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).

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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