intermediate neuroblasts defective

Promoter

A maternally established gradient of nuclear Dorsal protein is the first step in subdivision of the Drosophila neurectoderm into stripes of homeodomain gene expression. Dorsal in combination with the EGF and TGFβ signaling pathways are key regulators of the expression of the genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) in the developing neurectoderm. These three genes encode homeodomain transcription factors that can repress each other, which ensures adjacent, non-overlapping expression domains. Expression of vnd, ind, and msh is maintained after decline in EGF and TGFβ signaling, but the relevant positive transcriptional regulators have not yet been defined. This study shows that Ind can bind DNA with the same sequence specificity (GC T/C A/C ATTA G/A) as its murine ortholog Gsh1. A novel upstream regulatory element was identified at the ind locus containing predicted Ind binding sites, and Ind activity was shown to be both necessary and sufficient for reporter gene expression from this element. It is concluded that Ind can act as a transcriptional activator, and that positive autoregulation of Ind is a mechanism for persistent ind expression within the developing embryonic nervous system (Van Ohlen, 2007a).

This study presents in vivo data suggesting that the Ind homeodomain protein can act as a transcriptional activator. Specifically, the data show that Ind activity is required to maintain ind expression and that this autoregulation takes place through a previously uncharacterized regulatory element located upstream of the ind coding sequence. It is entirely possible that an additional as yet unidentified positive regulator is required for the maintenance of Ind expression (Van Ohlen, 2007a).

These results suggest that Ind can act as a transcriptional activator. However, the possibility cannot be ruled out that loss of ind function causes derepression of a repressor. One possible repressor of ind could be Msh. It is thought that this is the case for three reasons. (1) It has been demonstrated that expression of Msh is expanded ventrally in ind mutant embryos. In ind mutant embryos Msh expression is expanded ventrally into the Ind domain at the time of initiation, around stage 6. However, ind mRNA in the RR108 mutant appears largely normal until stage seven and eight. (2) It has been found that Msh over-expression does not repress ind expression. It is possible there could be another as yet unidentified repressor. (3) Expression of ind does not expand dorsally in msh mutant embryos (Van Ohlen, 2007a).

Regulation of ind expression appears to involve to separable regulatory elements. The previously described element that is located downstream of the ind coding sequence is required for initiation. An additional element located upstream of the coding sequence appears to be dependent on Ind activity. A parallel regulation might also be possible for Vnd where the early neurectodermal enhancer is located within the first intron. However, elements controlling expression in neuroblasts are located upstream of the coding sequence. Moreover, Vnd can bind to the upstream element and regulate reporter gene expression from it. It should be noted, that these results are based on tissue culture reporter assays and not in vivo results. Nevertheless, these data do support the idea that similar to ind, vnd expression might be regulated by separable enhancer elements which control initiation and maintenance independently (Van Ohlen, 2007a).

The ability of Vnd to act as a transcriptional activator appears to be in part regulated by interaction with the HMG domain-containing protein Dichaete. Genetic data suggest that Ind and Vnd interact with Dichaete in a similar manner. Ind expression is normal in dichaete mutant embryos. Thus, it is hypothesized that expression of Ind is most likely initiated normally in dichaete mutants and despite the apparently normal expression of Ind protein in dichaete mutants an effect on expression might be seeb from the upstream regulatory element. However, following recombination of the GN4lacZ transgene onto the dichaete chromosome no loss of lacZ expression was seen. Therefore, it cannot be convincingly argued that Dichaete is involved in this aspect of Ind function (Van Ohlen, 2007a).

The data provide evidence that following initiation by global patterning signals, expression of the Ind homeodomain is maintained by the activity of Ind itself. This occurs through a newly identified regulatory element that is positioned upstream of the coding sequence and away from the element controlling initiation. A parallel type of regulation might occur for the Vnd homeodomain protein. However, additional work is required to confirm this hypothesis (Van Ohlen, 2007a).

Transcriptional Regulation

Although ind was identified in a screen for Tinman transcriptional targets, ind and Tinman are expressed in nonoverlapping, nonadjacent regions of the CNS and mesoderm, so it is unlikely that Tinman regulates ind directly. Furthermore, tinman mutant embryos have no change in ind expression. Therefore it was hypothesized that ind is transcriptionally regulated by Vnd, a homeodomain protein related closely to Tinman. Vnd is produced in the ventral neuroectoderm immediately adjacent to the ind-expression domain. Genetic and molecular data demonstrate that ind is transcriptionally repressed by Vnd. In wild-type embryos the two genes are expressed in adjacent but nonoverlapping portions of the neuroectoderm. vnd is expressed in the ventral column, whereas ind is expressed in the intermediate column. In vnd mutant embryos, ind expression is broader and encompasses what would normally be the vnd-expression domain. This can be observed clearly in lateral views of whole-mount embryos as well as in embryo cross sections. These genetic experiments show that vnd is required to repress ind expression within the ventral column neuroectoderm (Weiss, 1998).

To determine whether Vnd regulates ind transcription directly, bacterially expressed Vnd protein was used to perform electrophoretic mobility-shift and footprinting assays with the genomic ind DNA fragment identified in the initial screen for Tinman regulated proteins. Vnd specifically binds the fragment of ind genomic DNA isolated in the screen. Three specific binding sites of roughly equal affinity can be identified using footprinting assays. The three sites protected in the footprinting assay each contain one copy of the sequence GTGAACT (Weiss, 1998), which has been found to be a recognition sequence for both Vnd and the Tinman-related Nkx2.5 vertebrate protein (Chen, 1995 and Gruschus, 1997).

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). The origins of DV patterning within the Drosophila CNS have been investigated. The earliest sign of neural DV patterning is the expression of three homeobox genes in the neuroectoderm -- ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) -- which are expressed in ventral, intermediate, and dorsal columns of neuroectoderm, respectively. Previous studies have shown that the Dorsal, Decapentaplegic (Dpp), and EGF receptor (Egfr) signaling pathways regulate embryonic DV patterning, as well as aspects of CNS patterning. This study describes the earliest expression of each DV column gene (vnd, ind, and msh), the regulatory relationships between all three DV column genes, and the role of the Dorsal, Dpp, and Egfr signaling pathways in defining vnd, ind, and msh expression domains. The vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report vnd is found not to be regulated by Dpp signaling. ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and abnormally high Dpp can repress ind expression. The msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. It is concluded that the initial diversification of cell fates along the DV axis of the CNS is coordinately established by Dorsal, Dpp, and Egfr signaling pathways. Understanding the mechanisms involved in patterning vnd, ind, and msh expression is important, because DV columnar homeobox gene expression in the neuroectoderm is an early, essential, and evolutionarily conserved step in generating neuronal diversity along the DV axis of the CNS (Von Ohlen, 2000).

Early stage 5 embryos express vnd in a narrow domain similar to its final width; ind and msh are not detected. By the end of stage 5, both vnd and ind are expressed with a one to two cell wide gap; again, this expression is seen in domains similar to their final widths. The gap fills in during development resulting in the precise juxtaposition of the vnd and ind domains. Expression of msh in the trunk is not detected until stage 7. Thus, the timing of gene expression progresses from ventral to dorsal: vnd is detected first, ind appears soon after, and msh is observed last (Von Ohlen, 2000).

There is a gap between the initial vnd and ind domains, suggesting that each gene is independently activated at a precise DV position. Subsequently, ind can be expressed in the ventral domain, but this is normally prevented by vnd-mediated repression. Because ind is capable of repressing vnd expression, if ind were to be expressed first in both the ventral and the intermediate columns, it might fully inhibit the expression of vnd. Thus, the temporal pattern of vnd and ind expression is likely to be important for establishing their final spatial pattern of gene expression. The activation and borders of vnd expression appear to be wholly dependent on the Dorsal morphogen gradient. High levels of Dorsal in the mesoderm/mesectoderm anlagen can activate twist, snail, and vnd, but Snail activity represses vnd expression. Intermediate levels of Dorsal are sufficient to activate vnd, but not snail, thus establishing the ventral column of neuroectoderm. It is unclear how the dorsal border of vnd is positioned, but it may be dependent on the concentration of nuclear Dorsal, because if Dorsal levels are increased in dorsal cells, there is a corresponding expansion of the vnd domain. In contrast to a previous report, no evidence has been found that Dpp signaling establishes the dorsal border of the vnd domain. No change was observed in the width of the vnd domain in dpp embryos, and repression of vnd in ectopic Dpp embryos was not observed. In fact, elevated Dpp activity in the neuroectoderm (in sog 4xdpp embryos) gives a slight expansion of the vnd domain, and even higher levels of Dpp (in brk;sog embryos) still fail to repress vnd expression, despite eliminating much of the remaining CNS. The reason the vnd domain is expanded in sog 4xdpp embryos remains unclear; however, it is felt that the combined results clearly demonstrate that Dpp signaling does not repress vnd and therefore cannot position the dorsal border of vnd. All existing data are consistent with Dorsal acting as a direct, concentration-dependent activator of vnd expression. In contrast, the Egfr and Dpp signaling pathways have no role in establishing the correct vnd expression pattern, although Egfr is required to maintain vnd expression later in embryogenesis (Von Ohlen, 2000 and references therein).

Initiation and maintenance of ind expression require both Dorsal and Egfr signaling pathways, but not Dpp activity. The ventral border of ind expression is established by the dorsal limit of vnd expression. The dorsal border of ind expression has more complex regulation. Dpp repression does not establish the dorsal border of ind, since the ind domain is normal in dpp embryos. In contrast, both Dorsal and Egfr are required to activate ind and set its dorsal border. In wild-type embryos, the domains of ind and activated Egfr have identical dorsal borders. When Egfr activity is increased throughout the embryo, ind expression shows a partial dorsal expansion, showing that the dorsal border of Egfr activity sets the precise dorsal border of ind expression. Ectopic Dorsal activity can also expand the ind domain (without affecting the Egfr activation domain), showing that sufficiently high levels of nuclear Dorsal protein can independently activate ind expression. As expected, when Egfr activity and nuclear Dorsal levels are simultaneously increased there is a complete dorsal expansion of the ind domain. The data presented here suggest that ind expression is activated by both Dorsal and Egfr pathways, limited ventrally by vnd, and limited dorsally by lack of Dorsal and Egfr activity. The data do not distinguish between a linear pathway in which Egfr signaling activates or potentiates Dorsal to allow ind transcription and a parallel pathway in which Dorsal and Egfr signaling act independently to activate ind expression (Von Ohlen, 2000).

Although Dpp is not required for any aspect of ind expression in wild type embryos, ectopic Dpp signaling in the neuroectoderm can repress ind expression. This shows that Dpp signaling must be kept low in the intermediate column to allow ind transcription and raises the possibility that the loss of ind expression seen in dorsal embryos is an indirect effect, due to the de-repression of Dpp activity within the neuroectoderm. dorsal;dpp double mutants fail to express ind, however, proving that loss of ind expression in dorsal mutants is not due to de-repression of Dpp within the neuroectoderm. It is proposed that Dorsal must both activate ind expression and repress Dpp signaling to allow ind expression (Von Ohlen, 2000).

msh is expressed in a DV domain that has low Vnd, Ind, and Dpp activity. Overexpression of any of these genes will repress msh expression, and dorsal;dpp embryos that lack all vnd, ind, and dpp expression show ectopic msh expression around the DV axis. Thus, the borders of the msh domain are defined by repression: Vnd and Ind ventrally, and Dpp dorsally. What activates msh expression? msh expression could be activated by 'basal' transcription factors present uniformly in the early embryo. Alternatively, msh expression may be induced by a low level of ubiquitous TGFbeta activity, similar to the observed activation of zebrafish msh homologs. The screw gene encodes a TGFbeta-like protein expressed at low levels throughout the embryo, and although it has no striking CNS phenotype, it would be interesting to see if screw;dpp embryos lose dorsal msh expression, or whether screw;dorsal;dpp embryos lose global msh expression (Von Ohlen, 2000).

Sox proteins form a family of HMG-box transcription factors related to SRY, the mammalian testis determining factor. Sox-mediated modulation of gene expression plays an important role in various developmental contexts. Drosophila SoxNeuro, a putative ortholog of the vertebrate Sox1, Sox2 and Sox3 proteins, is one of the earliest transcription factors to be expressed pan-neuroectodermally. SoxNeuro is essential for the formation of the neural progenitor cells in the central nervous system. Loss of function mutations of SoxNeuro are associated with a spatially restricted hypoplasia: neuroblast formation is severely affected in the lateral and intermediate regions of the central nervous system, whereas ventral neuroblast formation is almost normal. Evidence is presented that a requirement for SoxNeuro in ventral neuroblast formation is masked by a functional redundancy with Dichaete, a second Sox protein whose expression partially overlaps that of SoxNeuro. SoxNeuro/Dichaete double mutant embryos show a severe neural hypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. Genetic interactions of SoxNeuro and the dorsoventral patterning genes ventral nerve chord defective (vnd) and intermediate neuroblasts defective (ind) underlie ventral and intermediate neuroblast formation. Expression of the Achaete-Scute gene complex suggests that SoxNeuro acts upstream and in parallel with the proneural genes. The finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo (Buescher, 2002 and Overton, 2002).

The loss of one copy of vnd or ind in a SoxN homozygous mutant background dominantly enhances the SoxN phenotype, suggesting that SoxN genetically interacts with vnd and ind. Since the expression of Vnd and Ind does not require SoxN function, it is concluded that SoxN does not act upstream of vnd and ind, but rather in parallel. In ind mutant embryos, Ac expression in the NE is derepressed in the intermediate region. Nevertheless, NBs fail to form within this region. vnd is required for Ac expression in the ventral NE. However, there seems to be no causal relationship between the loss of Ac expression and the subsequent loss of NBs, since ectopic expression of Ac does not rescue NB formation. Thus, it appears that expression of the genes of the AS-C can confer neural potential to the NE only when SoxN, vnd and ind expression is intact (Buescher, 2002).

It is presumed that the differences between Dichaete and SoxN may well reflect interactions between each Sox protein and a different partner mediated by protein domains outside the highly conserved DNA-binding domain. In accordance with this, it has been suggested that, in the neuroectoderm, Dichaete interacts with the product of the ind gene to mediate repression of ac. Since ind is specifically expressed within the intermediate neuroectoderm, it is tempting to speculate that this protein might interact specifically with Dichaete to repress ac while it does not interact with SoxN in the same way if indeed at all. However, Zhao (2002) provide evidence for interactions between Dichaete and both ind and vnd in the context of NB specification. Since the data suggest that SoxN and Dichaete function is at least redundant within the vnd-positive medial row, it is very likely that Vnd interacts with SoxN as well as Dichaete (Overton, 2002).

Sequential patterns of vnd, ind, and msh expression respond to distinct thresholds of the Dorsal gradient

A nuclear concentration gradient of the maternal transcription factor Dorsal establishes three tissues across the dorsal-ventral axis of precellular Drosophila embryos: mesoderm, neuroectoderm, and dorsal ectoderm. Subsequent interactions among Dorsal target genes subdivide the mesoderm and dorsal ectoderm. The subdivision of the neuroectoderm by three conserved homeobox genes, ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh) has been investigated. These genes divide the ventral nerve cord into three columns along the dorsal-ventral axis. Sequential patterns of vnd, ind, and msh expression are established prior to gastrulation and evidence is presented that these genes respond to distinct thresholds of the Dorsal gradient. Maintenance of these patterns depends on cross-regulatory interactions, whereby genes expressed in ventral regions repress those expressed in more dorsal regions. This 'ventral dominance' includes regulatory genes that are expressed in the mesectoderm and mesoderm. At least some of these regulatory interactions are direct. For example, the misexpression of vnd in transgenic embryos represses ind and msh, and the addition of Vnd binding sites to a heterologous enhancer is sufficient to mediate repression. The N-terminal domain of Vnd contains a putative eh1 repression domain that binds Groucho in vitro. Mutations in this domain diminish Groucho binding and also attenuate repression in vivo. The significance of ventral dominance is discussed with respect to the patterning of the vertebrate neural tube, and ventral dominance is compared with the previously observed phenomenon of posterior prevalence, which governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos (Cowden, 2003).

The ability of Vnd to repress msh in addition to ind raises the possibility that transcriptional repressors expressed in ventral regions of the embryo can inhibit repressors active in more dorsal regions. Support for this hypothesis came from using the Krüppel enhancer to misexpress both ind and msh along the anterior-posterior axis. Ectopic Ind failed to repress vnd expression, while ectopic Msh did not repress either vnd or ind expression. To determine if 'ventral dominance' is restriced to the neuroectoderm, the mesodermal repressor snail was misexpressed in transgenic embryos using the even-skipped (eve) stripe 2 enhancer. The stripe2-snail transgene creates an ectopic domain of snail along the anterior-posterior axis. This ectopic expression leads to a gap in the sim expression pattern. The transgene also causes a gap in the vnd pattern, confirming the model that Snail excludes vnd expression in the ventral mesoderm and restricts expression to the neuroectoderm. The stripe2-snail transgene also creates a gap in the ind pattern. These results support the ventral dominance model, whereby repressors located in ventral regions inhibit repressors expressed in more dorsal regions. Consistent with this 'directionality' of repression, ectopic expression of Vnd, Ind, or Msh does not repress snail (Cowden, 2003).

Further support for ventral dominance of the Snail repressor was obtained by analyzing mutant embryos derived from CtBP germline clones. CtBP is a maternally deposited corepressor protein essential for snail-mediated repression. Removal of this corepressor results in ventral derepression of sim and vnd into the presumptive mesoderm due to loss of Snail mediated repression. However, this ventral expansion of vnd does not result in a transformation of mesoderm into medial neuroblasts. Instead, the expanded vnd pattern is lost at slightly later stages, and expression becomes restricted to lateral regions, similar to the endogenous expression pattern. This lateral restriction is consistent with the observation that neuroblasts are formed in lateral regions of CtBP^- mutants, and not in ventral regions that normally form the mesoderm. Neuroblast segregation can be visualized using a snail antisense RNA probe, which stains all neuroblasts following gastrulation. Sim may be responsible for the late repression of vnd, because vnd expands into the ventral midline of sim mutant embryos. Repression of vnd by Sim is probably indirect because a Krüppel-sim transgene does not alter vnd expression in the lateral neuroectoderm. Perhaps Sim activates an unknown repressor that ultimately inhibits vnd expression in the midline (Cowden, 2003).

It is conceivable that the cross-regulatory interactions among the Snail, Vnd, Ind, and Msh repressors are indirect. For example, perhaps Vnd activates an unknown repressor, which in turn inhibits the expression of ind and msh in medial neuroblasts. Several experiments were done to determine whether Vnd functions as a transcriptional repressor. The first examined whether Vnd binding sites mediate activation or repression in transgenic embryos (Cowden, 2003).

The IAB5 enhancer drives the expression of a lacZ reporter gene in a series of three adjacent bands in the presumptive abdomen of cellularizing embryos. This staining pattern is maintained through gastrulation and germ band elongation. Vnd binding sites were introduced into this IAB5-lacZ transgene by inserting a 220 bp genomic DNA fragment between the IAB5 enhancer and lacZ reporter. This genomic fragment is located 3' of the ind gene and contains three Vnd binding sites. Insertion of this fragment caused a ventrolateral gap in the IAB5-lacZ staining pattern. This gap coincides with the endogenous vnd expression pattern and is maintained during germ band elongation. At this stage, there is a clear loss of lacZ expression in medial regions of the developing ventral nerve cord. The importance of the Vnd binding sites in mediating this repression was examined by mutagenizing all three sites within the 220 bp DNA fragment. Each site was converted from the 5'-CAAGTG-3' consensus to 5'-CCCGGG-3'. The mutagenized IAB5-lacZ transgene exhibits expanded expression in medial regions of the presumptive nerve cord. This observation suggests that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

Further evidence that Vnd is a repressor was obtained using an in vivo repression assay in transgenic embryos. The N-terminal region of Vnd contains a putative eh1 Groucho-interaction motif, FxIxxIL. This eh1 motif is present in two known transcriptional repressors, Engrailed and Goosecoid. It is also found in the Ind and Msh proteins. GST pull-down assays suggest that this motif mediates interaction between Vnd and Groucho. A GST-VEH1 fusion protein containing amino acid residues 183 to 226 from Vnd binds S³⁵-labeled Groucho protein produced via in vitro translation. This binding is lost when the GST-Vnd fusion protein is mutagenized to replace the phenylalanine in the FxIxxIL motif with an alanine. Various positive and negative controls were included in these experiments. For example, Groucho does not bind a GST-Ind fusion protein containing the Ind homeodomain. Weak binding is observed with a GST-Eve fusion protein containing the FKPY Groucho-interaction motif (Cowden, 2003 and references therein).

A Gal4-Vnd fusion gene containing the Gal4 DNA binding domain and the N-terminal 543 codons of Vnd was placed under the control of the Krüppel 5' regulatory region. The resulting fusion gene is expressed in central regions of cellularizing embryos. Similar levels of expression were obtained with a mutagenized version of the fusion gene that contains multiple alanine substitutions in the FxIxxIL motif. The regulatory activities of the two Gal4-Vnd fusion proteins were monitored with a lacZ reporter gene that contains a modified version of the rhomboid NEE lateral stripe enhancer. The modified NEE enhancer contains three Gal4 binding sites (UAS) and lacks Snail repressor sites. The reporter gene is expressed in ventral regions, including the mesoderm and portions of the lateral neuroectoderm (Cowden, 2003).

The unmutagenized Gal4-Vnd fusion protein containing an intact FxIxxIL motif attenuates expression of the NEE-lacZ reporter gene. This result suggests that the fusion protein binds UAS sites in the modified NEE enhancer and mediates transcriptional repression, either by direct repression of the core promoter, or quenching Dorsal and other activators within the NEE. In contrast, the mutagenized Gal4-Vnd fusion protein (DeltaVEH1) fails to repress expression from the lacZ reporter gene. This result suggests that the FxIxxIL motif is essential for the repression activity of the normal Gal4-Vnd fusion protein. Altogether, these experiments, along with the analysis of Vnd binding sites, suggest that Vnd functions as a sequence-specific transcriptional repressor that might recruit the Groucho corepressor protein (Cowden, 2003).

Thus the Dorsal gradient directly subdivides the neuroectoderm into separate dorsal-ventral compartments through the differential regulation of three conserved homeobox genes, vnd, ind, and msh. Maintenance of sequential patterns of gene expression depends on cross-regulatory interactions, whereby repressors expressed in ventral regions inhibit repressors active in more dorsal regions. This ventral dominance is evocative of the posterior prevalence phenomenon that governs sequential patterns of Hox gene expression across the anterior-posterior axis of metazoan embryos. At least one of the cross-regulatory interactions is direct and evidence was presented that Vnd functions as a sequence-specific transcriptional repressor (Cowden, 2003).

The Dorsal gradient establishes at least three thresholds of gene expression across the dorsal-ventral axis of early embryos. High concentrations activate target genes such as twist and snail in ventral regions that form the mesoderm. Intermediate concentrations activate the rhomboid gene in ventral regions of the neuroectoderm. Finally, low levels of the gradient activate the sog gene in both ventral and dorsal regions of the neuroectoderm. The same low levels of Dorsal repress target genes important for the differentiation of the dorsal ectoderm, including dpp, zen, and tolloid (Cowden, 2003).

Mutant embryos lacking Dorsal fail to activate early expression of either vnd or ind. Conversely, ectopic Dorsal activity leads to a corresponding dorsal shift in the vnd and ind expression patterns. The lateral stripes of vnd expression encompass ventral regions of the neuroectoderm, similar to the rhomboid (rho) pattern. rho is a direct Dorsal target gene that is expressed in the neuroectoderm and encodes a membrane-associated protease that processes the EGFR ligand spitz. Like rho, vnd appears to be a direct target of the Dorsal gradient: an intronic enhancer containing clustered Dorsal and Twist binding sites directs lateral stripes of expression in transgenic embryos. The ind lateral stripes appear to straddle the region between the vnd/rhomboid ventrolateral stripes and the broad sog lateral stripes, and previous studies suggest that ind may be regulated in a different manner from vnd. The regulation of ind relies on both the Dorsal gradient and the EGF signaling pathway. Removal of either Dorsal or the EGF receptor results in the loss of ind expression from the neuroectoderm. It is unclear whether Dorsal directly activates ind or simply establishes a domain of EGF signaling through the regulation of rhomboid (rho). However, given the early onset of ind expression and the misexpression of ind by ectopic Dorsal, it is likely that Dorsal is essential for its regulation. Consistent with the possibility that early ind expression pattern might reflect a threshold readout of the Dorsal gradient is the finding that the low levels of Dorsal present in Toll^rm9/Toll^rm10 embryos are sufficient to activate ind, but not msh. Moreover, the ind lateral stripes do not extend beyond the sog expression pattern, which is known to be directly activated by vanishingly low levels of the Dorsal gradient. Finally, a 3' ind enhancer that encompasses the three Vnd binding sites used in this study contains optimal Dorsal and Twist binding sites, suggesting that it is directly regulated by the Dorsal and Twist gradients (Cowden, 2003).

The initial compartmentalization of the neuroectoderm appears to depend on threshold readouts of the Dorsal gradient. This strategy is different from the subdivision of the other two primary embryonic tissues, the mesoderm and dorsal ectoderm. Patterning the mesoderm depends on interactions between twist and dpp. The Snail repressor establishes the limits of mesoderm invagination, while the localized expression of Dpp restricts induction of the lateral mesoderm to dorsal-lateral regions. Similarly, subdivision of the dorsal ectoderm depends on the differential regulation of the Dorsal target genes sog and dpp. Both genes respond to the same low levels of the Dorsal gradient, but sog is activated by Dorsal, while dpp is repressed. Subsequent protein-protein interactions between Sog and Dpp establish a broad Dpp signaling gradient in the dorsal ectoderm (Cowden, 2003).

Transcriptional repression of ind by Vnd was predicted from previous genetic studies but lateral repression of msh was somewhat unexpected. Previous studies have shown that ectopic Vnd represses msh expression in the procephalic neuroectoderm, where the vnd and msh expression patterns overlap. This result was extended in the present study using a Krüppel-vnd transgene. It would appear that Vnd represses both ind and msh to specify medial neuroblasts. A similar result was seen using the eve stripe 2 enhancer to misexpress snail. Previous studies have shown that Snail acts as a transcriptional repressor to create the boundary between mesoderm and neuroectoderm. As expected, ectopic snail repressed vnd expression but surprisingly, ind was also repressed. These results suggest that the Dorsal gradient separates domains along the dorsal-ventral axis by activating a series of localized transcriptional repressors. According to this model, repressors located in ventral regions selectively repress those located more dorsally, while dorsal repressors do not inhibit ventral repressors. For example, ectopic Vnd represses ind but not snail, while ectopic Ind fails to repress vnd or snail. According to this model, ectopic Ind should repress msh expression. However, because none of the transgenic Krüppel-ind lines persisted until germband elongation when msh expression is uniform, it was not possible to determine if ectopic Ind repressed msh. Similarly, while ectopic Msh failed to repress snail, vnd, or ind expression, the lack of early target genes that are regulated by Msh prevents any definitive conclusions regarding its role as a transcriptional repressor. Both Ind and Msh contain putative eh1 domains, suggesting that they may function as Groucho dependent repressors and previous work supports such a role for Ind and Msh in the ventral nerve cord (Cowden, 2003).

'Ventral dominance' might govern the patterning of the ventral nerve cord in older embryos, in addition to the prepatterning of the neuroectoderm in pregastrulating embryos. Sim might exclude vnd, ind, and msh expression in the ventral midline. In embryos lacking maternal CtBP products, Snail fails to act as a repressor, allowing the ventral expansion of sim and vnd into the presumptive mesoderm. However, vnd expression is ultimately lost from ventral regions, while sim expression persists. As a result, ventral regions form an expanded mesectoderm, while neuroblasts arise from lateral regions. These observations suggest that Sim excludes vnd expression from ventral regions in CtBP mutants, either directly by acting through a CNS specific enhancer or indirectly by activating an unknown repressor. This putative repressor probably does not rely on the CtBP corepressor, as it is still capable of repressing vnd in CtBP germ line clones. According to a ventral dominance scenario, the misexpression of this unknown repressor should inhibit the expression of vnd, ind, and msh in the ventral midline. One potential target for the indirect repressor could be the EGF pathway. The ventral midline is a well-characterized source of EGF signaling and both vnd and ind rely upon EGF signaling for maintenance of expression. By eliminating EGF activation, this midline repressor could prevent vnd and ind expression (Cowden, 2003).

It is conceivable that the ventral dominance model governing cross-regulatory interactions among Vnd, Ind, Msh, Snail, and possibly sim, also applies to the patterning of the vertebrate neural tube. The vertebrate homolog of vnd, Nkx2.2, is expressed in ventral regions of the neural tube, while the homologs of ind (Gsh) and msh (Msx) are expressed in intermediate and dorsal regions, respectively. These neural tube expression patterns match the dorsal-to-ventral positions of vnd, ind, and msh in the ventral nerve cord of Drosophila. Furthermore, the vertebrate homolog of Vnd, Nkx2.2, also functions as a Groucho-dependent transcriptional repressor. A clear prediction of this study is that the misexpression of Nkx2.2 throughout the vertebrate neural tube should lead to the repression of both Gsh and Msx. In contrast, the misexpression of Gsh should repress Msx, but not Nkx2.2. Thus, a cascade of homologous localized transcriptional repressors could subdivide both the vertebrate and invertebrate CNS (Cowden, 2003).

Threshold-dependent BMP-mediated repression: a model for a conserved mechanism that patterns the neuroectoderm

Subdivision of the neuroectoderm into three rows of cells along the dorsal-ventral axis by neural identity genes is a highly conserved developmental process. While neural identity genes are expressed in remarkably similar patterns in vertebrates and invertebrates, previous work suggests that these patterns may be regulated by distinct upstream genetic pathways. This study asked whether a potential conserved source of positional information provided by the BMP signaling contributes to patterning the neuroectoderm. This question was addressed in two ways: (1) it was asked whether BMPs can act as bona fide morphogens to pattern the Drosophila neuroectoderm in a dose-dependent fashion, and (2), whether BMPs might act in a similar fashion in patterning the vertebrate neuroectoderm was examined. In this study, it was shown that graded BMP signaling participates in organizing the neural axis in Drosophila by repressing expression of neural identity genes in a threshold-dependent fashion. Evidence is also provided for a similar organizing activity of BMP signaling in chick neural plate explants, which may operate by the same double negative mechanism that acts earlier during neural induction. It is proposed that BMPs played an ancestral role in patterning the metazoan neuroectoderm by threshold-dependent repression of neural identity genes (Mazutani, 2006; full text of article).

The neural identity genes vnd, ind, and msh are expressed in a series of non-overlapping DV domains in the Drosophila embryo. These genes are expressed in a highly dynamic fashion and are activated in a ventral-to-dorsal sequence. The BMP antagonist Sog is expressed throughout the neuroectoderm; prior to the activation of neural identity gene expression and fades dorsally as the Dorsal gradient collapses. By the time msh is expressed in a single contiguous dorsal stripe, sog expression is largely lost from these dorsal-most cells. During this same period, the BMP2/4 homolog Dpp is expressed in adjacent dorsal cells, where it represses the expression of neural genes and acts in a graded fashion to pattern the non-neural ectoderm. It is possible that Dpp also signals to the neuroectoderm, although previous single and double mutant analyses of the dpp pathway have not resolved whether Dpp acts in a graded fashion to help establish the order of the neural domains. In none of these studies, was it possible to sort out the contribution of BMP signaling from that of the Dorsal gradient. To answer whether Dpp acts as a morphogen to pattern the Drosophila neuroectoderm, a system was developed for selectively analyzing its effects in the absence of other DV cues (Mazutani, 2006).

In order to separate the potential patterning effect of BMP signaling in Drosophila from that imposed by the Dorsal gradient, a genetic system was designed that allowed replacement of the normal ventral-to-dorsal gradient of nuclear Dorsal with a uniform neuroectodermal level of Dorsal along the entire DV axis of the embryo. These lateralized embryos were created by first eliminating polarized DV maternal patterning acting upstream of Toll signaling and then adding back uniform adjusted levels of Dorsal across the entire DV axis using activated alleles of the Toll receptor. Uniform maternal Toll signaling was adjusted to specific levels using activated Toll alleles of differing strengths and by altering the dose of maternal Dorsal. In such lateralized embryos, the response was then tested of neural genes to an ectopic BMP gradient formed along the AP axis. This BMP gradient was created by expressing dpp under the control of the even-skipped stripe 2 enhancer of dpp (st2-dpp) construct (Mazutani, 2006).

In lateralized embryos, pan-neuroectodermal markers such as sog are expressed around the entire circumference of the embryo. As expected from the threshold-dependent activity of Dorsal, mesodermal, and dorsal ectodermal markers are absent in these same embryos. The consistent and uniform amounts of Dorsal produced in these lateralized embryos correspond to mid-neuroectodermal levels as revealed by expression of ind along the full DV axis and the absence of vnd expression. The AP limits of ind expression are similar to those in wild-type embryos. Within this domain, msh expression is not detectable, presumably because Ind is acting in a ventral-dominant fashion to repress it. However, in more anterior cells abutting the ind domain, where msh expression normally extends further than ind, msh is expressed in a ring around the embryo. These initial studies indicate that both ind and msh can be expressed in mid-neuroectodermal lateralized embryos, and that Ind efficiently excludes msh from its domain (Mazutani, 2006).

Once conditions were established for reliably producing lateralized embryos, whether it was possible to induce a graded Dpp response by crossing a st2-dpp construct into the lateralized background was tested. The sole source of dpp expression in these embryos is provided by st2-dpp, except at the poles where endogenous dpp expression is independent of Dorsal regulation. The expected pattern of BMP pathway activation in such embryos, assessed by in situ phosphorylation of the signal transducer, phosphorylated form of Mothers against dpp (pMAD), is a broad band centered over the st2-dpp stripe. Expression of the epidermal Dpp target gene u-shaped (ush) was also tested as a second marker for BMP activation. Because lateralized embryos ubiquitously express the BMP inhibitor sog, neither pMAD nor ush expression could be detected near the stripe of dpp expression. However, when sog function was eliminated in st2-dpp lateralized embryos, pMAD was activated in a broad domain extending approximately eight cell diameters beyond the narrower dpp stripe. In addition, ush expression was also activated in this region. These results indicate that Dpp diffusing from a sharp stripe can elicit a graded response over significant distances (Mazutani, 2006).

The effect of graded Dpp activity on the relative patterns of ind and msh expression was examined. Multiplex in situ hybridization methods were used to examine the simultaneous expression of msh, ind, and ush, while scoring for the sog+ versus sog− genotype of the embryos. These experiments revealed a clear dose-dependent repression of ind expression characterized by strong repression near the source of dpp and graded reduction in expression extending approximately 20 cell diameters posteriorly. In contrast, the opposite effect was observed with regard to msh expression, resulting in its activation in cells expressing the lowest levels of ind. In control sog+ lateralized embryos, where BMP signaling is blocked, st2-dpp had no discernable effect on the pattern or intensity of either msh or ind expression. These results can be understood if Dpp signaling preferentially represses expression of ind in sog−; st2-dpp lateralized embryos, thereby relieving ind-mediated repression of msh in cells near the Dpp source. The induction of msh expression near the Dpp stripe followed by a zone of ind expression mimics the wild-type configuration of gene expression and provides the first evidence that BMP signaling can influence the pattern of neuroectodermal gene expression in the absence of other DV cues such as the Dorsal gradient. Similar long-range inhibition of ind and short-range induction of ectopic msh expression can be observed in sog−; eve2-dpp embryos with an intact Dorsal gradient, indicating that ind is also likely to be more sensitive than msh to BMP-mediated repression in wild-type embryos. The fact that the zone of ind repression extends considerably further from the dpp stripe than the region of msh activation indicates that msh is not responsible for ind repression, consistent with existing evidence that msh does not regulate ind. It seems likely, therefore, that BMP signaling acts directly to repress ind expression. These data support the prevailing ventral-dominant model for cross-regulation of neural identity genes, and exclude an alternative model in which Dpp signaling activates msh, which in turn inhibits ind (Mazutani, 2006).

Previous studies of the ventral-most neural identity gene, vnd, reported only a mild expansion of its expression domain in dpp− mutants, or no consistent effect. The sensitive lateralized system was exploited to re-examine the BMP response of vnd in order to resolve these existing ambiguities. st2-dpp was expressed in embryos with uniform levels of Dorsal corresponding to the ventral neuroectoderm, which are sufficient to induce ubiquitous expression of vnd. In such “ventro-lateralized” embryos, both ind and msh expression are absent, presumably due to repression by vnd. Elimination of sog function in these embryos resulted in activation of BMP signaling as judged by the localized activation of the epidermal marker ush; however, vnd expression remained unaltered. When the function of both sog and the transcriptional repressor of BMP signaling, brinker (brk), was eliminated, stronger and expanded expression of ush and potent repression of vnd was observed in a broad zone centered over st2-dpp. These results indicate that vnd is indeed sensitive to BMP-mediated repression and that Brk can block the repressive as well as activating functions of BMP signaling. In analogy to what was observed in mid-lateralized embryos, it might have been expected that relief of Vnd repression in ventro-lateralized embryos would result in activation of ind in cells lacking vnd expression. However, no expression of either ind or msh was detected in these embryos, even near the edges of the vnd repression domain. These data suggest that the high levels of Dpp signaling generated under these experimental conditions are sufficient to repress vnd, as well as ind and msh. Such strong BMP signaling, which is similar to that acting in the non-neural ectoderm of wild-type embryos, may obscure potential differences in the relative sensitivities of these genes to BMP-mediated repression by repressing expression of all neural genes. Although it remains to be determined what the relative sensitivity of vnd is to BMP repression, the fact that vnd is subject to such repression raises the possibility that Dpp might also regulate vnd expression along its dorsal border in wild-type embryos, despite the low levels of Dpp that diffuse into that region. Since the concentration of Dorsal is limiting with regard to activating vnd in cells along this border, these cells would be expected to be the most susceptible to BMP-mediated repression (Mazutani, 2006).

This analysis of BMP signaling in lateralized embryos showed that Dpp can regulate the expression of ind and msh in a dose-dependent fashion along the AP axis, and can also repress vnd expression. To test whether Dpp plays a similar dosage-sensitive role in the regulation of neural identity genes along the DV axis in the presence of an intact gradient of nuclear Dorsal, an experiment was devised to locally inhibit the response of neural genes to Dpp within the neuroectoderm of embryos with normal DV polarity. Because Brk can suppress BMP-mediated repression of vnd, it was reasoned that mis-expression of brk with the eve-st2 enhancer might also relieve BMP repression of ind and msh. This localized expression of the st2-brk construct has the advantage of providing an internal comparison of gene expression domains within the same embryo. In embryos carrying the st2-brk construct, all three neural domains shifted dorsally at the site of brk over-expression. msh expression was de-repressed in a stripe dorsally as has been observed previously in dpp minus mutants, and the border between msh and ind shifted dorsally by approximately 4-6 cells. The dorsal shift in ind expression was observed prior to initiation of msh expression, consistent with their normal ventral-to-dorsal sequence of activation. In addition, a modest but consistent dorsal shift of 1-2 cells was observed in the ind/vnd border within the zone of st2-brk expression. The domains of msh and ind expression also shift in other situations where BMP signaling is altered in the context of an intact Dorsal gradient, which reinforces the view that BMP signaling plays a role in determining the positions and extents of these expression domains in wild-type embryos (Mazutani, 2006).

The results described above indicate that graded Dpp activity normally plays an important role in establishing the position of the border between the msh and ind domains, and to a lesser degree influences the ind/vnd border, which forms 10-12 cells from the dorsal source of Dpp. The co-ordinate shifts in the borders of neural identity gene expression in st2-brk embryos are consistent with the known ventral-dominant chain of repression among vnd, ind, and msh. This analysis also provides additional support for cis-acting vnd sequences being sensitive to BMP repression and suggests that the dorsal border of vnd expression is normally determined by balancing the opposing influences of Dorsal activation and BMP-mediated repression. It is noted that the dorsal expansion of vnd expression in st2-brk embryos does not necessarily imply that vnd is more sensitive to BMP-mediated repression than ind or msh, but instead that at limiting levels of Dorsal, even low levels of BMP signaling can exert a repressive effect on vnd expression (Mazutani, 2006).

Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila central nervous system

The Drosophila embryonic CNS develops from the ventrolateral region of the embryo, the neuroectoderm. Neuroblasts arise from the neuroectoderm and acquire unique fates based on the positions in which they are formed. Previous work has identified six genes that pattern the dorsoventral axis of the neuroectoderm: Drosophila epidermal growth factor receptor (Egfr), ventral nerve cord defective (vnd), intermediate neuroblast defective (ind), muscle segment homeobox (msh), Dichaete and Sox-Neuro (SoxN). The activities of these genes partition the early neuroectoderm into three parallel longitudinal columns (medial, intermediate, lateral) from which three distinct columns of neural stem cells arise. Most of the knowledge of the regulatory relationships among these genes derives from classical loss of function analyses. To gain a more in depth understanding of Egfr-mediated regulation of vnd, ind and msh and investigate potential cross-regulatory interactions among these genes, loss of function was combined with ectopic activation of Egfr activity. Ubiquitous activation of Egfr expands the expression of vnd and ind into the lateral column and reduces that of msh in the lateral column. This work has identified the genetic criteria required for the development of the medial and intermediate column cell fates. ind appears to repress vnd, adding an additional layer of complexity to the genetic regulatory hierarchy that patterns the dorsoventral axis of the CNS. This study also demonstrates that Egfr and the genes of the achaete-scute complex act in parallel to regulate the individual fate of neural stem cells (Zhao, 2007b).

The Dorsal gradient initiates patterning of the CNS via the transcriptional regulation of the expression vnd, rhomboid and zen. Dorsal-mediated activation of rhomboid, the rate-limiting factor in Egfr-signaling and vnd establishes the initial expression domains of two of the earliest positive activators of CNS patterning along the DV axis. Similarly, Dorsal-mediated repression in the ventral and ventrolateral ectoderm limits the expression of zen and decapentaplegic (dpp) to the dorsal ectoderm. Dpp functions as a morphogen and defines via a repressive mechanism the lateral limit of the developing CNS (Zhao, 2007b).

Within the CNS, vnd and rhomboid exhibit differential sensitivity to the dorsal gradient with vnd being activated solely within the medial column and rhomboid in both the intermediate and medial columns. Since rhomboid is the limiting factor in Egfr signaling, its presence activates Egfr-signaling activity in the medial and intermediate columns. In wild-type embryos, Egfr activity maintains vnd expression in the medial column and is necessary to promote ind expression in the intermediate column. The ability of vnd to repress ind expression explains the restriction of ind expression to the intermediate column. vnd expression persists throughout most of the medial column until the end of embryogenesis; in contrast, ind expression is extinguished in the intermediate column neuroectoderm by stage 10 after the first two (of five) waves of NB segregation (Zhao, 2007b).

This work adds a new regulatory relationship into the genetic regulation of CNS patterning, since it was found that ind helps establish the lateral limit of vnd expression. ind could perform this function via the direct repression of vnd, a possibility supported by gain-of-function and loss-of-function experiments. If this model is correct, the mutual repression of vnd and ind would bear striking similarity to the reciprocal repressive interactions observed for the class I and class II homeodomain proteins that pattern the DV axis of the vertebrate CNS. In this context, it is important to note that the vertebrate ortholog of vnd, Nkx2.2., is a class II protein that plays a key role in patterning some of the ventral-most regions of the vertebrate CNS. Alternatively or additionally, vnd and ind could establish their mutual sharp boundary indirectly via the regulation of other factors. For example, differential regulation of homophilic cell-adhesion molecules could account for the observed phenotype. Differential expression of cell-adhesion molecules on medial versus intermediate column cells would cause these cells to associate preferentially with cells from the same column and result in a sharp boundary between the two cell populations that minimized interaction. Loss of such differences would reduce the requirement to minimize interactions and likely result in a jagged boundary. Additional work is necessary to identify the precise mechanism through which ind helps establish the lateral limit of vnd expression. Previous work has shown that misexpression of ind along the anterior-posterior axis using the Kruppel enhancer failed to repress vnd expression in the medial column. However, this is not contradictory to the current findings of this study. This work suggests that ind can repress vnd in the intermediate and lateral columns but not in the medial columns. It is likely that some factors that are present in the intermediate and lateral columns but are absent in the medial column help ind to repress vnd (Zhao, 2007b).

In addition, this work demonstrates that Egfr and vnd are sufficient to confer medial fate and that Egfr and ind are sufficient to confer intermediate fate. Although loss-of- function studies have shown that both Egfr and vnd are necessary for NBs to acquire medial fate, it is not clear whether Egfr functions solely through vnd. It has been shown that ectopic vnd expression results in partial transformation of lateral column into medial column. The current work shows that ectopic Egfr activity can induce the expression of vnd and together Egfr and vnd fully transform the lateral column into the medial column. Therefore, Egfr likely plays additional roles in determining medial cell fate other than maintaining vnd expression in the neuroectoderm. However, it remains unclear whether Egfr contributes to the intermediate column NB fate determination other than through its regulation of ind and whether ind by itself is sufficient to confer intermediate fate. Further studies are necessary to dissect the regulatory mechanisms that control intermediate column NB fate specification. In addition, while this work did not address the roles of Dichaete and Sox-Neuro, it has been reported that ubiquitous EGFR signaling activates Dichaete expression throughout the neuroectoderm. Because Dichaete and SoxNeuro cooperates with vnd in the mediate column and ind in the intermediate column in NB fate specification, they are likely to act as co-factors with Vnd and Ind in embryos expressing Egfr over a prolonged period to specify NB fate in the lateral column (Zhao, 2007b).

These experiments also underline the importance of temporal regulation of gene expression during CNS patterning. This is most notable with respect to the dynamic regulation of ind and vnd expression by Egfr signaling. Previous work suggested that the spatial dynamics of Egfr activity in the CNS account for the transient nature of ind expression in the intermediate column. Prior to NB formation Egfr activity is present in the intermediate column and activates ind expression in this domain. Once NBs begin to form Egfr activity disappears from the intermediate column and ind expression is also lost from intermediate column neuroectodermal cells. These data supported a simple regulatory relationship in which the presence of Egfr activity is necessary for ind expression in the intermediate column. However, while Egfr is necessary to activate ind in the intermediate column and sufficient to activate ind in the entire CNS, this study finds that ind expression turns over at its normal time even in the presence of ubiquitous and prolonged Egfr activity in the CNS. Thus, even though Egfr activity is necessary and sufficient for the activation of ind, once activated ind expression in the CNS appears to become independent of Egfr activity and other factors must regulate its temporally precise downregulation in the CNS (Zhao, 2007b).

Similarly, vnd also exhibits differential sensitivity to Egfr activity as a function of time. In contrast to ind, Egfr activity is not necessary to activate vnd expression in the medial column, however, Egfr activity is required later to maintain vnd expression in this domain. Thus, vnd and ind exhibit opposite responses to the Egfr signaling -- ind is activated but not maintained by Egfr activity while vnd is maintained but not activated by this pathway. It is interesting to note that vnd becomes competent to respond to Egfr signaling about the time ind loses its ability to respond to this signal. While the differential competency of the vnd and ind promoters to Egfr signaling is essential for proper DV patterning of the CNS, the molecular bases of these differences remain unknown. Some of the specificity likely resides within the promoters or regulatory regions of the genes themselves. However, since both promoters are Egfr-responsive albeit at different times additional levels of regulation appear necessary to explain the complexity in regulation. Alteration to higher order chromatin structure is known to play a key role in controlling the competency of different promoters to respond to specific signals and is a clear candidate to help mediate the differential responses of ind and vnd to Egfr-activity. However, how chromatin structure affects the ability of ind and/or vnd to respond to Egfr-activity remains unexplored. Future work that addresses the influence of modulation of chromatin structure on the ability of these and other genes to respond differentially to the same inputs should shed light on basic principles of gene regulation during development (Zhao, 2007b).

Genetic studies indicate that the activities of Egfr and the ac/sc genes converge to specify the fate of MP2 and possibly other NBs. Additional work on genes that regulate NB fate suggests that distinct convergent signals may play a general role in NB specification. For example, the transcription factor Huckebein is expressed in NB 4-2 and its associated proneural cluster and helps promote the fate of some of the neurons that develop in the 4-2 lineage. However, in the absence of huckebein function, the 4-2 lineage retains many of its wild-type characteristics. Thus additional intrinsic and extrinsic cues likely converge with huckebein to control the fate of NB4-2 and enable it to elaborate its proper cell lineage. Similar, albeit less detailed observations, have been made for runt and msh. These genes are expressed in specific NBs and the cell clusters from which they delaminate. Each gene appears to regulate only a subset of the distinguishing characteristics of the neuronal lineages that arise from their respective NBs yet none of them appears deterministic for a specific NB fate. Thus, it is speculated that convergent regulation of NB fate by multiple intrinsic and extrinsic factors is a general theme in CNS development and that classical double and triple mutant analyses will be essential to reveal convergent pathways involved in NB as well as neuronal specification (Zhao, 2007b).

Targets of Activity

muscle segment homeobox is normally expressed in the lateral column of neuroblasts. ind represses transcription of msh either directly or indirectly within intermediate column neuroectoderm. Normally the ind and msh expression domains are adjacent but nonoverlapping, consistent with negative regulation of msh by ind. achaete is normally in rows 3 and 7 of the neuroectoderm, with expression restricted to the ventral and dorsal columns and excluded from the intermediate column. ind expression in the intermediate column abuts these clusters of achaete-expressing cells precisely without overlapping them. In ind mutant embryos, derepression of achaete expression is observed within the intermediate column of neuroectoderm. It is concluded that ind represses achaete expression directly or indirectly, and that ind is necessary for establishing proper intermediate-column identity within the neuroectoderm (Weiss, 1998).

Snail, a zinc-finger transcriptional repressor, is a pan-neural protein, based on its extensive expression in neuroblasts. Previous results have demonstrated that Snail and related proteins, Worniu and Escargot, have redundant and essential functions in the nervous system. The Snail family of proteins control central nervous system development by regulating genes involved in asymmetry and cell division of neuroblasts. Whether the neuroblast expression of snail and worniu is regulated by proneural genes was examined. Such a result would place the snail family in the well established genetic hierarchy that controls early neuroblast differentiation. The scute^B57 deletion mutant uncovers the three pro-neural genes: achaete, scute and lethal of scute. In this mutant, the expression of worniu in neuroblasts is significantly reduced. Only a few neuroblasts within each segment exhibit staining, and the expression level is substantially lower than in the wild type. The expression of worniu is also regulated by vnd and ind, such that in these mutant embryos the whole ventral and intermediate columns of staining are missing. In the msh^Delta68 mutant, no abnormal expression of worniu was detected. Previous results have shown that the neuroblast expression of snail is slightly affected in achaete-scute and vnd mutants but is not affected in a daughterless mutant. In ind and msh mutants, Snail protein expression was observed in many neuroblasts but the spatial pattern was rather disorganized. In summary, most of the proneural genes tested have profound effects on the expression of worniu, and have detectable but lesser effects on that of snail. The predominant expression of snail and worniu in neuroblasts and their regulation by proneural genes suggests that the snail family genes may have important functions within neuroblasts (Ashraf, 2001).

Linking pattern formation to cell-type specification: Dichaete and Ind directly repress achaete gene expression in the Drosophila CNS

Mechanisms regulating CNS pattern formation and neural precursor formation are remarkably conserved between Drosophila and vertebrates. However, to date, few direct connections have been made between genes that pattern the early CNS and those that trigger neural precursor formation. Drosophila has been used to link directly the function of two evolutionarily conserved regulators of CNS pattern along the dorsoventral axis, the homeodomain protein Ind and the Sox-domain protein Dichaete, to the spatial regulation of the proneural gene achaete (ac) in the embryonic CNS. A minimal achaete regulatory region that has been identified that recapitulates half of the wild-type ac expression pattern in the CNS; multiple putative Dichaete-, Ind-, and Vnd-binding sites have been found within this region. Consensus Dichaete sites are often found adjacent to those for Vnd and Ind, suggesting that Dichaete associates with Ind or Vnd on target promoters. Consistent with this finding, Dichaete can physically interact with Ind and Vnd. Finally, the in vivo requirement of adjacent Dichaete and Ind sites in the repression of ac gene expression has been demonstrated in the CNS. These data identify a direct link between the molecules that pattern the CNS and those that specify distinct cell-types (Zhao, 2007b).

Sox-domain proteins physically associate with other transcription factors to regulate gene transcription. Thus, the identification that Dichaete genetically interacts with Vnd and Ind suggested that Dichaete associates with Vnd and Ind to regulate gene expression in the CNS. To test this model, it was asked whether Dichaete can interact with Ind or Vnd in the yeast two-hybrid assay. Control experiments revealed that the full-length Dichaete protein as well as the region C-terminal to the high-mobility-group (HMG) DNA-binding domain (amino acids 221–384) activate transcription on their own when fused to the Gal4 DNA-binding domain, suggesting that the C-terminal region contains transcriptional activation activity. As a result, a number of distinct Dichaete fusion constructs were tested for self-activation of transcription and four were identified that were transcriptionally inert. One of these contained the HMG domain and the C-terminal region, indicating that the presence of the HMG domain may mask the transactivation properties of the C-terminal region. A prior study mapped a transactivation domain to the N-terminal region of Dichaete (Ma, 1998), yet no transactivation properties of this domain were identified in this study. Consistent with a transactivation domain residing in the C-terminal region of Dichaete, all other identified transactivation domains in Sox-family proteins map C-terminal to the HMG domain (Zhao, 2007b).

By using the four Dichaete bait constructs, it was found that the N-terminal region of Dichaete (amino acids 1–141) specifically interacted with full-length Ind protein. In a reciprocal manner, the ability of the Dichaete N-terminal region to interact with two different regions of Ind was tested: the region N-terminal to the homeodomain (amino acids 1–302) and the region including the homeodomain and all residues C-terminal to it (296–391). Both regions of Ind interacted strongly with the Dichaete N-terminal region, suggesting that this region of Dichaete can interface with two distinct regions of Ind (Zhao, 2007b).

In a similar manner, two distinct regions of Dichaete, the regions N-terminal (amino acids 1–141) and C-terminal (amino acids 221–384) to the HMG domain, interact with the full-length Vnd protein. Three different Vnd prey constructs were used to localize the regions of Vnd that interact with Dichaete. It was determined that the region of Vnd located between the TN domain (a domain common to Tinman/NK-2 proteins) and the homeodomain (amino acids 217–536) interacts with the Dichaete N-terminal domain. This result confirms and extends those of Yu (2005) who found that Vnd and Dichaete coprecipitate and that a Vnd deletion lacking the first 408 amino acids interacts with Dichaete. It was not possible to define the region of Vnd that interacts with the Dichaete C-terminal region, perhaps because the constructs interrupt the domain to which the C-terminal region of Dichaete binds or disrupt the general topology of this domain. Nonetheless, the yeast two-hybrid results indicate that Dichaete can interact with Ind and Vnd consistent with the model that Dichaete complexes with Ind and Vnd on target gene promoters to regulate transcription in the CNS (Zhao, 2007b).

A molecular understanding of how Dichaete, Ind, and Vnd pattern the CNS requires the identification and characterization of the regulatory regions of candidate direct target genes. One such candidate is the ac gene. Prior studies on ac suggested that regulatory regions important for its spatial regulation exist both 5' and 3' to the ac gene. Thus, an 8.15-kb minigene was generated that contains the ac transcription unit as well as ~4.8 kb of DNA 5' to the transcription start and ~2.4 kb of DNA 3' to the polyadenylation site and its ability to drive ac expression in an In (1)y^3PLsc^8R mutant background was tested. This genetic background carries a deletion of ac and also deletes the regulatory regions necessary to drive sc expression in row 3. Thus, it allows visualization of ac expression as driven by the minigene in the absence of endogenous ac/sc gene expression in row 3. The ac minigene drives ac expression in half of its wild-type CNS pattern because ac is expressed normally in the medial and lateral clusters of row 3 but is not expressed in row 7. The dynamics of ac expression as driven by the minigene in row 3 mirror those of endogenous ac expression because ac expression in each cluster quickly becomes restricted to a single cell, the presumptive neuroblast, which then delaminates into the interior of the embryo and extinguishes ac gene expression before its first division. Thus, the DNA contained within the minigene is sufficient to activate ac in its wild-type expression pattern in row 3 and to mediate the Notch-dependent restriction of ac to the presumptive neuroblast (Zhao, 2007b).

By creating a series of 5' and 3' deletions of the initial minigene, the regulatory regions sufficient to drive ac expression in row 3 was delimited to a 2.84-kb genomic fragment (pG7), which is referred to as the row 3 element. This element contains the ac transcription unit, 1.34 kb of DNA 5' to the start of transcription and 542 base pairs of DNA 3' to the end of the transcription unit. ac minigenes were characterized for their ability to respond to the functions of Dichaete, ind, and vnd and for the presence and in vivo relevance of putative binding sites for these factors (Zhao, 2007b).

In support of Dichaete, Vnd, and Ind acting directly on the row 3 element to regulate ac expression, loss of Dichaete, vnd, or ind function affects ac expression as driven by ac-pG4 or ac-pG7 in the same way, and these defects are identical to those observed for endogenous ac expression in these mutant backgrounds. For example, loss of ind or Dichaete causes, respectively, strong or modest derepression of ac expression in the intermediate column, whereas loss of vnd results in the absence of ac expression in the medial column (Zhao, 2007b).

To see whether Dichaete, Ind, or Vnd act directly on the row 3 element to control ac expression, this element was searched for perfect matches to the consensus Vnd [CAAGTG], Sox-domain [(A/T)(A/T)CAA(A/T)G and homeodomain (TAATGG) binding sites. The canonical Sox-domain and homeodomain binding site sequences were used because the consensus sites for Dichaete and Ind have not been determined. This search identified one match for Vnd (V) and three each for Dichaete (S1, S3, and S4) and Ind (H1, H3, and H4). Notably, predicted Dichaete/Sox-binding sites tend to reside close to predicted Vnd or Ind sites, consistent with Dichaete acting with Vnd and Ind to regulate ac expression. The sole exception is the Ind site (H1) located upstream of the transcriptional start site of ac. However, gel-shift assays identify a Dichaete-binding site 11 bp 5' of this Ind site (S2) (Zhao, 2007b).

Because the precise binding specificity of Ind is unknown, whether Ind can bind the predicted sites was tested by using gel-shift assays. Focused was placed on the predicted Ind site located upstream of the transcription start site because it is the only location where Dichaete and Ind sites are found adjacent to each other. It was found that Ind specifically binds this site in vitro. During these experiments, a second Ind-binding site (TAAATG) 8 bp 3' to this site was found, that differs slightly from the consensus homeodomain site. Thus, Ind can bind to two sites located within 1 kb of the ac promoter, suggesting a possible molecular mechanism for Ind-dependent repression of ac (Zhao, 2007b).

The initial search for Dichaete-binding sites required a perfect match to the consensus Sox-binding site. However, bona fide transcription factor-binding sites often differ from the experimentally defined consensus by a few base pairs, indicating that the search likely underpredicted possible Dichaete-binding sites. Because of this, gel-shift assays were used to search for Dichaete-binding sites throughout the entire row 3 element (pG7). Three sites were identified to which Dichaete bound specifically. Two of these correspond to sites identified in the consensus sequence search (sites S1 and S3); whereas the third resides 11 bp 5' of the first of the two Ind sites near the transcriptional start of ac (S2); this site (GACAATG) differs from the consensus by one base pair. No binding was detected of Dichaete to one predicted Sox site (S4). Because Dichaete and ind are known to repress ac expression, the three binding sites for Ind and Dichaete upstream of the ac promoter identify a likely site of action through which these factors repress ac (Zhao, 2007b).

The clustering of binding sites for Dichaete, Vnd, and Ind, together with the ability of Dichaete to interact with Vnd and Ind, supports the idea that Dichaete acts with these factors to regulate ac expression in the CNS. To test this model directly, the in vivo relevance was assayed of the adjacent Vnd and Dichaete sites as well as the adjacent Dichaete and Ind sites on ac expression. ac expression was unaltered when the Vnd-binding site, the adjacent Dichaete site, or both sites were mutated. Thus, vnd either does not regulate ac expression directly or other Vnd binding sites in the row 3 element compensate for the loss of this site (Zhao, 2007b).

The relevance of the three Dichaete- and Ind-binding sites located ~850 bp upstream of the start of ac transcription was assayed. Mutating any single site or any combination of two sites had no effect on ac expression. However, mutating all three sites derepressed ac expression in the intermediate column, a phenotype similar to that found in embryos mutant for ind or Dichaete. This result provides direct link between genes that pattern the CNS and those that specify distinct cell types. Because the derepression of ac is less severe than that observed in ind mutant embryos, Ind and Dichaete likely act through additional sites in this element to repress ac expression fully in the intermediate column (Zhao, 2007b).

Unexpectedly, derepression of ac expression posterior to row 3 was observed upon mutation of the three sites. This posterior expansion of ac mimics the effect that removal of gooseberry function has on the expression of ac, suggesting that Gooseberry, another homeodomain protein, may bind the same sites as Ind and act with Dichaete to repress ac expression in its expression domain (Zhao, 2007b).

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