InteractiveFly: GeneBrief

intermediate neuroblasts defective: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

Gene name - intermediate neuroblasts defective

Synonyms -

Cytological map position - 71A

Function - transcription factor

Keywords - CNS

Symbol - ind

FlyBase ID: FBgn0025776

Genetic map position -

Classification - homeodomain protein

Cellular location - nuclear

NCBI link: Entrez Gene

ind orthologs: Biolitmine
Recent literature
Samee, M. A., Lim, B., Samper, N., Lu, H., Rushlow, C. A., Jimenez, G., Shvartsman, S. Y. and Sinha, S. (2015). A systematic ensemble approach to thermodynamic modeling of gene expression from sequence data. Cell Syst 1: 396-407. PubMed ID: 27136354
To understand the relationship between an enhancer DNA sequence and quantitative gene expression, thermodynamics-driven mathematical models of transcription are often employed. These 'sequence-to-expression' models can describe an incomplete or even incorrect set of regulatory relationships if the parameter space is not searched systematically. This study focused on an enhancer of the Drosophila gene ind and demonstrate how a systematic search of parameter space can reveal a more comprehensive picture of a gene's regulatory mechanisms, resolve outstanding ambiguities, and suggest testable hypotheses. An approach is described that generates an ensemble of ind models; all of these models are technically acceptable solutions to the sequence-to-expression problem in light of wild-type data, and some represent mechanistically distinct hypotheses about the regulation of ind. This ensemble can be restricted to biologically plausible models using requirements gleaned from in vivo perturbation experiments. Biologically plausible models make unique predictions about how specific ind enhancer sequences affect ind expression; these predictions were validated in vivo through site mutagenesis in transgenic Drosophila embryos.
Salomone, J., Qin, S., Fufa, T. D., Cain, B., Farrow, E., Guan, B., Hufnagel, R. B., Nakafuku, M., Lim, H. W., Campbell, K. and Gebelein, B. (2021). Conserved Gsx2/Ind homeodomain monomer versus homodimer DNA binding defines regulatory outcomes in flies and mice.. Genes Dev 35(1-2): 157-174. PubMed ID: 33334823
How homeodomain proteins gain sufficient specificity to control different cell fates has been a long-standing problem in developmental biology. The conserved Gsx homeodomain proteins (ind in Drosophila) regulate specific aspects of neural development in animals from flies to mammals, and yet they belong to a large transcription factor family that bind nearly identical DNA sequences in vitro. This study showa that the mouse and fly Gsx factors unexpectedly gain DNA binding specificity by forming cooperative homodimers on precisely spaced and oriented DNA sites. High-resolution genomic binding assays revealed that Gsx2 binds both monomer and homodimer sites in the developing mouse ventral telencephalon. Importantly, reporter assays showed that Gsx2 mediates opposing outcomes in a DNA binding site-dependent manner: Monomer Gsx2 binding represses transcription, whereas homodimer binding stimulates gene expression. In Drosophila, the Gsx homolog, Ind, similarly represses or stimulates transcription in a site-dependent manner via an autoregulatory enhancer containing a combination of monomer and homodimer sites. Integrating these findings, this study tested a model showing how the homodimer to monomer site ratio and the Gsx protein levels defines gene up-regulation versus down-regulation. Altogether, these data serve as a new paradigm for how cooperative homeodomain transcription factor binding can increase target specificity and alter regulatory outcomes.
Linskens, A., Doe, C. and Lee, K. (2022). Developmental origin of the Pair1 descending interneuron. MicroPubl Biol 2022. PubMed ID: 36606082
Pair1 is part of a Drosophila larval locomotor circuit that promotes backward locomotion by inhibiting forward locomotion. It was hypothesized that lineage related neurons may function in neuronal circuits together. Testing this hypothesis requires knowing the progenitor of each neuron within this locomotor circuit, and this study focussed exclusively on Pair1. During Drosophila melanogaster embryogenesis, unique neuroblasts form by inheriting the spatial transcription factors (TFs) expressed in their birth location within the neuroectoderm. The Pair1 neurons were examined using immunofluorescence to determine which neuroblast the Pair1s derive from. The results show that Pair1 is derived from gnathal neuroblast 5-3 which expresses Gooseberry (Gsb) and Intermediate neuroblasts defective (Ind). When Gsb or Ind were overexpressed in the Pair1 lineage, extra neurons formed with similar Pair1 morphology.

The Drosophila homeobox gene intermediate neuroblasts defective (ind) was a serendipitous discovery in a search for promoter elements that bind Tinman. It turns out that the promoter binding sequence recognized by Tinman is identical to that recognized by the related protein Ventral nervous system defective (Vnd). ind turns out to be a target of Vnd and not Tinman (Weiss, 1998).

The early neuroblasts of Drosophila form an orthogonal grid of four rows along the anterior-posterior (AP) axis and three columns (ventral, intermediate, and dorsal) along the dorsoventral (DV) axis. Subsequently, each neuroblast expresses a characteristic combination of genes and contributes a stereotyped family of neurons and glia to the CNS. Thus the earliest steps in patterning the CNS are the formation and specification of neuroblasts. While the proneural achaete-scute genes and the neurogenic genes of the Notch pathway are widely understood as being essential for early neurogenesis, equally important are several homeobox genes that act in the neuroectoderm to determine the identity of the three neuroblast columns along the DV axis. ind is expressed specifically in the intermediate column of neuroblasts cells prior to delamination and is essential for intermediate column development. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, and ind represses another homeobox gene, muscle segment homeobox, in the intermediate column. Whereas vnd determines the identity of the ventral most column of neuroblasts, msh determines the identity of the lateral column. The DV control genes vnd, ind, and msh, together with the AP-patterning genes, constitute a Cartesian cell-fate determination system for the developing CNS. Vertebrate genes closely related to vnd (Nkx2.1 and Nkx2.2), ind (Gsh1 and Gsh2), and msh (Msx1 and Msx3) are expressed in corresponding ventral, intermediate, and dorsal domains during vertebrate neurogenesis, raising the possibility that dorsoventral patterning within the central nervous system is evolutionarily conserved (Weiss, 1998).

ind mutations were isolated by a mutagenesis screen for altered even-skipped (eve) expression in the CNS (J. Skeath and C.Q. Doe, unpubl.). In addition, three ind alleles were obtained by mobilizing a P element located next to the ind locus. The earliest ind mutant phenotype is observed in stage 7 embryonic neuroectoderm, when msh expression occurs both in its normal locations in the dorsal columns and in the adjacent intermediate columns. Thus ind represses transcription of msh 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. During the earliest stage of neurogenesis (stage 8 of development), wild-type embryos show expression of the proneural gene achaete 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 precisely abuts these clusters of achaete-expressing cells without overlapping them. In ind mutant embryos, derepression of achaete expression is observed within the intermediate column of neuroectoderm in rows 3 and 7 . This is consistent with a transformation of intermediate to dorsal neuroectoderm msh marker. It is concluded that ind represses msh and achaete gene expression directly or indirectly, and that ind is necessary for establishing proper intermediate-column identity within the neuroectoderm (Weiss, 1998).

In stage 9 wild-type embryos, five neuroblasts constitute the intermediate column in each hemisegment. In ind mutant embryos, at most one intermediate-column neuroblast is observed in each hemisegment, whereas the normal number of ventral and dorsal column neuroblasts form. What causes ind mutants to have reduced neuroblast formation? One possibility is that ind activates proneural gene expression in the intermediate column of neuroblasts. The only proneural gene known to be expressed in this domain is lethal of scute, which was not assayed in this study. However, the ectopic expression of the proneural gene achaete that is observed in the intermediate-column neuroectoderm in ind mutants should promote, rather than reduce, neuroblast formation. Two alternative explanations for the failure to generate intermediate column neuroblasts in ind mutant embryos are suggested. (1) Proneural clusters of the dorsal column expand to include cells of the intermediate column, but still produce one, single dorsal column neuroblast per row. This is consistent with achaete expression in the intermediate-column neuroectoderm. (2) Intermediate-column neuroectoderm assumes a novel cell fate that is incompatible with neuroblast formation. This hypothesis (Weiss, 1998) is supported by data showing that alterations in neuroectoderm cell fate along the AP axis can lead to reduced neuroblast formation without affecting proneural gene expression (Chu-LaGraff, 1993).

ind may act in parallel to the known proneural genes to promote neuroblast formation in the intermediate column. Similarly, vnd is thought to promote neuroblast formation by proneural-dependent and proneural-independent pathways (Jimenez, 1995 and McDonald, 1998). Vnd and Ind could promote neuroblast formation by transcriptionally activating known or novel proneural genes; by transcriptionally repressing neurogenic genes (e.g., Notch), or by regulating genes currently unlinked to the proneural or neurogenic pathways (Weiss, 1998).

Conserved properties of the Drosophila homeodomain protein, Ind

Ind-Gsh-type homeodomain proteins are critical to patterning of intermediate domains in the developing CNS; yet, the molecular basis for the activities of these homeodomain proteins is not well understood. This study identifies domains within the Ind protein that are responsible for transcriptional repression, as well as those required for its interaction with the co-repressor, Groucho. To do this, a combination of chimeric transient transfection assays, co-immunoprecipitation and in vivo expression assays are utilized. IndÂ’s candidate Eh1 domain is shown to be essential to the embryonic repression activity of this protein, and that Groucho interacts with Ind via this domain. However, when activity is assayed in transient transfection assays using Ind-Gal4 DNA binding domain chimeras to determine domain activity, the repression activity of the Eh1 domain is minimal. This result is similar to previous results on the transcription factors, Vnd and Engrailed. Furthermore, the Eh1 domain is necessary, but not sufficient, for binding to Groucho; the C terminus of Ind, including the homeodomain also affects the interaction with this co-repressor in co-immunoprecipitations. Finally, this study shows that aspects of the cross-repressive activities of Ind/Gsh2-Ey/Pax6 are evolutionarily conserved. Taken together, these results point to conserved mechanisms used by Gsh/Ind-type homeodomain protein in regulating the expression of target genes (Van Ohlen, 2007b).

The data presented in this study indicate that the capacity of ind to repress target gene expression is conferred not only by its ability to interact with Groucho through its Eh1 domain, but also by secondary domains, which include the C terminus of the protein, wherein resides the homeodomain. Indeed, deletion of Ind's C terminus affects the repressor activity of Ind in Gal4-Ind chimeric assays in tissue culture. Ind's physical interaction with Groucho suggests that this transcription factor uses redundant protein-protein interactions to exert maximal repressor activity (Van Ohlen, 2007b).

Ind's candidate Eh1 domain is required for Ind-mediated repression in embryos and in vitro. Apart from the homeodomain, this is the only Ind region that is highly conserved between flies and vertebrates. Moreover, the co-immunoprecipitation data indicate that Ind's secondary structure is important for efficient Groucho binding. The fact that the full requirement for Ind's Eh1 domain is masked in the transient transfection assay can be explained by this observation, which coincidentally parallels previous findings for the Eh1 domain of Engrailed, Nkx6, and Vnd using chimeric transfection assays. Previous studies have shown that the sequestering of Groucho to its DNA-bound transcription factor target, Dorsal, requires secondary DNA binding proteins, including Dead Ringer and Cut. Potentially, the binding of Ind to DNA via the Gal4 DBD, rather than the homeodomain, results in an altered Ind conformation relative to when the native protein contacts its DNA target via its homeodomain. This could in turn result in less efficient Groucho binding to the chimeric Gal4-Ind proteins in the transfection assay. Indeed, the transcription factor, Pax 2, must be bound to its bone fide Pax 2 target for Groucho recruitment (Van Ohlen, 2007b).

Dichaete and Sox neuro interact genetically with ind. An ac enhancer represses expression of that gene, when tested in a reporter assay in transgenic embryos. It contains 3 Ind binding sites adjacent to a single Dichaete binding site. When all four sites are mutated, the reporter is partially de-repressed relative to the wild-type reporter in transgenic embryos. In addition, Ind physically interacts with Dichaete in a yeast two-hybrid expression assay. These results, and the demonstration that Ind interacts with the co-repressor, Groucho, possibly explain the relatively weak Ind over-expression phenotype, despite strong expression of the transgene. Perhaps the limited (wild-type) availability of Groucho and Dichaete in cells that over-express Ind leads to the titration, and depletion, of these essential co-repressors, such that some ectopic Ind molecules cannot exert their regulatory effects maximally (Van Ohlen, 2007b).

A major function of Ind/Gsh-type transcription factors is the restriction of the expression domains of proneural genes to distinct subsets of progenitors. The proneural gene, ac, is ectopically expressed in ind mutants, and this ectopic expression of ac expression leads to the loss of intermediate neuroblasts. This study shows that over-expression of ind causes down-regulation of ac in both ventral and lateral neuroblasts. Similarly, the proneural genes, neurogenenin 1 and 2, are ectopically expressed in gsh2 mutants. Moreover, just as Gsh2 represses Pax 6 in an adjacent domain, it was similarly found that Ind can repress eyeless, the Drosophila Pax 6 homologue. Ind and its vertebrate homologues differ however in their capacity to repress msh/msx genes. Whereas, the ability of ind to repress msh expression is critical to maintaining the tri-columnar organization of the neuroectoderm in Drosophila , Msx 1 expression is unaffected in gsh1; gsh2 double mutants, and the expression domains of these two proteins overlap. Thus, Ind shares many common properties with its vertebrate homologues, but also has repression targets that are not evolutionarily conserved. The non-conserved repression domains identified in Ind, additional to the Eh1 domain, may explain the divergence in the capacity of Ind/Gsh homeodomain proteins to repress Msx-msh gene expression. Further work is required to address whether the secondary repression domains in Ind are functionally significant in the embryo. In addition, whether primary protein structure alone accounts for some of the divergent activities of ind and gsh1 or gsh2 needs to be addressed, by determining whether ind's vertebrate homologues can functionally substitute for ind function in the Drosophila embryo (Van Ohlen, 2007b).

Role of en and novel interactions between msh, ind, and vnd in dorsoventral patterning of the Drosophila brain and ventral nerve cord

Subdivision of the neuroectoderm into discrete gene expression domains is essential for the correct specification of neural stem cells (neuroblasts) during central nervous system development. This study extends knowledge on dorsoventral (DV) patterning of the Drosophila embryonic brain and uncovers novel genetic interactions that control expression of the evolutionary conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind), and muscle segment homeobox (msh). Cross-repression between Ind and Msh was shown to stabilize the border between intermediate and dorsal tritocerebrum and deutocerebrum, and both transcription factors are competent to inhibit vnd expression. Conversely, Vnd segment-specifically affects ind expression; it represses ind in the tritocerebrum but positively regulates ind in the deutocerebrum by suppressing Msh. These data provide further evidence that in the brain, in contrast to the trunk, the precise boundaries between DV gene expression domains are largely established through mutual inhibition. Moreover, it was found that the segment-polarity gene engrailed (en) regulates the expression of vnd, ind, and msh in a segment-specific manner. En represses msh and ind but maintains vnd expression in the deutocerebrum, is required for down-regulation of Msh in the tritocerebrum to allow activation of ind, and is necessary for maintenance of Ind in truncal segments. These results indicate that input from the anteroposterior patterning system is needed for the spatially restricted expression of DV genes in the brain and ventral nerve cord (Seibert, 2010)

The spatial and temporal order in which the DV genes (vnd, ind, and msh) are activated in neuromeres of the brain differs from their appearance in the trunk neuroectoderm, and those differences seem to be basic for the segment-specific regulation of vnd, ind, and msh expression. In the early trito- and deutocerebrum, Vnd is expressed not only in the ventral but also in the intermediate neuroectoderm, where cross-repression between Vnd and dorsally expressed Msh establishes the border between intermediate and dorsal neuroectoderm. Since Msh was found to be an ind repressor, the repression of msh via Vnd is a prerequisite for ind to become activated in the intermediate tritocerebrum (anterior) and deutocerebrum. In the trunk, ind expression in the intermediate neuroectoderm starts before that of msh in the dorsal neuroectoderm, and msh and vnd domains do not abut; accordingly, repressive interaction between Msh and Vnd is not required (Seibert, 2010)

In the tritocerebrum, Vnd not only acts as repressor of msh but also of ind, in contrast to the deutocerebrum. When the level of Vnd protein in the intermediate tritocerebrum declines with time (down-regulated through the activity of Ems), ind becomes subsequently activated. In the trito- and deutocerebrum, instead of Vnd, increasing levels of Ind, together with the recently uncovered msh-repressor Nkx6, still keep msh expression limited to the dorsal neuroectoderm. Since it was found that Nkx6 expression starts earlier and persists longer than that of ind in both brain neuromeres, and additionally, that msh is expanded into the intermediate neuroectoderm in Nkx6 but not in ind mutants, it is proposes that Nkx6 represses msh more efficiently (Seibert, 2010)

The most striking difference in DV gene regulation leads to the question how vnd and ind can be co-expressed in the anterior deutocerebrum (during stages 6–9), if Vnd is a repressor of ind and, vice versa, Ind is also capable of preventing vnd expression in the neuroectoderm. It has been reported recently that the repressor activity of Ind on vnd seems to be stage-specific, not taking place before stage 9. By contrast, Vnd repression of ind seems independent of the developmental period. In this context, it is interesting that activity of Vnd can be modified by EGFR signalling, which is supposed to affect the selective interaction of Vnd with co-factors necessary to mediate repression or activation of target genes. Availability of co-factors might also account for the specific situation of vnd and ind co-expression in the anterior deutocerebrum that was observed specifically during early stages of development (Seibert, 2010)

Involvement of en, which can act as transcriptional repressor as well as activator, has been implicated in diverse developmental processes in Drosophila such as compartmentalization in the early embryo, modulation of Hox gene expression, or regulation of molecules that directly govern axon growth (e.g. frazzled). This study demonstrates a novel function for En in the early embryo, that is to control the spatially restricted expression of the DV genes in the neuromeres of the posterior brain (trito- and deutocerebrum) and ventral nerve cord. In the posterior compartment of the deutocerebrum, En represses expression of msh and ind, but maintains expression of vnd. Since it was found that Ind (later) becomes a vnd repressor, this indicates that En maintains expression of vnd by repressing ind. In the posterior compartment of the tritocerebrum, En is also required for down-regulation of Msh, but opposite to the deutocerebrum, En is necessary for activation of ind. This study shows that Msh is an ind repressor, its repression by En seems to allow for activation of ind; yet, it cannot be excluded that En in addition directly activates ind expression. Similar to the situation in the tritocerebrum, En seems to negatively regulate expression of msh and to positively regulate expression of ind (as a maintenance factor) in the neuroectoderm of the ventral nerve cord. Together, these data suggest that the AP patterning gene engrailed is crucially involved in fine-tuning the regionalized expression of distinct DV genes in the posterior compartment of neuromeres in the brain and ventral nerve cord. En may act as a positive or negative transcriptional regulator depending on the gene that is regulated and on the segmental context. For DV genes it is known that they control formation and specification of brain neuroblasts. Since all the genetic interactions between En and DV genes take place during the period when neuroblasts develop, it is likely that En, via regulation of DV genes, controls formation and fate specification of neuroblasts in the brain (Seibert, 2010)

It was observed that cross-repressive interaction between pairs of DV gene factors in the brain (i.e. in trito- and deutocerebrum) is essential for the establishment and maintenance of discrete DV gene expression domains. Early, cross-repression between Ems/Vnd pre-patterns the ventral and intermediate neuroectoderm in both neuromeres. Mutual repression between Msh/Nkx6 and Msh/Ind maintains the dorsal/intermediate neuroectodermal border in trito- and deutocerebrum, and between Ind/Vnd the intermediate/ventral border in the tritocerebrum. All these genetic interactions, and the observation that Msh and Vnd act as mutual repressors, are not in compliance with the concept of ventral dominance (as proposed in the neuroectoderm of the ventral nerve cord where the more ventral gene represses the gene expressed more dorsally) but rather support the model that in the brain cross-repression between DV factors is crucial for stabilizing these borders (Seibert, 2010)

However, despite the ability of Msh and Ind to repress vnd, neither factor seems to be sufficient to define the dorsal border of vnd expression in trito- and deutocerebrum, as has been shown for Ind in the ventral nerve cord (from stage 9 onwards). Instead of reinforcing this border through repressive interaction, vnd expression in the brain could also be limited by a (too) low concentration or absence of an activator, like Dorsal (as has been speculated for the trunk), or be regulated by BMP signalling in a dosage-dependent fashion. Neuromere-specific differences are also observed regarding limitation of ind and msh expression domains along the DV axis. Vnd establishes the ventral border of ind expression in the trunk and tritocerebrum, but not in the deutocerebrum or protocerebrum (where the expression domains of ind and vnd do not abut). ind expression was found to be limited dorsally by repression through Msh in the trito- and deutocerebrum, but not in the protocerebrum (where msh is not expressed before stage 11) or trunk, although evidence is available that Msh might act in rendering the dorsal border of ind expression more precisely in the ventral nerve cord. Taking into account that ind expression does not expand into the complete dorsal neuroectoderm of trito- and deutocerebrum in msh mutants, this may also indicate an involvement of the nuclear Dorsal gradient, possibly in concert with graded activity of EGFR (as was shown for the trunk neuroectoderm), or BMP (which can repress ind in the trunk neuroectoderm), in establishing a rough dorsal border of ind expression that is further defined and stabilized via repression by Msh. Whereas Vnd is initially responsible for keeping msh expression confined to the dorsal neuroectoderm in trito- and deutocerebrum, it is only indirectly involved in defining the ventral border of msh expression in the trunk neuroectoderm. Later in development Ind helps to maintain repression of msh in trito- and deutocerebrum (together with Nkx6), which is in contrast to the trunk where Ind directly establishes the ventral limit of msh expression from the beginning (Seibert, 2010)

DV neuroectodermal and corresponding stem cell domains in the Drosophila brain become established and maintained through cross-repressive regulation, and it has been speculated that such genetic interactions are more common in the fly brain. This study has presented further examples supporting this hypothesis. Notably, this is a feature that bears similarity to DV patterning in the neural tube of vertebrates where cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains (Seibert, 2010)

All interactions between DV genes in the brain identified so far are based on the interplay of transcriptional repressors. Likewise, this study shows that Vnd does not act as a direct activator to positively regulate ind, but according to a double-negative mechanism, it suppresses the ind-repressor Msh. It has been shown previously, that interactions of the AP patterning gene ems with the DV genes (vnd, ind, msh, and Nkx6) are indispensable for proper development of the trito- and deutocerebrum. This study demonstrates that the segmentation gene en is significantly involved in regionalization of DV gene expression domains, thus representing a further example of an AP patterning gene integrating into the DV gene regulatory network that patterns the brain. This study has shown that En acts differently on the respective DV genes, but no evidence is available that, vice versa, DV genes control en, as has been observed for expression of ems. DV genes, as well as En and Ems, all contain an Eh1 repressor domain and are able to interact with the co-repressor Groucho (Gro), and thus are capable of mediating repression on target genes (including each other). But how could it be possible that all DV genes interact with the same co-factor to stabilize expression domains by conferring repression onto genes expressed in neighboring domains? In the first place, the DV genes display spatio-temporal differences in their respective expression. In addition, conformational changes of the protein seem to be necessary to enable binding of Gro which has been at least shown for Nkx6. It has been observed that Vnd can be phosphorylated by activated MAPK and is present in different isoforms in the developing embryo, which most likely leads to a change in its binding partners. Another critical point could be inactivation of the co-repressor, in case of Gro also through phosphorylation by activated MAPK, or modification of target genes so that binding of the repressor complex is impaired. Still, that the DV genes are able to interact with Groucho, does not exclude that their repressor activity is Gro-independent, since also other repressor domains have been reported for these genes, as well as activator domains, at least for Vnd, Ind, and Nkx6. Whether the DV gene products function as repressors or activators seems to depend on co-factor availability as well as on the respective target gene, since not only the presence of a transcriptional binding site, but also its accessibility is limiting in this context (Seibert, 2010)



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).

Lateral gene expression in Drosophila early embryos is supported by Grainyhead-mediated activation and tiers of dorsally-localized repression

The general consensus in the field is that limiting amounts of the transcription factor Dorsal establish dorsal boundaries of genes expressed along the dorsal-ventral (DV) axis of early Drosophila embryos, while repressors establish ventral boundaries. Yet recent studies have provided evidence that repressors act to specify the dorsal boundary of intermediate neuroblasts defective (ind), a gene expressed in a stripe along the DV axis in lateral regions of the embryo. This study shows that a short 12 base pair sequence ('the A-box') present twice within the ind CRM is both necessary and sufficient to support transcriptional repression in dorsal regions of embryos. To identify binding factors, affinity chromatography using the A-box element was conducted and a number of DNA-binding proteins and chromatin-associated factors were found using mass spectroscopy. Only Grainyhead (Grh), a CP2 transcription factor with a unique DNA-binding domain, was found to bind the A-box sequence. The results suggest that maternally expressed Grh acts as an activator to support expression of ind, which was surprising since this factor was identified using an element that mediates dorsally-localized repression. Grh and Dorsal both contribute to ind transcriptional activation. However, another recent study found that the repressor Capicua (Cic) also binds to the A-box sequence. While Cic was not identified through the A-box affinity chromatography, utilization of the same site, the A-box, by both factors Grh (activator) and Cic (repressor) may also support a 'switch-like' response that helps to sharpen the ind dorsal boundary. Furthermore, the results also demonstrate that TGF-beta signaling acts to refine ind CRM expression in an A-box independent manner in dorsal-most regions, suggesting that tiers of repression act in dorsal regions of the embryo (Garcia, 2011).

Other studies have shown combinatorial interactions are necessary to support patterns of gene expression along the DV axis. For instance, one study showed Dorsal and Zelda function together to produce the broad lateral domain of sog. Mutation of either the Dorsal sites or the Zelda sites in the sog CRM produced a pattern that was narrower than the wild-type expression pattern. It was concluded that both Dorsal and Zelda must be present to produce a proper Sog pattern. It is also well appreciated that Dorsal can act cooperatively with the bHLH transcription factor Twist to support expression in ventral and ventrolateral regions of the embryo. It is proposed that Grh and Dorsal act together to support the ind expression pattern. While the ind CRM containing a mutant Dorsal site did support some expression, the expression pattern contained a gap and was weaker in posterior regions; in contrast, in Dorsal mutants, ind expression is completely absent. This result may be explained if both indirect as well as direct functions for Dorsal are required to support ind expression. For instance, Dorsal has other target genes including rho, which is required to support Egfr signaling. Furthermore, mutation of the A-box/Grh binding site within the ind CRM caused expression of the reporter that was expanded dorsally and weak, suggesting this site mediates repression and also activation. Similar to Dorsal mutants, the phenotype observed when the A-box sites were mutated is different than the phenotype in the Grh mutants, thus it cannot be ruled out that Grh may act through other sites as well as the A-box and/or that Grh may act indirectly to influence ind expression by regulating the expression of other transcription factors. A model is proposed that is most consistent with the current data which is that ind is activated in regions where Dorsal is present as well as optimal levels of Grh; it is then refined by Snail and Vnd in ventral regions and Cic and Schnurri/Mad/Medea (SMM) in dorsal regions (Garcia, 2011).

grh and cic genes are both maternal and ubiquitously expressed, thus, another input is necessary to explain how localized expression of ind is supported. This positional information could be provided in part by competition between Grh and Cic proteins for the A-box binding site and in part by ventrolaterally-localized Egfr signaling. A model in which Egfr signaling supports activation of ind via inhibition of a ubiquitous repressor (e.g. Cic) is supported by the results which demonstrate that A-box mediated repression is expanded in Egfr mutants. A recent study also showed expanded expression of an ind CRM fragment reporter in ras cic double mutants in which neither Egfr signaling or Cic repressor is present, suggesting that Egfr may function by inhibition of an 'inhibitor' to promote activation. This data suggests that the putative A-box repressor, Cic, may not be dorsally localized but that its activity is regulated by Egfr signaling which provides the positional information necessary for a sharp boundary. However, the domain of dpERK activation (as detected by anti-dpERK, an antibody to the dual-phosphorylated from of ERK) does not exactly overlap with the ind expression domain at cellularization, as would be expected in the simplest model (Garcia, 2011).

Ajuria (2011) suggested that Egfr signaling supports ind expression through inhibition of Cic, and it is added that it is also plausible Egfr signaling impacts activation of ind through Grh. In fact, a recent study showed that Grh activity during wound response is modulated by ERK signaling. Specifically, both unphosphorylated and phosphorylated Grh were shown to be able to bind DNA and act as an activator. The former is used during normal development of the epidermal barrier and the latter is used to overcome a semi-dormant state during wound response. Another study showed the tyrosine kinase Stitcher activates Grh during epidermal wound healing. In the early embryo Grh may be phosphorylated by Egfr signaling to support activation of ind through the A-box binding site. It is suggested that phosphorylation of both Grh as well as Cic by Egfr signaling can act as a switch to help fine-tune the expression of ind (Garcia, 2011).

Whether a relationship between Grh activation and Cic repression was used in regulation of other genes containing A-box or Cic binding sites was investigated. One other Cic target gene, hkb, was unaffected in Grh mutants. As the A-box site (WTTCATTCATRA) is larger than the Cic consensus binding sequence [T(G/C)AATGAA, complement TTCATT(G/C)A] defined by Ajuria (2011) it is possible that Grh needs the full A-box site to bind. The full A-box sequence is not present in the hkb CRM, but Cic binding may be facilitated by a partial sequence (i.e., TGAATGAA). Alternatively, it is possible that a role for Grh and/or Cic at the A-box is context dependent. For instance, Grh-mediated activation may be a necessary input to support ind expression but not for the support of hkb, which also receives activation input from Bicoid and Hunchback transcriptional activators and is expressed in the pre-cellularized embryo (Garcia, 2011).

Other studies have suggested that Grh acts to repress transcription of fushi tarazu (ftz), dpp, and tll in the Drosophila embryo, but this study is the first to identify a role for Grh-mediated gene activation in the early embryo, in support of dorsoventral patterning. Previous studies had shown that Grh can function as an activator at later embryonic stages. One analysis identified Grh (also called NTF-1 or Efl-1) biochemically using an element from the dpp early embryonic CRM, however the dpp expression domain was unchanged in the grh mutants (Garcia, 2011).

Another recent study also showed Grh binds to sites that are similar to Zelda binding sites (Harrison, 2010). Zelda and Grh each showed stronger affinity for different variations of the shared consensus sequence, but in vitro studies showed they also competed for binding. Harrison (2010) proposed that as levels of Zelda increase it is able to compete against Grh for binding sites and cause activation of the first zygotic genes. Competition at the same binding sites results in a cascading effect in which ubiquitous activators regulate genes in a temporally related manner. It was proposed that Grh functions first to silence gene expression; while, alternatively, the current data is more consistent with a model in which Grh mediated activation follows that of Zelda. ind is considered a 'late' response gene as it appears at mid stage 5 (nc 14), at the onset of cellularization, whereas Zelda was shown to support gene expression earlier at nc 10 (Garcia, 2011).

It is possible that Grh competes for binding to a variety of sites (not only those recognized by Zelda), and that this competition influences gene activation/repression. At the A-box sequence, Cic and Grh may compete to help establish a sharp boundary; unfortunately, the Cic binding to the A-box sequence demonstrated previously in vitro was quite weak (Ajuria1, 2010), so this competition is best examined in vivo in future studies (Garcia, 2011).

This study found there is yet another tier of repression activity that is independent of the A-box mediated repression. Analysis of the eve.stripe3/7-ind-mutant-A-box reporter construct revealed that, while dorsal-lateral repression was lost, there was still repression in the dorsal-most part of the embryo. This led to the idea that other binding sites in the ind CRM, independent of the A-box binding site, mediate repression. Previous research showed ectopic TGF-beta/Dpp signaling can repress ind expression, and therefore it is hypothesized that the repression activity observed in dorsal-most regions of the embryo may be regulated by Dpp signaling (Garcia, 2011).

The results suggested that the Dpp dependent repression supports repression in the dorsal most part of the embryo and not in dorsal lateral regions of the embryo. An expansion of the ind domain in the mutants affecting only this dorsal-most repressor would not be expected, thus the SMM site was mutated in the context of two mutant A-boxes and it was found that the expression pattern was expanded into dorsal regions of the embryo. However, when the A-box sites were mutated, expansion of ind more dorsally into dorsal-lateral regions was seen, but expression was absent in dorsal-most regions. It is possible the embryo can tolerate a slight expansion of ind into dorsal lateral regions of the embryo but expansion of ind into the non-neurogenic ectoderm is detrimental. Thus, two tiers of repression have developed to insure that expression of ind is limited to the neurogenic ectoderm. It is suggested that partially redundant repressor mechanisms are more common than appreciated, because in contrast to activation it is difficult to track repression activity (Garcia, 2011).

Epigenetic changes to DNA and chromatin remodeling have been shown to be vital in repression and activation of genes that define structures in late stages of Drosophila development. For example, Polycomb group genes silence the homeotic genes of the Bithorax complex, which control differentiation of the abdominal segments. To date, little is known regarding how/if chromatin factors play a role in early development of Drosophila embryos. This study has presented evidence that several chromatin-related factors bound an A-box affinity column but did not bind a column containing the mutant A-box element. Although several of these factors did not bind to the A-box element alone when tested by EMSA, it is possible that they bind indirectly via a larger complex. One of these factors Psq has been implicated in both silencing and activation via the Polycomb/Trithorax response elements. Independently, Psq was recently found to positively regulate the Torso/RTK signaling pathway in the germline, while being epistatic to cic a negative regulator of the Torso signaling. It is possible that some of these factors play a role in regulating ind via the A-box element, which would suggest a role for chromatin remodeling early in development - an avenue which is worth pursuing in future studies (Garcia, 2011).

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 Tollrm9/Tollrm10 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).

A Myc-Groucho complex integrates EGF and Notch signaling to regulate neural development

Integration of patterning cues via transcriptional networks to coordinate gene expression is critical during morphogenesis and misregulated in cancer. Using DNA adenine methyltransferase (Dam)ID chromatin profiling, protein-protein interaction between the Drosophila Myc oncogene and the Groucho corepressor was identified that regulates a subset of direct dMyc targets. Most of these shared targets affect fate or mitosis particularly during neurogenesis, suggesting the dMyc-Groucho complex may coordinate fate acquisition with mitotic capacity during development. An antagonistic relationship was found between dMyc and Groucho that mimics the antagonistic interactions found for EGF and Notch signaling: dMyc is required to specify neuronal fate and enhance neuroblast mitosis, whereas Groucho is required to maintain epithelial fate and inhibit mitosis. The results suggest that the dMyc-Groucho complex defines a previously undescribed mechanism of Myc function and may serve as the transcriptional unit that integrates EGF and Notch inputs to regulate early neuronal development (Orian, 2007).

Gro is a downstream transducer of several signaling pathways and was placed at the crossroads of the Notch and EGF signaling pathways during patterning of the Drosophila nervous system, where EGF-induced site-specific phosphorylation of Gro attenuates it repression activity. During embryonic stage 9, the CNS matures in three bilaterally symmetrical longitudinal rows of neuroblasts, with the homeobox transcription factors, Vnd, Ind, and Msh, specifying the medial (ventral), intermediate, and lateral rows, respectively. EGF regulates the expression of both Vnd and Ind and is thus required for the formation of the ventral and intermediate rows. Interestingly, both Vnd and Ind are among the 38 dMyc-Gro shared targets identified in this study. Gro and dMyc, but not dMnt, are expressed in neuroblasts of stage 9 embryos. Because dMyc-Gro targets are associated with both neuroblast fate and mitosis, it is hypothesized that EGF and Notch coregulate cell fate and mitosis within the developing neuroectoderm via dMyc-Gro antagonism. Vnd expression (a shared Myc-Gro target whose expression overlaps with and is required for establishment of S1 neuroblasts), the overall number of neuroblasts, and mitotic activity in wild-type embryos were compared to groe47 loss-of-function (LOF) mutants (in which the maternal contribution of Gro is removed), Egfr2, or Notch55e11 [note that dMyc LOF embryos cannot be generated]. These parameters were also evaluated in embryos overexpressing either dMyc or Gro using the conditional Gal4/upstream activating sequence (UAS) expression system. Vnd expression is stronger and expanded in both Notch and gro LOF embryos, as well as in embryos overexpressing dMyc when compared with wild type. These mutants also show neuroblast hyperplasia and elevated mitotic activity. Furthermore, Egfr LOF or Gro-overexpressing embryos show reduced Vnd expression, neuronal hypoplasia, and reduced mitotic activity, consistent with the molecular nature of the dMyc-Gro common targets (Orian, 2007).

Myc proteins are required for both cell growth/size and cell proliferation. The model in which Myc functions are mediated by heterodimerization with Max and antagonized by Mxd (Mad/Mnt) proteins has been well established. However, recent studies suggest that a set of interactions outside the canonical Myc/Max/Mxd network also regulate some of Myc's functions. Interestingly, the current studies point to a subset of dMyc direct targets that are not shared with either dMax or dMnt. Furthermore, dMnt-Dam and dMax-Dam were not recruited to these dMyc targets even in experiments where the Dam fusions were coexpressed in the presence of high levels of dMax or dMyc, respectively, suggesting that previously uncharacterized mechanisms may mediate Myc's recruitment to DNA, and proteins other than dMnt may antagonize its transcriptional activity on this set of targets. This study reports the identification of Gro as the first component in a pathway that antagonizes dMyc function independent of dMnt and operates during Drosophila neurogenesis (Orian, 2007).

Transcriptionally, dMyc was found to be positively required for the expression of dMyc-Gro targets, activity that is antagonized by Gro. Importantly, dMyc is not a Gro target, and reducing Gro levels does not affect dMyc protein levels. Furthermore, Gro antagonism is limited only to the dMyc-Gro subset of shared targets and does not involve dMnt: there is no overlap between genes bound by dMnt or Gro, dMnt is not expressed in cells where the dMyc-Gro interaction is observed, RNAi to dMnt does not affect Myc-Gro shared target expression, and overexpression of dMnt does affect PNS development (Orian, 2007).

Although the possibility that dMyc-Gro targets are coregulated by individual dMyc and Gro complexes cannot be excluded, the results suggest that dMyc and Gro are part of a single larger protein complex. First, the observation that RNAi to dMyc results in reduction of target expression and is restored by coreducing Gro suggests that other activators coregulate shared target expression along with dMyc. Second, biochemical purification, binding data, and DNA adenine methyltransferase (Dam)ID Southern analyses support the idea that both proteins physically interact with one another yet associate with DNA through distinct binding sites. Third, Gro does not bind directly to DNA but must be recruited to targets by sequence-specific DNA-binding transcription factors. Fourth, most of the dMyc-Gro targets lack E-box sequences associated with canonical Myc network targets, suggesting that dMyc and Gro may be recruited to shared targets via a novel mechanism or by other protein(s) yet to be identified. Candidates for recruiting Gro may be the E(spl) proteins that convey the Notch signal, antagonize the EGF pathway, interact with Gro, and exhibit similar phenotypes. Thus, the identification of the entire dMyc-Gro complex and its regulation will be an important next step (Orian, 2007).

Gro's role as a downstream transducer of Notch signaling during neurogenesis is well documented, and mounting evidence supports Myc as a key player in progenitor cell proliferation. This study has identified a previously undescribed role for dMyc, together with Gro, during Drosophila early neuronal development. dMyc and Gro are required to directly regulate key fate controlling genes such as the homeodomain proteins vnd and ind that are downstream targets of EGF signaling. Because Vnd was identified as a regulator of the proneural gene complex, the differential regulation of vnd by dMyc and Gro implicates them as antagonistic regulators upstream of proneural genes. Thus, it is proposed that dMyc is transiently required within the neuroectoderm, where it promotes specific fate acquisition and allows mitotic expansion of committed neuronal cells (Orian, 2007).

Phenotypically, it was observed that, similar to EGF, dMyc promotes neurogenesis both in the PNS and CNS, whereas Gro and Notch inhibit neuroblast formation and mitosis. This is a different role than that previously ascribed to dMyc, because it is usually associated with regulation of cell size and organismal growth, functions that are antagonized by dMnt. Consistent with this, a recent study identified EGF-induced phosphorylation of c-Myc, Max, and TLE proteins in mammalian cells. The antagonistic relationship of Myc/EGF to Gro/Notch is likely to be highly dependent on the developmental context and the specific progenitor niche. For example, in cellular contexts in which Notch promotes proliferation, such as during the development of T cells in acute leukemia, Myc is a direct target of mutated Notch1 and is required for T cell proliferation and development. The current findings also fit well with observations that N-Myc is required during mouse progenitor development, and that the fly tumor suppressor Brat regulates dMyc levels posttranscriptionally in larval neuroblasts resulting in a 'tumorous' phenotype (Orian, 2007).

Taken together, the snapshot provided by DamID data leads to the suggestion of a model in which changes in neuronal progenitor fate and mitosis are determined by the balance between EGF and Notch signaling that is likely transcriptionally mediated by the dMyc-Gro complex. During epithelial development, Notch, like Gro, is required to specify and maintain epithelial fate. It is proposed that Gro sequesters dMyc in an inactive multiprotein complex formed by associating with dMyc, preventing the activation of dMyc-Gro shared targets. Upon EGF signaling, a molecular switch takes place whereby Gro is phosphorylated, and its repression is attenuated. dMyc, as part of an as-yet-to-be-identified activation complex, is then liberated to activate zygotic transcription of a subset of targets that determines neuronal fate and enhances mitosis. One of these targets is dMax, which is specifically expressed in the neuroectoderm. Activation of dMax would be expected to establish a feed-forward loop required for the subsequent activation of (E box-containing) Myc targets to promote cell growth. As development progresses, the dMnt gene would be induced, and dMnt-dMax complexes would replace dMyc-Max complexes, thereby promoting cellular differentiation (Orian, 2007).

Finally, both EGF/dMyc and Notch/Gro misregulation and mutation are intimately involved in hematological, epithelial, and neuroectodermal cancers. Thus, identification of a dMyc-Gro complex that could serve as a molecular junction to integrate EGF and Notch signaling inputs is highly relevant for both developmental biology and cancer (Orian, 2007).

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).

Ems and Nkx6 are central regulators in dorsoventral patterning of the Drosophila brain

In central nervous system development, the identity of neuroblasts critically depends on the precise spatial patterning of the neuroectoderm in the dorsoventral (DV) axis. This study has uncovered novel gene regulatory network underlying DV patterning in the Drosophila brain; the cephalic gap gene empty spiracles (ems) and the Nk6 homeobox gene (Nkx6) encode key regulators. The regulatory network implicates novel interactions between these and the evolutionarily conserved homeobox genes ventral nervous system defective (vnd), intermediate neuroblasts defective (ind) and muscle segment homeobox (msh). Msh cross-repressively interacts with Nkx6 to sustain the boundary between dorsal and intermediate neuroectoderm in the tritocerebrum (TC) and deutocerebrum (DC), and Vnd positively regulates Nkx6 by suppressing Msh. Remarkably, Ems is required to activate Nkx6, ind and msh in the TC and DC, whereas later Nkx6 and Ind act together to repress ems in the intermediate DC. Furthermore, the initially overlapping expression of Ems and Vnd in the ventral/intermediate TC and DC resolves into complementary expression patterns due to cross-repressive interaction. These results indicate that the anteroposterior patterning gene ems controls the expression of DV genes, and vice versa. In addition, in contrast to regulation in the ventral nerve cord, cross-inhibition between homeodomain factors (between Ems and Vnd, and between Nkx6 and Msh) is essential for the establishment and maintenance of discrete DV gene expression domains in the Drosophila brain. This resembles the mutually repressive relationship between pairs of homeodomain proteins that pattern the vertebrate neural tube in the DV axis (Seibert, 2009).

This study shows that the evolutionarily conserved homeodomain protein Ems is an integral component of the gene regulatory network that governs DV patterning in the posterior brain neuromeres, the TC and DC. This novel function is surprising because ems has hitherto been exclusively connected with patterning functions along the AP axis. It has been proposed that the combined activities of the gap genes ems, buttonhead and orthodenticle (ocelliless - FlyBase) generate head segments and that ems mutants exhibit defects in the formation of the intercalary and antennal segment as well as in the corresponding TC and DC in accordance with the early pattern of ems expression. ems probably also has a homeotic function in specifying aspects of intercalary segment identity. This study provides evidence that another crucial function of Ems is its cross-repressive interaction with Vnd. Previously, it was shown that vnd expression is dynamic and exhibits specific differences in the TC and DC. This study demonstrates that Ems is involved in the regulation of brain-specific differences in vnd expression, and that Vnd acts to repress ems in complementary parts of the TC and DC. These interactions help to refine the pattern into mutually exclusive domains at the onset of neurogenesis, which is important as both genes provide positional information that subsequently specifies the identity of individual brain NBs. Depending on the context, Vnd/Nkx2 can act as a transcriptional activator or repressor, as determined by physical interaction with the co-repressor Groucho, which enhances repression. Interestingly, it was observed that Ems also regulates the expression of two Nkx genes in an opposing manner: it represses vnd/Nkx2 but is necessary to activate Nkx6. The repressor function of Ems most likely also depends on Groucho; Ems has been reported to bind Groucho in vitro (Seibert, 2009).

In ems mutants, defects in proneural gene expression (lethal of scute and achaete) are restricted to NE regions where ems is normally expressed during early neurogenesis, leading to the loss of a subset of NBs in the TC and DC. This contrasts with the phenotype of the late embryonic ems mutant brain, which exhibits a severe reduction, or entire elimination, of the TC and DC, suggesting that the proper development of a larger NE domain and/or fraction of NBs in the TC and DC must be affected. However, in ems mutants the organization of the early procephalic NE appears normal until stages 9/10 and apoptosis is not detected. A possible explanation for the subsequent complete loss of TC and DC is that in ems mutants, vnd becomes derepressed in the ventral/intermediate NE of both neuromeres, and expression of msh, ind and Nkx6 is not activated. It has been shown that ectopic vnd prevents the expression of many NB identity genes. Indeed, the expression of a number of molecular markers has been reported to be absent in the ems mutant brain. It is therefore conceivable that in the TC and DC of ems mutants, as a consequence of lacking ems and ectopic vnd (and the absence of proneural gene activation), some NBs do not form. Additionally, owing to mis-specification of the NE (where neural identity gene expression is absent or altered), the other NBs and their progeny might still form but degenerate at later stages (Seibert, 2009).

It has been largely unclear how expression of Nkx6 is regulated in the brain NE, although Vnd has been suggested to act as a positive regulator. At the blastodermal stage, coexpression of ems and vnd is only observed in the intermediate and ventral NE of the TC and DC, which might account for early Nkx6 expression being limited to the respective NE in the brain and absent from the trunk. The data indicate that Ems and Vnd together facilitate the activation of Nkx6. Ems expression closely prefigures the domain of Nkx6 expression in the TC and DC, and together with the fact that Nkx6 is completely abolished in ems mutants, this suggests that Ems might act as a direct activator to regulate the extension of the Nkx6 domain along the AP axis. Vnd indirectly regulates the enlargement of the Nkx6 domain along the DV axis by repressing the Nkx6-repressor Msh. That DV patterning in the brain NE integrates AP signals is additionally supported by the fact that Ems is also necessary for activation of ind and msh, indicating that ems is a key regulator in DV patterning of the TC and DC. Evidence is also provided for a negative-feedback control in the DV regulatory network, in which Ems is needed to activate its own later-stage repressors, Nkx6 and Ind. Together, these data suggest not only that Ems regulates the expression of all DV genes (activating Nkx6, ind, msh and repressing vnd), but also that DV factors (Nkx6, Ind and Vnd) control expression of ems, indicating that integration of DV and AP patterning signals takes place at different levels in the DV genetic network (Seibert, 2009).

Nkx6 has been identified as specifically involved in DV patterning of the TC and DC. In addition to later suppression of ems (in concert with Ind), a further pivotal function of Nkx6 is to maintain the suppression of msh in the intermediate/ventral TC and DC that was initiated by Vnd. Since in both neuromeres the expression of Nkx6 starts before and persists longer than that of ind, and because msh is ventrally derepressed in Nkx6 but not in ind mutants, this implies that Nkx6 (but not Ind) is the major msh suppressor necessary to prevent intermediate/ventral NE and the descending NBs from adopting dorsal fates. Consequently, Nkx6 indirectly regulates the proper specification of brain NB identity by suppressing msh (and ems). Further experiments are required to show whether Nkx6 is also more directly involved in the fate specification of NBs and progeny cells in the brain, as has been shown in the VNC, where Nkx6 promotes the fate of ventrally projecting, and represses the fate of dorsally projecting, motoneurons (Seibert, 2009).

Additionally, cross-inhibitory interactions were observed between Nkx6 and Msh. It is assumed that this mutually repressive regulation in the TC and DC is necessary to stabilize the boundary between dorsal and intermediate NE, and to ensure the regionalized expression of msh and Nkx6 over time. It is likely that Nkx6 and Msh/Msx interact with the co-repressor Groucho to repress each other at the transcriptional level. Interestingly, aspects of the genetic interactions between Nkx6 and Msh/Msx seem to be evolutionarily conserved, since Msx1, which is expressed in the vertebrate midbrain and functions as a crucial determinant in the specification of dopamine neurons, represses Nkx6.1 in ventral midbrain dopaminergic progenitors of mice (Seibert, 2009).

It had not been shown until now that domains of DV gene expression in the Drosophila brain become established through cross-repressive regulation, and it is possible that such genetic interactions are more common than previously thought (e.g. Ind and Msh act as mutual inhibitors). This suggests that in the fly brain, cross-inhibition between pairs of homeodomain transcription factors is fundamental for establishing and maintaining DV neuroectodermal and corresponding stem cell domains. By contrast, in the NE of the VNC, where DV patterning is much better understood, cross-repressive interactions of homeobox genes are largely omitted. There, DV patterning is proposed to be conducted by a strict ventral-dominant hierarchy according to which ventral genes repress more-dorsal genes. However, one exception to the rule seems to be the cross-inhibitory interaction between Vnd and Ind. Interestingly, in the developing vertebrate neural tube, cross-repressive interactions of homeodomain proteins are common and indeed crucial for the establishment of discrete DV progenitor domains. This bears a marked resemblance to the mutually antagonistic relationship between pairs of homeodomain proteins that dorsoventrally pattern the fly brain (Seibert, 2009).

A predominant feature of the brain-specific DV genetic network described in this study, and a general design feature of gene regulatory networks, is the extensive use of transcriptional repression to regulate target gene expression in spatial and temporal dimensions. All factors involved in the network operate as repressors (except Ems, which may also serve as an activator), via mutual repression (between Ems and Vmd, and between Nkx6 and Msh), a double-negative mechanism (Vnd represses Msh, which represses Nkx6), and a negative-feedback loop (Ems is needed to activate Nkx6 and Ind, which in turn repress Ems). The spatial and temporal complexity of the regulatory interactions that have been deciphered implies similar complexity in the underlying cis-regulatory control of these factors. For example, the domain of msh expression is regulated by the input of at least two transcriptional repressors acting in subsequent time windows (Vnd early and Nkx6 late), and the input of at least three repressors regulates the dynamics of ems expression (Vnd early, Ind and Nkx6 late). The brain-specific DV patterning network probably comprises further genes in addition to those that have been identified, and it is likely that interactions with other putative regulators (e.g. Dorsal, Egfr, Dpp) will complement the present model. Altogether, these data provide the basis for a systematic comparison of the genetic processes underlying DV patterning of the brain between different animal taxa at the level of gene regulatory networks (Seibert, 2009).

The genetic factors considered in this study in the developing fly brain are expressed in similar NE domains from early embryonic stages onwards in the anterior neural plate in vertebrates. Emx2, for example, is expressed in the laterodorsal region, and Nkx2 genes in the ventral region, of the early vertebrate forebrain. At the four-somite stage (~E8), these two domains exhibit a common border, similar to that observed in Drosophila after Ems and Vnd have, through cross-repression, regulated their mutually exclusive expression domains. Moreover, whereas Msx genes are mainly expressed in dorsal regions of the posterior forebrain, midbrain and hindbrain, expression of Nkx6 genes is reported in more lateroventral regions, overlapping ventrally with the expression of Nkx2 genes. However, even though these patterns of gene expression exhibit certain similarities between insects and vertebrates, it remains to be shown whether their genetic interactions are also conserved (Seibert, 2009).

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 scuteB57 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 mshDelta68 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)y3PLsc8R 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).



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).

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

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

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

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

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


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 flbIK35 (referred to as Egfr mutant embryos). ind expression is absent in Egfr mutant embryos, indicating that Egfr activates ind expression in the intermediate column. By contrast, vnd expression in Egfr mutant embryos appears normal through the onset of stage 8. However, during stage 8, vnd expression begins to dissipate in medial column cells, and by early stage 10 these cells no longer express vnd. Conversely, medial column NBs that form in Egfr mutant embryos express vnd normally and retain vnd expression throughout embryogenesis. Thus, Egfr functions to maintain vnd expression in the neuroectoderm but is dispensable for vnd expression in NBs. These data indicate that Egfr resides atop the genetic hierarchy known to subdivide the neuroectoderm along the DV axis (Zhao, 2002).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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


The conservation of developmental functions exerted by Antp-class homeoproteins in protostomes and deuterostomes has suggested that homologs with related functions are present in diploblastic animals, in particular, in Hydra. Phylogenetic analyses show that Antp-class homeodomains belong either to non-Hox or to Hox/paraHox families. See Phylogenetic relationships among 200 Antp-class genes. Among the 13 non-Hox families, 9 reported here have diploblastic homologs: Msx, Emx, Barx, Evx, Tlx, NK-2, and Prh/Hex, Not, and Dlx. Among the Hox/paraHox, poriferan sequences are not found, and the cnidarian sequences form at least five distinct cnox families. Cnox-1 shows some affinity to paralogous group (PG) 1; this group includes Drosophila Labial. Cnox-2 is related to Drosophila Intermediate neuroblast defective. Cnox-3 and 5 show some affinity to PG9-10; this group includes Drosophila AbominalB. Cnox-4 has no counterparts in Drosophila or vertebrates. Intermediate Hox/paraHox genes (PG 3 to 8 and lox) do not have clear cnidarian counterparts. In Hydra, cnox-1, cnox-2, and cnox-3 are not found chromosomally linked within a 150-kb range and display specific expression patterns in the adult head. During regeneration, cnox-1 is expressed as an early gene whatever the polarity, whereas cnox-2 is up-regulated later during head but not foot regeneration. Finally, cnox-3 expression is reestablished in the adult head once the head is fully formed. These results suggest that the Hydra genes related to anterior Hox/paraHox genes are involved at different stages of apical differentiation. However, the positional information defining the oral/aboral axis in Hydra cannot be correlated strictly to that characterizing the anterior-posterior axis in vertebrates or arthropods (Gauchat, 2000)

The chordate central nervous system has been hypothesized to originate from either a dorsal centralized, or a ventral centralized, or a noncentralized nervous system of a deuterostome ancestor. In an effort to resolve these issues, the hemichordate Saccoglossus kowalevskii was examined and the expression of orthologs of genes that are involved in patterning the chordate central nervous system was examined. All 22 orthologs studied are expressed in the ectoderm in an anteroposterior arrangement nearly identical to that found in chordates. Domain topography is conserved between hemichordates and chordates despite the fact that hemichordates have a diffuse nerve net, whereas chordates have a centralized system. It is proposed that the deuterostome ancestor may have had a diffuse nervous system, which was later centralized during the evolution of the chordate lineage (Lowe, 2003).

The adult S. kowalevskii has tripartite, tricoelomic organization. At the anterior is the muscular proboscis or prosome, used for burrowing and collecting food particles. It contains the heart, kidney, a section of the dorsal nerve cord, and the protocoel. The middle region, which is the collar or mesosome, contains the mouth, a section of dorsal nerve cord formed by neurulation, the paired mesocoels, and the base of the stomochord, which projects forward into the prosome. The posterior region or metasome contains the gill slits, the remainder of the dorsal nerve cord, the entire ventral nerve cord, paired metacoels, gonads, a long through-gut, and terminal anus. At juvenile stages, a ventral post-anal extension (called a tail or sucker) is present (Lowe, 2003).

Gastrulation entails uniform and simultaneous inpocketing of the vegetal half of the hollow blastula. As the blastopore closes, a gumdrop-shaped gastrula is formed. As the embryo lengthens, two circumferential grooves indent and divide the length into prosome, mesosome, and metasome regions. Mesodermal coeloms outpouch from the gut anteriorly and laterally. The first gill slit pair appears externally by day 5, and the animal bends from the dorsal side. The hatched juvenile elongates and adds further pairs of gill slits successively. The animal is nearly bilaterally symmetric, except that the prosome excretory pore (the proboscis pore) from the kidney is reliably on the left, defining a left-right asymmetry (Lowe, 2003).

The hemichordate adult nervous system is not centralized but is a diffuse intraepidermal, basiepithelial nerve net. Nerve cells are interspersed with epidermal cells and account for 50% or more of the cells in the proboscis and collar ectoderm and a lower percentage in the metasome. Axons form a meshwork at the basal side of the epidermis. The two nerve cords are through-conduction tracts of bundled axons and are not enriched for neurogenesis. This general organizational feature of the nervous system has been largely underemphasized in recent literature that focuses on possible homologies between chordate and hemichordate nerve cords (Lowe, 2003).

Twenty-two full-length coding sequences of orthologs associated with neural patterning in chordates were isolated. These genes are probably present as single copies in S. kowalevskii because orthologs of most of them are present as single copies in lower chordates and echinoderms, and many of the genes were recovered multiple times in the EST analysis without finding any closely related sequences (Lowe, 2003).

Using full-length probes for in situ hybridization, all 22 genes were found to be expressed strongly in the ectoderm as single or multiple bands around the animal, in most cases without dorsal or ventral differences (rx, hox4, nkx2-1, en, barH, lim1/5, and otx are exceptions). Circumferential expression is consistent with diffuse neurogenesis in the ectoderm. The domains resemble the circumferential expression of orthologs in Drosophila embryos. In chordates, by contrast, most of these neural patterning genes are expressed in stripes or patches only within the dorsal neurectoderm and not in the epidermal ectoderm. Also, in chordates, the domains are often broader medially or laterally within the neurectoderm, and there are usually additional expression domains in the mesoderm and endoderm. In most of the 22 cases in S. kowalevskii, the ectodermal domain is the only expression domain (six3, otx, gbx, otp, nkx2-1, dbx, hox11/13, and irx are exceptions) (Lowe, 2003).

Although each of the 22 genes has a distinct expression domain along the anteroposterior dimension of the chordate body, attempts were made to divide them into three broad groups to facilitate the comparison with hemichordates: anterior, midlevel, and posterior genes. Anterior genes are those which in chordates are expressed either throughout or within a subdomain of the forebrain. Midlevel genes are those expressed at least in the chordate midbrain, having anterior boundaries of expression in the forebrain or midbrain, and posterior boundaries in the midbrain or anterior hindbrain. Posterior genes are those expressed entirely within the hindbrain and spinal cord of chordates. Many of the chordate genes have additional domains of expression elsewhere in the nervous system and in other germ layers, but comparisons were restricted to domains involved in specifying the neuraxis in the anteroposterior dimension. Taking these groups of genes one at a time, it was asked where the orthologous genes are expressed in S. kowalevskii. In all comparisons, no morphological homology is implied between the subregions of the chordate and hemichordate nervous systems (Lowe, 2003).

Six posterior neural domain genes were examined: gastrulation brain homeobox (gbx), hox1, 3, 4, 7/8, and 11/13. All of these genes are expressed in chordate neurectoderm in major domains entirely within the hindbrain and spinal cord regions of the nervous system. gbx was chosen in chordates because it has a role in forming the midbrain-hindbrain boundary and in establishing the site of the isthmic organizer (in vertebrates) by way of a mutual antagonism of gbx and otx expression. Its domain in chordates extends from the midbrain-hindbrain boundary back into the spinal cord. In Drosophila, it may serve an analogous function, delineating a neural boundary and antagonizing otx expression. This does not necessarily imply a structural homology between central nervous systems but, merely, a homologous use in anteroposterior patterning. In S. kowalevskii, the gbx ortholog is initially expressed in the entire metasome except for the ciliated telotroch region. Later, the anterior metasome becomes the site of strong expression, and posterior expression diminishes. An additional domain of gbx expression is detected only in early stages in the endoderm, with its anterior limit extending into the mesosome, beyond the anterior limit of the ectodermal domain. The ectodermal domain of gbx overlaps anteriorly with both the en and otx domains, whereas in chordates gbx overlaps en partially, but not otx (which it antagonizes). irx expression overlaps gbx, en, and otx expression in both chordates and hemichordates. Thus, the contiguity of ectodermal domains of gbx, en, otx, and irx resembles that in chordates, though with some differences of overlap (Lowe, 2003).

The 22 expression domains of orthologs of chordate neural patterning genes of S. kowalevskii correspond strikingly to those in chordates. There are differences such as the extent of overlap of edges of domains of otx, en, and gbx and other midlevel genes that are critical for forming boundaries within the chordate brain, but the relative domain locations are nonetheless very similar. This similar topography of domains is most parsimoniously explained by conservation in both lineages of a domain arrangement (a map) already present in the common ancestor, the ancestor of deuterostomes (Lowe, 2003).

At least 14 of the 22 conserved domains have similar locations in one or more protostome groups. Such similarities are most parsimoniously explained as a conservation of domains from the ancestral bilaterian. In the case of the hox genes, otx, emx, pax6, six3, gbx, and tll, there is strong evidence for such conservation, but less so for the others (barH and rx). At least four of the chordate-hemichordate conserved domains may not be shared by protostomes. Namely, three of these genes (dbx, vax, and hox11/13) are absent from the Drosophila genome and have not been cloned from other protostome groups. Also, one gene, engrailed, has no clear corresponding domain of expression known in protostomes. In Drosophila, en is expressed in the posterior compartments of 14 body segments and at three or more sites in the head that probably derive from ancient preoral segments. This pattern for en appears very different from the single ectodermal band in deuterostomes (Lowe, 2003).

The nerve net of hemichordates could represent the basal condition of the deuterostome ancestor, or it could represent the secondary loss of a central nervous system from an ancestor. Was the complex map of the ancestor associated with a complex diffuse nerve net or a central nervous system in the ancestor? It is suggested that the deuterostome ancestor may have had a diffuse basiepithelial nervous system with a complex map of expression domains, though not necessarily a diffuse net exactly like that of extant hemichordates. Hemichordates would then have retained a diffuse system in their lineage and early in the chordate lineage, centralization would have taken place. In this proposal, the domain map predates centralization and is carried into the nervous system. In this respect, the core questions of nervous system evolution would concern the modes of centralization utilized by the ancestor's various descendents rather than a dorsoventral inversion, per se. Thus, it is proposed that in chordates, especially vertebrates, the major innovation may have been the formation of a large contiguous nonneural (epidermogenic) region (Lowe, 2003).

Gsh-2 is a novel murine dispersed homeobox gene. Analysis of its cDNA sequence, including the full open reading frame, reveals an encoded homeodomain that is surprisingly similar to the homeodomains of the Antennapedia-type clustered Hox genes. In addition, the encoded protein includes polyhistidine and polyalanine tracts, as observed for several other genes of developmental significance. In situ hybridizations show Gsh-2 expression in the developing central nervous system, including the ganglionic eminences of the forebrain, the diencephalon, which gives rise to the thalamus and hypothalamus, and in the hindbrain. A random oligonucleotide selection and PCR amplification procedure was used to define a target DNA binding sequence, CNAATTAG, as a first step toward the identification of downstream target genes (Hsieh-Li, 1995).

Gsh-1, a novel murine homeobox gene, produces a transcript of approximately 2 kb present during development at embryonic day 10.5, 11.5, and 12.5. The cDNA sequence encodes a proline rich motif, a polyalanine tract, and a homeodomain with strong homology to those homeodomains encoded by the clustered Hox genes. The Gsh-1 expression pattern has been determined for days E8.5 to E13.5 by whole mount and serial section in situ hybridizations. Gsh-1 transcription is restricted to the central nervous system. Expression is present in the neural tube and hindbrain as two continuous, bilaterally symmetrical stripes within neural epithelial tissue. In the mesencephalon, expression is seen as a band across the most anterior portion. There is also diencephalon expression in the anlagen of the thalamus and the hypothalamus as well as in the optic stalk, optic recess, and the ganglionic eminence. Through the use of fusion proteins containing the Gsh-1 homeodomain, the consensus DNA binding site of the Gsh-1 homeoprotein has been determined to be GCT/CA/CATTAG/A (Valerius, 1995).

The anterior pituitary regulates the function of multiple organ systems as well as body growth; in turn, it is controlled by peptides released by the hypothalamus. Mutation of the Gsh-1 homeobox gene results in pleiotropic effects on pituitary development and function. Homozygous mutants exhibit extreme dwarfism, sexual infantilism and significant perinatal mortality. The mutant pituitary is small in size and hypocellular, with severely reduced numbers of growth hormone- and prolactin-producing cells. Moreover, the pituitary content of a subset of pituitary hormones, including growth hormone, prolactin and luteinizing hormone, is significantly decreased. The hypothalamus, although morphologically normal, is also perturbed in mutants. The gsh-1 gene is essential for growth hormone-releasing hormone (GHRH) gene expression in the arcuate nucleus of the hypothalamus. Further, sequence and electrophoretic mobility shift data suggest that the Gsh-1 and GHRH genes are potential targets regulated by the Gsh-1-encoded protein. The mutant phenotype indicates a critical role for Gsh-1 in the genetic hierarchy of the formation and function of the hypothalamic-pituitary axis (Li, 1996).

The Gsh-2 nonclustered homeobox gene is expressed within the developing forebrain, midbrain, and hindbrain. Gsh-2 transcripts are shown to be particularly abundant in the hindbrain and within the developing ganglionic eminences of the forebrain. Homozygous Gsh-2 mutant mice uniformly fail to survive more than 1 day following birth. At the physiologic level the mutants experienced apnea and reduced levels of hemoglobin oxygenation. Histologically, the mutant brains have striking alterations of discrete components. In the forebrain, the lateral ganglionic eminence is reduced in size. In the hindbrain, the area postrema, an important cardiorespiratory chemosensory center, is absent. The contiguous nucleus tractus solitarius, involved in integrating sensory input to maintain homeostasis, is also severely malformed in mutants. Immunohistochemistry was used to examine the mutant brains for alterations in the distribution of markers specific for serotonergic and cholinergic neurons. In addition, in situ hybridizations were used to define expression patterns of the Dlx 2 and Nkx 2.1 homeobox genes in Gsh-2 mutant mice. The mutant lateral ganglionic eminences show an abnormal absence of Dlx 2 expression. These results better define the genetic program of development of the mammalian brain, support neuromeric models of brain development, and further suggest similar patterning function for homeobox genes in phylogenetically diverse organisms (Szucsik, 1997).

Screening of a medaka (Oryzias latipes) adult brain cDNA library, with a degenerated probe corresponding to the most conserved region of helix III of the homeodomain, led to the isolation of a gene homologous to a murine orphan Hox gene, named Gsh-1. This gene has been termed Ol-Gsh 1 (Oryzias latipes-Gsh 1). Molecular analysis of the Ol-Gsh 1 putative protein points to potential functional domains that are highly conserved between fish and mouse genes. Whole-mount in situ hybridization shows that Ol-Gsh 1 is expressed in several waves during embryonic development. Transcripts are found in many regions of the central nervous system: the spinal cord, dorsal rhombencephalon, optic tectum, dorsal diencephalon, hypothalamus anlagen and rostral telencephalon. This multimodal expression pattern, strikingly conserved between fish and mammals, is reminiscent of both clustered and orphan homeobox genes. In addition, each expression wave is initiated in the fish embryo earlier than in the mammalian embryo, relative to the time scale defined by somitogenesis. It is proposed that Ol-Gsh 1 may be involved in conserved developmental pathways and in particular may be linked to proliferation events. Mouse Gsh-1 has been shown to participate in neuro-endocrine functions of the hypothalamus. From late developmental stages onward, Ol-Gsh 1 expression is also restricted to the hypothalamus. The expression pattern in this structure raises interesting questions concerning a fully or partially conserved function for these genes (Deschet, 1998).

Described here is the successful application of a strategy that potentially provides for an efficient and universal screen for downstream gene targets. The promoter of the Gsh-1 homeobox gene was used to drive expression of the SV40 T-antigen gene in transgenic mice. The Gsh-1 homeobox gene is expressed in discrete domains of the ganglionic eminences, diencephalon, and hindbrain during brain development. Gsh-1-SV40 T transgenic mice show cellular hyperplasia in regions of the brain coincident with Gsh-1 expression. The Gsh-1-SV40 T transgene was introduced, by breeding, into Gsh-1 homozygous mutant mice, and Gsh-1 -/- cell lines were made. Clonal cell lines were generated and analyzed by Northern blot hybridizations and Affymetrix GeneChip probe arrays to determine gene expression profiles. The results indicate that the cell lines remain representative of early developmental stages. Further, immunocytochemistry shows uniformly high levels of nestin expression, typical of central nervous system progenitor cells, and the absence of terminal differentiation markers of neuronal cells. One clonal cell line, No. 14, was then stably transfected with a tet-inducible Gsh-1 expression construct and subcloned. The starting clone 14, together with the uninduced and induced subclones, provided cell populations with varying levels of Gsh-1 expression. Differential display and Affymetrix GeneChip probe arrays were then used to identify transcript differences that represent candidate Gsh-1 target genes. Of particular interest, the drm and gas1 genes, which repress cell proliferation, were observed to be activated in Gsh-1-expressing cells. These observations support models predicting that homeobox genes function in the regulation of cell proliferation (Li, 1999).

Inactivation of Gbx2 produces Hox-like phenotypes in the axial skeleton

Expression of Hoxa10 in the presomitic mesoderm is sufficient to confer a Hox group 10 patterning program to the somite, producing vertebrae without ribs, an effect not achieved when Hoxa10 is expressed in the somites. In addition, Hox group 11-dependent vertebral sacralization requires Hoxa11 expression in the presomitic mesoderm, while their caudal differentiation requires that Hoxa11 is expressed in the somites. Therefore, Hox gene patterning activity is different in the somites and presomitic mesoderm, the latter being very prominent for Hox gene-mediated patterning of the axial skeleton. This is further supported by the finding that inactivation of Gbx2, a homeobox-containing gene expressed in the presomitic mesoderm but not in the somites, produces Hox-like phenotypes in the axial skeleton without affecting Hox gene expression (Carapuco, 2005).

Gsh2 is required for the repression of Ngn1 and specification of dorsal interneuron fate in the spinal cord

The molecular programs that specify progenitors in the dorsal spinal cord remain poorly defined. The homeodomain transcription factor Gsh2 is expressed in the progenitors of three dorsal interneuron subtypes, dI3, dI4 and dI5 neurons, whereas Gsh1 is expressed only in dI4 and dI5 progenitors. Mice lacking Gsh2 exhibit a selective loss of dI3 interneurons that is accompanied by an expansion of the dI2 progenitor domain. In Gsh2 mutant embryos, expression of the proneural bHLH protein Mash1 is downregulated in dI3 neural progenitors, with Mash1 mutants exhibiting a concordant reduction in dI3 neurons. Conversely, overexpression of Gsh2 and Mash1 leads to the ectopic production of dI3 neurons and a concomitant repression of Ngn1 expression. These results provide evidence that genetic interactions involving repression of Ngn1 by Gsh2 promote the differentiation of dI3 neurons from class A progenitors (Kirks, 2005).

Conserved and novel roles for the Gsh2 transcription factor in primary neurogenesis

The Gsx genes encode members of the ParaHox family of homeodomain transcription factors, that are expressed in the developing central nervous system in members of all major groups of bilaterians. The Gsx genes in Xenopus show similar patterns of expression to their mammalian homologues during late development. However, they are also expressed from early neurula stages in an intermediate region of the open neural plate where primary interneurons form. The Gsx homologue in the protostome Drosophila is expressed in a corresponding intermediate region of the embryonic neuroectoderm, and is essential for the correct specification of the neuroblasts that arise from it, suggesting that Gsx genes may have played a role in intermediate neural specification in the last common bilaterian ancestor. This study shows that manipulation of Gsx function disrupts the differentiation of primary interneurons. Despite their similar expression patterns, the uni-directional system of interactions between homeodomain transcription factors from the Msx, Nkx and Gsx families in the Drosophila neuroectoderm is not conserved between their homologues in the Xenopus open neural plate. Finally, the identification is reported of Dbx1 (see Drosophila Dbx) as a direct target of Gsh2-mediated transcriptional repression, a series of cross-repressive interactions, reminiscent of those that exist in the amniote neural tube, are shown to act between Gsx, Dbx and Nkx transcription factors to pattern the medial aspect of the central nervous system at open neural plate stages in Xenopus (Winterbottom, 2010).

Gbx2 and the development of the midbrain

The anterior neural ridge (ANR), and the isthmic organizer (IsO) represent two signaling centers possessing organizing properties necessary for forebrain (ANR) as well as midbrain and rostral hindbrain (IsO) development. An important mediator of ANR and IsO organizing property is the signaling molecule FGF8. Previous work has indicated that correct positioning of the IsO and Fgf8 expression in this domain is controlled by the transcription factors Otx2 and Gbx2. In order to provide novel insights into the roles of Otx2 and Gbx2, mutant embryos carrying different dosages of Otx2, Otx1 and Gbx2 were studied. Embryos deficient for both OTX2 and GBX2 proteins (hOtx12/hOtx12; Gbx2-/-) show abnormal patterning of the anterior neural tissue, that is evident at the presomite-early somite stage prior to the onset of Fgf8 neuroectodermal expression. Indeed, hOtx12/hOtx12; Gbx2-/- embryos exhibit broad co-expression of early forebrain, midbrain and rostral hindbrain markers such as hOtx1, Gbx2, Pax2, En1 and Wnt1 and subsequently fail to activate forebrain and midbrain-specific gene expression. In this genetic context, Fgf8 is expressed throughout the entire anterior neural plate, thus indicating that its activation is independent of both OTX2 and GBX2 function. Analysis of hOtx12/hOtx12; Gbx2-/- and Otx1+/-; Otx2+/- mutant embryos also suggests that FGF8 cannot repress Otx2 without the participation of GBX2. Embryos carrying a single strong hypomorphic Otx2 allele (Otx2lambda) in an Otx2 and Gbx2 null background (Otx2lambda/-; Gbx2-/-) recover both the headless phenotype exhibited by Otx2lambda/- embryos and forebrain- and midbrain-specific gene expression that is not observed in hOtx12/hOtx12; Gbx2-/- mutants. Together, these data provide novel genetic evidence indicating that OTX2 and GBX2 are required for proper segregation of early regional identities anterior and posterior to the mid-hindbrain boundary (MHB) and for conferring competence to the anterior neuroectoderm in responding to forebrain-, midbrain- and rostral hindbrain-inducing activities (Martinez-Barbera, 2001).

The isthmic organizer, which patterns the anterior hindbrain and midbrain, is one of the most studied secondary organizers. In recent years, new insights have been reported on the molecular nature of its morphogenetic activity. Studies in chick, mouse and zebrafish have converged to show that mutually repressive interactions between the homeoproteins encoded by Otx and Gbx genes position this organizer in the neural primordia. Evidence is presented that equivalent (in addition to novel) interactions between these and other genes operate in Xenopus embryos to position the isthmic organizer. Use was made of fusion proteins in which Otx2 or Gbx2 homeodomains were combined with the E1A activation domain or the EnR repressor element; these were then injected into embryos. Otx2 and Gbx2 are likely to be transcriptional repressors, and these two proteins repress each other's transcription. The interaction between these two proteins is required for the positioning of the isthmic organizer genes Fgf8, Pax2 and En2. A novel in vitro assay has been developed for the study of the formation of this organizer. Conjugating animal caps previously injected with Otx2 and Gbx2 mRNAs recreates the interactions required for the induction of the isthmic organizer. This assay was used to determine which cells produce and which cells receive the Fgf signal. A novel genetic element, Xiro1, which encode another homeoprotein, was added to this process. Xiro1 expression domain overlaps with territories expressing Otx2, Gbx2 and Fgf8. By expressing wild-type or dominant negative forms of Xiro1, this gene is shown to activate the expression of Gbx2 in the hindbrain. In addition, Xiro1 is required in the Otx2 territory to allow cells within this region to respond to the signals produced by adjacent Gbx2 cells. Moreover, Xiro1 is absolutely required for Fgf8 expression at the isthmic organizer. A model is discussed where Xiro1 plays different roles in regulating the genetic cascade of interactions between Otx2 and Gbx2 that are necessary for the specification of the isthmic organizer (Glavic, 2002).

Development and differentiation of the vertebrate caudal midbrain and anterior hindbrain are dependent on the isthmic organizer signals at the midbrain/hindbrain boundary (MHB). The future MHB forms at the boundary between the Otx2 and Gbx2 expression domains. Recent studies in mice and chick have suggested that the apposition of Otx2- and Gbx2-expressing cells is instrumental for the positioning and early induction of the MHB genetic cascade. Otx2 and Gbx2 perform different roles in this process. Ectopically expressed Otx2 on its own can induce a substantial part of the MHB genetic network, namely En2, Wnt1, Pax-2, Fgf8 and Gbx2, in a concentration-dependent manner. This induction does not require protein synthesis and ends during neurulation. In contrast, Gbx2 is a negative regulator of Otx2 and the MHB genes. Based on the temporal patterns of expression of the genes involved, it is proposed that Otx2 might be the early inducer of the isthmic organizer genetic network while Gbx2 restricts Otx2 expression along the anterior-posterior axis and establishes an Otx2 gradient (Tour, 2002a).

Anterior-posterior patterning of the embryo requires the activity of multiple homeobox genes, among them Hox, caudal (Cdx, Xcad) and Otx2. During early gastrulation, Otx2 and Xcad2 establish a cross-regulatory network, which is an early event in the anterior-posterior patterning of the embryo. As gastrulation proceeds and the embryo elongates, a new domain forms, which expresses neither Otx2 nor Xcad2 genes. Early transcription of the Xenopus Gbx2 homolog, Xgbx2a, is spatially restricted between Otx2 and Xcad2. When overexpressed, Otx2 and Xcad2 repress Xgbx2a transcription, suggesting their role in setting the early Xgbx2a expression domain. Homeobox genes have been shown to play crucial roles in the specification of the vertebrate brain. The border between the transcription domains of Otx2 and Gbx2 is the earliest known marker of the region where the midbrain/hindbrain boundary (MHB) organizer will develop. Xgbx2a is a negative regulator of Otx2 and a weak positive regulator of Xcad2. Using obligatory activator and repressor versions of Xgbx2a, it has been demonstrated that during early embryogenesis, Xgbx2a acts as a transcriptional repressor. In addition, taking advantage of hormone-inducible versions of Xgbx2a and its antimorph, it has been shown that the ability of Xgbx2a to induce head malformations is restricted to gastrula stages and correlates with its ability to repress Otx2 during the same developmental stages. It is therefore suggested that the earliest known step of the MHB formation, the establishment of Otx2/Gbx2 boundary, takes place via mutual inhibitory interactions between these two genes and this process begins as early as midgastrulation (Tour, 2002b).

The organizer at the midbrain-hindbrain boundary (MHB) forms at the interface between Otx2 and Gbx2 expressing cell populations, but how these gene expression domains are set up and integrated with the remaining machinery controlling MHB development is unclear. The isolation, mapping, chromosomal synteny and spatiotemporal expression of gbx1 and gbx2 in zebrafish is reported. Focus was placed on the expression of these genes during development of the midbrain-hindbrain territory. The results suggest that these genes function in this area in a complex fashion, as evidenced by their highly dynamic expression patterns and relation to Fgf signaling. Analysis of gbx1 and gbx2 expression during formation of the MHB in mutant embryos for pax2.1, fgf8 and pou2 (noi, ace, spg), as well as Fgf-inhibition experiments, show that gbx1 acts upstream of these genes in MHB development. In contrast, gbx2 activation requires ace (fgf8) function, and in the hindbrain primordium, also spg (pou2). It is proposed that in zebrafish, gbx genes act repeatedly in MHB development, with gbx1 acting during the positioning period of the MHB at gastrula stages, and gbx2 functioning after initial formation of the MHB, from late gastrulation stages onwards. Transplantation studies furthermore reveal that at the gastrula stage, Fgf8 signals from the hindbrain primordium into the underlying mesendoderm. Apart from the general involvement of gbx genes in MHB development reported also in other vertebrates, these results emphasize that early MHB development can be divided into multiple steps with different genetic requirements with respect to gbx gene function and Fgf signaling. Moreover, these results provide an example for switching of a specific gene function of gbx1 versus gbx2 between orthologous genes in zebrafish and mammals (Rhinn, 2003).

The cerebellum develops from the rhombic lip of the rostral hindbrain and is organized by fibroblast growth factor 8 (FGF8) expressed by the isthmus. Irx2, a member of the Iroquois (Iro) and Irx class of homeobox genes is expressed in the presumptive cerebellum. When Irx2 is misexpressed with Fgf8a in the chick midbrain, the midbrain develops into cerebellum in conjunction with repression of Otx2 and induction of Gbx2. During this event, signaling by the FGF8 and mitogen-activated protein (MAP) kinase cascade modulates the activity of Irx2 by phosphorylation. These data identify a link between the isthmic organizer and Irx2, thereby shedding light on the roles of Iro and Irx genes, which are conserved in both vertebrates and invertebrates (Matsumoto, 2004).

Gsh2 and the telencephalon

The genetic mechanisms that regulate dorsal-ventral identity in the embryonic mouse telencephalon and, in particular, the specification of progenitors in the cerebral cortex and striatum have been examined. The respective roles of Pax6 and Gsh2 in cortical and striatal development were studied in single and double loss-of-function mouse mutants. Gsh2 gene function is essential to maintain the molecular identity of early striatal progenitors and in its absence the ventral telencephalic regulatory genes Mash1 and Dlx are lost from most of the striatal germinal zone. In their place, the dorsal regulators Pax6, neurogenin 1 and neurogenin 2 are found ectopically. Conversely, Pax6 is required to maintain the correct molecular identity of cortical progenitors. In its absence, neurogenins are lost from the cortical germinal zone and Gsh2, Mash1 and Dlx genes are found ectopically. These reciprocal alterations in cortical and striatal progenitor specification lead to the abnormal development of the cortex and striatum observed in Pax6 (small eye) and Gsh2 mutants, respectively. In support of this, double homozygous mutants for Pax6 and Gsh2 exhibit significant improvements in both cortical and striatal development compared with their respective single mutants. Taken together, these results demonstrate that Pax6 and Gsh2 govern cortical and striatal development by regulating genetically opposing programs that control the expression of each other as well as the regionally expressed developmental regulators Mash1, the neurogenins and Dlx genes in telencephalic progenitors (Toresson, 2000).

The Gsh1/2 homolog, intermediate neuroblasts defective (ind), is expressed in selected areas of the fly CNS including the intermediate region of the ventral nerve cord. ind mutants show a reduction in the number of neuroblasts in this region of the ventral nerve cord. The remaining neuroblasts, by all markers tested, appear to have acquired a dorsal fate. This phenotype is reminiscent of the mouse Gsh2 mutant phenotype where the progenitors in the LGE (i.e. the intermediate region of the telencephalon) have lost the expression of certain ventral genes and instead express the dorsal genes Pax6, neurogenin 1 and neurogenin 2. These findings therefore indicate that at least some aspects of Gsh/ind function in dorsal-ventral specification of the developing nervous system have been conserved throughout evolution (Toresson, 2000).

The telencephalon and diencephalon, which comprise the vertebrate forebrain, arise from the anterior-most region of the neuraxis. The telencephalon is subdivided into the pallial and subpallial domains. The pallium gives rise to dorsal structures, including the cerebral cortex, while the subpallium gives rise to ventral structures, including the globus pallidus and the striatum, which in combination form the majority of the basal ganglia. The basal ganglia arise from two major protrusions in the wall of the ventral telencephalon known as the medial ganglionic eminence (MGE) and the lateral ganglionic eminence (LGE), which primarily gives rise to the globus pallidus and the striatum, respectively. The appearance of these eminences occurs sequentially during development, with the more-medial MGE arising subsequent to neural-tube closure and the more lateral LGE arising shortly thereafter. The MGE and LGE are additionally hypothesized to be the source of a majority of the interneurons found in the olfactory bulb and the cerebral cortex (Corbin, 2000 and references therein).

Homeobox genes are important for the proper patterning of the mammalian telencephalon. One of these genes is Gsh2, whose expression in the forebrain is restricted to the ventral domain. Expression of Gsh2 is restricted in the mouse forebrain to the ventral domain. Gsh2 is first detected between E9 and E10 in the forebrain and is expressed in the later developing ventral thalamus, hypothalamus, MGE, LGE and caudal ganglionic eminence (CGE). To better understand the localization of Gsh2, its expression was assayed at a variety of stages of telencephalic development. At E9.5, Gsh2 mRNA expression is found the ventrolateral telencephalon, a region from where the LGE will putatively emerge. Interestingly, at E9.5, although a few GSH2-expressing cells appear to intermingle with those expressing NKX2.1, GSH2 and NKX2.1 are predominantly expressed in separate domains. By E10.5, coincident with the emergence of the MGE, GSH2 expression extends into the MGE and overlaps with NKX2.1 expression. By E12.5, Gsh2 is expressed in the MGE, LGE and septum. At E12.5, Gsh2 is also expressed in the CGE and the ventral diencephalon. Expression of Gsh2 mRNA is restricted to the VZ and is not expressed in the SVZ or differentiated structures of the developing ventral telencephalon (Corbin, 2000).

Gsh2 is a downstream target of sonic hedgehog and lack of Gsh2 results in profound defects in telencephalic development. Gsh2 mutants have a significant decrease in the expression of numerous genes that mark early development of the lateral ganglionic eminence, the striatal anlage. Accompanying this early loss of patterning genes is an initial expansion of dorsal telencephalic markers across the cortical-striatal boundary into the lateral ganglionic eminence. The homeodomain transcription factors Dlx1 and Dlx2 are expressed in the VZ and SVZ of multiple ventral forebrain structures, including the MGE, LGE, CGE and ventral diencephalon. At E12.5, in embryos lacking Gsh2 there is an absence of both Dlx1 and Dlx2 expression in all but the most ventral aspect of the LGE. Expression of Mash1, a bHLH transcription factor essential for proper striatal development, shows a similar reduced expression pattern. Furthermore, expression of Ebf1, a gene essential for the transition of cells from the SVZ to the striatal mantle, is significantly reduced in the mutant LGE, as is Gad67, the precursor enzyme that catalyzes the formation of the neurotransmitter GABA. Interestingly, as development proceeds, there is compensation for this early loss of markers that is coincident with a molecular re-establishment of the cortical-striatal boundary. Despite this compensation, there is a defect in the development of distinct subpopulations of striatal neurons. Moreover, while this analysis suggests that the migration of the ventrally derived interneurons to the developing cerebral cortex is not significantly affected in Gsh2 mutants, there is a distinct delay in the appearance of GABAergic interneurons in the olfactory bulb (Corbin, 2000).

At E18.5, a reduction of Enkephalin expression in the most ventral aspect of the nucleus accumbens and ventrolateral (perirhinal) region of the ventral telencephalon is observed in Gsh2 mutant embryos. Expression of Dopamine receptor 2 is also missing in both the nucleus accumbens and perirhinal regions at this age. There is also a partially penetrant loss of striatal marker DARPP32-positive cells in the striatal SVZ further suggesting a loss of subpopulations of early born striatal neurons. In contrast, development of later born neurons that comprise the striatal matrix appears unaffected. Ebf1 expression, although reduced at E15.5 and at E18.5, is present in the presumptive striatal matrix in Gsh2 mutants. Normal expression of calbindin within the striatal matrix further suggests this aspect of striatal development is unaffected by the loss of Gsh2. These results suggest that, despite the recovery of early patterning defects by later stages of telencephalic development, the generation of specific subpopulations of early, but not late born, striatal neurons is permanently affected. Taken together, these data support a model in which Gsh2, in response to sonic hedgehog signaling, plays a crucial role in multiple aspects of telencephalic development (Corbin, 2000).

The role of the two closely related homeobox genes, Gsh1 and Gsh2, in the development of the striatum and the olfactory bulb was examined. The neurons that comprise the striatum have their origin in two prominent elevations, called the medial and lateral ganglionic eminences (MGE and LGE, respectively). The GABAergic medium-sized spiny projection neurons, which constitute the vast majority of striatal neurons, are generated from the LGE. Conversely, the majority of striatal interneurons appear to be generated from the MGE. Gsh1 and Gsh2 are expressed in a partially overlapping pattern by ventricular zone progenitors of the ventral telencephalon. Gsh2 is expressed in both of the ganglionic eminences while Gsh1 is largely confined to the medial ganglionic eminence. Previous studies have shown that Gsh2-/- embryos suffer from an early misspecification of precursors in the lateral ganglionic eminence leading to disruptions in striatal and olfactory bulb development. This molecular misspecification is present only in early precursor cells while at later stages the molecular identity of these cells appears to be normalized. Concomitant with this normalization, Gsh1 expression is notably expanded in the Gsh2-/- LGE. While no obvious defects in striatal or olfactory bulb development were detected in Gsh1-/- embryos, Gsh1/2 double homozygous mutants displayed more severe disruptions than were observed in the Gsh2 mutant alone. Accordingly, the molecular identity of LGE precursors in the double mutant is considerably more perturbed than in Gsh2 single mutants. These findings, therefore, demonstrate an important role for Gsh1 in the development of the striatum and olfactory bulb of Gsh2 mutant mice. In addition, the data indicate a role for Gsh genes in controlling the size of the LGE precursor pools, since decreasing copies of Gsh2 and Gsh1 alleles results in a notable decrease in precursor cell number, particularly in the subventricular zone (Toresson, 2001).

Considerable data suggest that sonic hedgehog (Shh) is both necessary and sufficient for the specification of ventral pattern throughout the nervous system, including the telencephalon. The regional markers induced by Shh in the E9.0 telencephalon are dependent on the dorsoventral and anteroposterior position of ectopic Shh expression. This suggests that by this point in development regional character in the telencephalon is established. To determine whether this prepattern is dependent on earlier Shh signaling, the telencephalon was examined in mice carrying either Shh- or Gli3-null mutant alleles. This analysis revealed that the expression of a subset of ventral telencephalic markers, including Dlx2 and Gsh2, although greatly diminished, persists in Shh-/- mutants, and that these same markers are expanded in Gli3-/- mutants. To understand further the genetic interaction between Shh and Gli3, Shh/Gli3 and Smoothened/Gli3 double homozygous mutants were examined. Notably, in animals carrying either of these genetic backgrounds, genes such as Gsh2 and Dlx2, which are expressed pan-ventrally, as well as Nkx2.1, which demarcates the ventral most aspect of the telencephalon, appear to be largely restored to their wild-type patterns of expression. These results suggest that normal patterning in the telencephalon depends on the ventral repression of Gli3 function by Shh and, conversely, on the dorsal repression of Shh signaling by Gli3. In addition, these results support the idea that, in addition to hedgehog signaling, a Shh-independent pathways must act during development to pattern the telencephalon (Rallu, 2002).

Regional patterning of the mammalian telencephalon requires the function of three homeodomain-containing transcription factors, Pax6, Gsh2 and Nkx2.1. These factors are required for the development of the dorsal, lateral and medial domains of the telencephalon, respectively. Pax6 and Gsh2 have been shown to cross-repress one another in the formation of the border between dorsal and lateral region of the telencephalon. This study examines whether similar interactions are responsible for the establishment of other boundaries of telencephalic gene expression. Surprisingly, despite the fact that, at specific times in development, both Pax6 and Gsh2 maintain a complementary pattern of expression with Nkx2.1, in neither case are these boundaries maintained through a similar cross-repressive mechanism. Rather, as revealed by analysis of double-mutant mice, Nkx2.1 and Gsh2 act cooperatively in many aspects to pattern the ventral telencephalon. By contrast, as indicated by both loss- and gain-of-function analysis, Gsh2 expression in the medial ganglionic eminence after E10.5 may negatively regulate Nkx2.1 dependent specification of oligodendrocytes. Taken together with previous studies, a hierarchy of gene expression for producing interneurons and oligodendrocytes is becoming apparent. Initiating the generation of these cell types in ventral regions are extrinsic cues, including Shh. These cues result in the expression of homeodomain genes, including Nkx2.1 and Gsh2, that ensure the expression of pan-ventral transcription factors, such as Dlx1/2, Mash1 and Olig2, in the MGE and LGE. These genes, in turn, may act as key effectors in the generation of specific ventral cell types, such as interneurons, and distinct populations of oligodendrocytes (Corbin, 2003).

The role was examined of the homeobox gene Gsh2 in retinoid production and signaling within the ventral telencephalon of mouse embryos. Gsh2 mutants exhibit altered ventral telencephalic development, including a smaller striatum with fewer dopamine- and cyclic AMP-regulated phosphoprotein (DARPP-32)-immunoreactive neurons than wild types. The expression of the retinoic acid (RA) synthesis enzyme, retinaldehyde dehydrogenase 3 (Raldh3, also known as Aldh1a3), is reduced in the lateral ganglionic eminence (LGE) of Gsh2 mutants. Moreover, using a retinoid reporter cell assay, it was found that retinoid production in the Gsh2 mutants is markedly reduced. The striatal defects in Gsh2 mutants are thought to result from ectopic expression of Pax6 in the LGE. Removal of Pax6 from the Gsh2 mutant background improves the molecular identity of the LGE in these double mutants; however, Raldh3 expression is not improved. The Pax6;Gsh2 double mutants possess a larger striatum than the Gsh2 mutants, but the disproportionate reduction in DARPP-32 neurons is not improved. These findings suggest that reduced retinoid production in the Gsh2 mutant contributes to the striatal differentiation defects. Since RA promotes the expression of DARPP-32 in differentiating LGE cells in vitro, whether exogenous RA can improve striatal neuron differentiation in the Gsh2 mutants was examined. Indeed, RA supplementation of Gsh2 mutants, during the period of striatal neurogenesis, results in a significant increase in DARPP-32 expression. Thus, in addition to the previously described role for Gsh2 to maintain correct molecular identity in the LGE, these results demonstrate a novel requirement of this gene for retinoid production within the ventral telencephalon (Waclaw, 2004).

Distinct temporal requirements for the homeobox gene Gsx2 in specifying striatal and olfactory bulb neuronal fates

The homeobox gene Gsx2 (formerly Gsh2) is known to be required for striatal and olfactory bulb neurogenesis; however, its specific role in the specification of these two neuronal subtypes remains unclear. To address this, a temporally regulated gain-of-function approach in transgenic mice was employed and it was found that misexpression of Gsx2 at early stages of telencephalic neurogenesis favors the specification of striatal projection neuron identity over that of olfactory bulb interneurons. In contrast, delayed activation of the Gsx2 transgene until later stages exclusively promotes olfactory bulb interneuron identity. In a complementary approach, Gsx2 was conditionally inactivated in a temporally progressive manner. Unlike germline Gsx2 mutants, which exhibit severe alterations in both striatal and olfactory bulb neurogenesis at birth, the conditional mutants exhibited defects restricted to olfactory bulb interneurons. These results demonstrate that Gsx2 specifies striatal projection neuron and olfactory bulb interneuron identity at distinct time points during telencephalic neurogenesis (Waclaw, 2009).

The telencephalon represents the largest and most complex region of the mammalian brain. This region is charged with the task of complex neural processing that controls all cognitive processes and purposeful actions. Accordingly, the telencephalon exhibits the greatest amount of neuronal diversity of any portion of the CNS. Many groups have focused on the generation of neuronal diversity within the telencephalon. While neuronal progenitors in the dorsal telencephalon (also termed the pallium) are thought to give rise to the excitatory cortical projection neurons, the vast majority of cortical interneurons originate from progenitor domains located in the ventral telencephalon. Although limited pallial contributions to ventral telencephalic neuronal subtypes have recently been proposed, most of the neuronal diversity found in the mature telencephalon appears to derive from progenitor cells positioned in the ventral telencephalon during embryogenesis (Waclaw, 2009).

The lateral ganglionic eminence (LGE) represents one such ventral telencephalic progenitor region, which has been shown to generate the projection neurons of the striatum as well as interneurons in the olfactory bulb. Despite the fact that both striatal projection neurons and olfactory bulb interneurons derive from the LGE, they exhibit different temporal profiles of neurogenesis in the rodent; the striatal neurons are generated almost exclusively at embryonic time points, whereas the olfactory bulb interneurons begin their genesis at later embryonic time points and continue into the early postnatal period, when the vast majority are born. Recent studies have suggested that these two neuronal subtypes derive from separate progenitors located in distinct regions within the LGE, termed the ventral (v)LGE and dorsal (d)LGE, respectively. These two LGE subdivisions were first describe based on gene expression patterns at midgestation stages. The dLGE was characterized by high levels of Gsx2 and Er81 in progenitors of the ventricular zone (VZ), while the vLGE lacks Er81 expression and displays lower levels of Gsx2. These compartments can also be identified in the subventricular zone (SVZ) and mantle regions of the LGE. Islet1 (Isl1) is expressed in the vLGE SVZ and transiently in its striatal projection neuron derivatives whereas Er81 and Sp8 mark the dLGE SVZ and remain expressed in distinct subtypes of olfactory bulb interneurons. These two LGE progenitor domains are bordered dorsally by the ventral pallium (marked by Dbx1) and ventrally by the interganglionic sulcus, which is marked by Nkx6.2 expression. Dbx1- and Nkx6.2-expressing progenitors have been shown to contribute to amygdalar projection neurons and cortical (Waclaw, 2009).

Correct patterning of the vLGE and dLGE requires Gsx2 and Pax6 gene function. The loss of the pallial regulator Pax6 results in a dorsal expansion of dLGE markers, suggesting a role for the paired homeodomain factor in repressing dLGE identity within pallial progenitors. In the absence of Gsx2, the vLGE and dLGE as well as their derivatives, the striatal projection neurons and olfactory bulb interneurons, are severely reduced. The specific role of Gsx2 in patterning and specification of the vLGE and dLGE, however, remains somewhat unclear. So far, no evidence has been provided to support a role for Gsx2 in directly ventralizing telencephalic progenitors. Loss-of-function studies, however, suggested that Gsx2 indirectly controls LGE specification by repressing the expression of dorsal telencephalic regulators such as Pax6 in LGE progenitors. This appears to be a conserved function of Gsx2 since the Drosophila homolog Ind (intermediate neuroblasts defective) has also been shown to repress eyeless, the Pax6 homolog, in fly CNS development (Waclaw, 2009).

This study reexamined the role of Gsx2 in LGE specification using conditional gain-of-function and loss-of-function approaches in mice. These models afforded the analysis of temporally distinct roles for Gsx2 in the specification of vLGE and dLGE. The results demonstrate that Gsx2 can directly ventralize pallial progenitors and, depending on the developmental stage, specifies different neuronal fates. In particular, at early stages of telencephalic development, Gsx2 is necessary and sufficient to correctly specify the vLGE and its major derivatives, the striatal projection neurons; however, at later stages, high levels of Gsx2 specify LGE progenitors toward dLGE fates including olfactory bulb interneurons (Waclaw, 2009).


Search PubMed for articles about Drosophila intermediate neuroblasts defective

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Carapuco, M., Novoa, A., Bobola, N. and Mallo, M. (2005). Hox genes specify vertebral types in the presomitic mesoderm. Genes Dev. 19: 2116-2121. 16166377

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Cinnamon, E., et al. (2008), Multiple RTK pathways downregulate Groucho-mediated repression in Drosophila embryogenesis. Development 135: 829-837. PubMed Citation: 18216172

Corbin, J. G., et al. (2000). The Gsh2 homeodomain gene controls multiple aspects of telencephalic development. Development 127: 5007-5020. PubMed citation: 11060228

Corbin, J. G., et al. (2003). Combinatorial function of the homeodomain proteins Nkx2.1 and Gsh2 in ventral telencephalic patterning. Development 130: 4895-4906. 12930780

Cowden, J. and Levine, M. (2003). Ventral dominance governs sequential patterns of gene expression across the dorsal-ventral axis of the neuroectoderm in the Drosophila embryo. Dev. Biol. 262: 335-349. 14550796

Deschet, K., et al. (1998). Expression domains of the medaka (Oryzias latipes) Ol-Gsh 1 gene are reminiscent of those of clustered and orphan homeobox genes. Dev. Genes Evol. 208(5): 235-44. PubMed citation: 9683739

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Gauchat, D., et al. (2000). Evolution of Antp-class genes and differential expression of Hydra Hox/paraHox genes in anterior patterning. Proc. Natl. Acad. Sci. 97: 4493-4498. PubMed citation: 10781050

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Von Ohlen, T., Syu, L. J. and Mellerick, D. M. (2007b). Conserved properties of the Drosophila homeodomain protein, Ind. Mech. Dev. 124(11-12): 925-3417900877

Von Ohlen, T. L. and Moses, C. (2009). Identification of Ind transcription activation and repression domains required for dorsoventral patterning of the CNS. Mech. Dev. 126: 552-562. PubMed Citation: 19348939

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Waclaw, R. R., et al. (2009). Distinct temporal requirements for the homeobox gene Gsx2 in specifying striatal and olfactory bulb neuronal fates. Neuron 63(4): 451-65. PubMed Citation: 19709628

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Winterbottom, E. F., Illes, J. C., Faas, L. and Isaacs, H. V. (2010). Conserved and novel roles for the Gsh2 transcription factor in primary neurogenesis. Development 137(16): 2623-31. PubMed Citation: 20610487

Zhao, G. and Skeath, J. B. (2002). The Sox-domain containing gene Dichaete/fish-hook acts in concert with vnd and ind to regulate cell fate in the Drosophila neuroectoderm. Development 129: 1165-1174. 11874912

Zhao, G., Wheeler, S. R. and Skeath, J. B. (2007a). Genetic control of dorsoventral patterning and neuroblast specification in the Drosophila Central Nervous System. Int. J. Dev. Biol. 51(2): 107-15. Medline abstract: 17294361

Zhao, G., Boekhoff-Falk, G., Wilson, B. A. and Skeath, J. B. (2007b). Linking pattern formation to cell-type specification: Dichaete and Ind directly repress achaete gene expression in the Drosophila CNS. Proc. Natl. Acad. Sci. 104(10): 3847-52. Medline abstract: 17360441

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date revised: 22 May 2023

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