wide and runs from the procephalic region to the most posterior region of the embryo. During gastrulation, the ind expression domain narrows until each column is two cells wide. The position of each of the two symmetric ind columns is just dorsal to the domain of vnd transcription, in cells that will become the intermediate column of neuroectoderm. When neuroblast formation begins at late stage 8, IND mRNA is expressed in both the intermediate column neuroectoderm and in the S1 neuroblasts (Nbs) derived from the intermediate column (Nbs 3-2 and 5-3). By stage 9, IND mRNA is absent from the neuroectoderm, but is detectable in all of the neuroblasts in the intermediate column (Nbs 3-2, 4-2, 5-3, 6-2, and 7-2). ind is not expressed in any ventral or dorsal column neuroectoderm or neuroblasts. By stage 11, IND mRNA is detectable in just a single intermediate column neuroblast, Nb 6-2. After stage 11, IND mRNA was not detected anywhere in the embryo (Weiss, 1998).

Antibody staining confirms that Ind protein is restricted to the intermediate column neuroectoderm and neuroblasts. The Ind protein-containing cells are immediately adjacent but nonoverlapping with Vnd-containing cells of the ventral column neuroectoderm (McDonald, 1998). Double labeling with various neuroblast markers shows that Ind protein is detected in the same intermediate column neuroblasts as IND mRNA. In contrast to IND mRNA, Ind protein is detectable in all intermediate column neuroblasts at stage 13, after the mRNA is no longer detectable in most of these neuroblasts (Weiss, 1998).

ventral nervous system defective, muscle segment homeobox, and ind regulate dorsoventral patterning of the procephalic neuroectoderm. vnd, msh, and ind are each expressed in the procephalic ectoderm: Vnd in a ventral domain, Ind in three small clusters of cells at intermediate positions, and Msh in a dorsal domain. There are two differences in gene expression and regulation in the procephalic region compared with the thoracic and abdominal neuroectoderm: (1) Vnd and Msh share an extensive border, only interrupted by two small islands of Ind+ cells. In vnd embryos, Msh expands into the ventral domain of the procephalic neuroectoderm, showing that Vnd is required to repress msh expression in the head. Consistent with this result, misexpression of vnd leads to repression of msh. (2) The Ind+ anterior cell cluster 1 appears to coexpress Vnd; coexpression of Vnd and Ind is never observed in the thoracic and abdominal neuroectoderm. Surprisingly, vnd embryos show a loss of the Ind+ cluster 1, and misexpression of vnd does not affect Ind expression in cluster 1; thus, in this domain of the embryo, Vnd is required for the development of the Ind+ cluster 1. Because the Ind+ cells of cluster 1 are primarily restricted to neuroblasts, one possibility is that loss of vnd in the neuroectoderm leads to a failure of neuroblast formation and thus to a loss of Ind+ cells, rather than that Vnd directly activates ind transcription in this domain. The remaining two Ind+ cell clusters (2 and 3) are expressed and regulated in a manner consistent with the thoracic and abdominal neuroectoderm. Both Ind+ cell clusters 2 and 3 directly abut Vnd+ cells but do not express Vnd. In vnd embryos, the Ind+ cluster 3 expands ventrally into the domain normally expressing vnd, whereas Ind+ cluster 2 appears unaffected. Misexpression of vnd represses ind expression in clusters 2 and 3. Thus, vnd can both activate ind (cluster 1) or repress ind (clusters 2 and 3) depending on the position within the procephalic neuroectoderm (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).

Effects of Mutation or Deletion

In addition to regulating gene expression in the neuroectoderm, ind has an essential role in neuroblast formation and specification. In wild-type embryos, Hunchback staining reveals three columns of neuroblasts (ventral, intermediate, and dorsal). In ind mutant embryos, only 10% of the intermediate-column neuroblasts develop, although adjacent ventral and dorsal column neuroblasts form normally. The 10% of neuroblasts that develop in the intermediate column always express achaete, which is never detected in wild-type intermediate column neuroblasts (Weiss, 1998).

The infrequent neuroblasts that form in the intermediate column of ind mutant embryos were stained with anti-Msh and anti-Vnd antibodies to determine if there would be a transformation to either dorsal or ventral cell fates in these mutants. A total of 80 hemisegments were scored. In a wild-type embryo 400 intermediate neuroblasts would be expected. In the ind mutant embryo 32 were observed. Eleven expressed Vnd, 12 expressed Msh, and 9 expressed neither marker. Thus, ind is essential for the formation of intermediate column neuroblasts, and for the repression of ventral- and dorsal-specific genes within these neuroblasts (Weiss, 1998).

To test whether intermediate-column neuroblasts that form in ind mutant embryos have the potential to generate cell lineages characteristic of intermediate column neuroblasts, an assay was performed of the development of the Eve-containing RP2 neuron, which is a motor neuron derived from the intermediate-column neuroblast 4-2 . The pattern of Eve is a sensitive indicator for normal cell fates within neuroblast cell lineages. In wild-type embryos, a Eve-protein-containing RP2 is found in every hemisegment (100/100), whereas in ind mutant embryos the Eve-containing RP2 is never detected (0/96). This defect is caused in part by a failure in NB 4-2 formation, but is also likely to be caused by defects in NB 4-2 specification or cell division in the 10% of the NB 4-2s that appear to form. These data suggest that ind regulates intermediate column neuroblast-cell lineages (Weiss, 1998).

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 flb^IK35 (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).

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date revised: 20 December 2007

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