Regulated vnd expression is required for both neural and glial specification in Drosophila

The Drosophila embryonic CNS arises from the neuroectoderm, which is divided along the dorsal-ventral axis into two halves by specialized mesectodermal cells at the ventral midline. The neuroectoderm is in turn divided into three longitudinal stripes—ventral, intermediate, and lateral. The ventral nervous system defective, or vnd, homeobox gene is expressed from cellularization throughout early neural development in ventral neuroectodermal cells, neuroblasts, and ganglion mother cells, and later in an unrelated pattern in neurons. Here, in the context of the dorsal-ventral location of precursor cells, we reassess the vnd loss- and gain-of-function CNS phenotypes using cell specific markers. We find that over expression of vnd causes significantly more profound effects on CNS cell specification than vnd loss. The CNS defects seen in vnd mutants are partly caused by loss of progeny of ventral neuroblasts—the commissures are fused and the longitudinal connectives are aberrantly positioned close to the ventral midline. The commissural vnd phenotype is associated with defects in cells that arise from the mesectoderm, where the VUM neurons have pathfinding defects, the MP1 neurons are mis-specified, and the midline glia are reduced in number. vnd over expression results in the mis-specification of progeny arising from all regions of the neuroectoderm, including the ventral neuroblasts that normally express the gene. The CNS of embryos that over express vnd is highly disrupted, with weak longitudinal connectives that are placed too far from the ventral midline and severely reduced commissural formation. The commissural defects seen in vnd gain-of-function mutants correlate with midline glial defects, whereas the mislocalization of interneurons coincides with longitudinal glial mis-specification. Thus, Drosophila neural and glial specification requires that vnd expression by tightly regulated. © 2002 Wiley Periodicals, Inc. J Neurobiol 50: 118–136, 2002; DOI 10.1002/neu.10022Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/34482/1/10022_ftp.pd

Contextual interactions determine whether the Drosophila homeodomain protein, Vnd, acts as a repressor or activator

At the molecular level, members of the NKx2.2 family of transcription factors establish neural compartment boundaries by repressing the expression of homeobox genes specific for adjacent domains [Muhr et al. (2001) Cell, 104, 861–873; Weiss et al. (1998) Genes Dev., 12, 3591–3602]. The Drosophila homologue, vnd, interacts genetically with the high-mobility group protein, Dichaete, in a manner suggesting co-operative activation [Zhao and Skeath (2002) Development, 129, 1165–1174]. However, evidence for direct interactions and transcriptional activation is lacking. Here, we present molecular evidence for the interaction of Vnd and Dichaete that leads to the activation of target gene expression. Two-hybrid interaction assays indicate that Dichaete binds the Vnd homeodomain, and additional Vnd sequences stabilize this interaction. In addition, Vnd has two activation domains that are typically masked in the intact protein. Whether vnd can activate or repress transcription is context-dependent. Full-length Vnd, when expressed as a Gal4 fusion protein, acts as a repressor containing multiple repression domains. A divergent domain in the N-terminus, not found in vertebrate Vnd-like proteins, causes the strongest repression. The co-repressor, Groucho, enhances Vnd repression, and these two proteins physically interact. The data presented indicate that the activation and repression domains of Vnd are complex, and whether Vnd functions as a transcriptional repressor or activator depends on both intra- and inter-molecular interactions

Interaction of Vnd with the high-mobility protein, Dichaete, relieves Vnd–Gal4-mediated repression

<p><b>Copyright information:</b></p><p>Taken from "Contextual interactions determine whether the homeodomain protein, Vnd, acts as a repressor or activator"</p><p>Nucleic Acids Research 2005;33(1):1-12.</p><p>Published online 07 Jan 2005</p><p>PMCID:PMC546129.</p><p>© 2005, the authors © </p> The indicated Vnd–Gal4 fusion proteins were expressed transiently with increasing amounts of a pACT- expression plasmid. Expression of the VP16 activation domain fused to Dichaete from this construct relieves Vnd–Gal4-mediated repression of the luciferase reporter because of Vnd–Dichaete interactions. The baseline for each experimental series was set using pACT- with empty pBind. Because the Vnd domains have regulatory activity in the one-hybrid assay, the data are expressed as fold de-repression. Note that full de-repression of Vnd–Gal4 by Dichaete requires the presence of the homeodomain and sequences C-terminal to this domain