BIOLOGICAL OVERVIEW

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. Indís 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).

GENE STRUCTURE

PROTEIN STRUCTURE

Amino Acids - 320

Structural Domains

ind encodes a protein containing a homeodomain that is most closely related to the vertebrate Gsh1 and Gsh2 homeodomain proteins. There is 85% amino acid identity between Ind and either of the Gsh homeodomains. Ind, Gsh1, and Gsh2 also share a short amino-terminal region of homology. ind is expressed in two longitudinal stripes in sharply defined DV domains of the Drosophila CNS. This shows striking similarity to the expression of Gsh1 and Gsh2 in the developing murine CNS (Weiss, 1998).

date revised: 28 December 98

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