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. Here we identify 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, we utilized a combination of chimeric transient transfection assays, co-immunoprecipitation and in vivo expression assays. We show that Ind's candidate Eh1 domain is 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, we show 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.

Convergence of dorsal, dpp, and egfr signaling pathways subdivides the drosophila neuroectoderm into three dorsal-ventral columns.

An important question in neurobiology is how different cell fates are established along the dorsoventral (DV) axis of the central nervous system (CNS). Here we investigate the origins of DV patterning within the Drosophila CNS. 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. Here we describe 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. We confirm that the vnd domain is established by Dorsal and maintained by Egfr, but unlike a previous report we show that vnd is not regulated by Dpp signaling. We show that ind expression requires both Dorsal and Egfr signaling for activation and positioning of its dorsal border, and that abnormally high Dpp can repress ind expression. Finally, we show that the msh domain is defined by repression: it occurs only where Dpp, Vnd, and Ind activity is low. We conclude 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.

The ciD mutation encodes a chimeric protein whose activity is regulated by Wingless signaling.

The Drosophila cubitus interruptus (ci) gene encodes a sequence-specific DNA-binding protein that regulates transcription of Hedgehog (Hh) target genes. Activity of the Ci protein is posttranslationally regulated by Hh signaling. In animals homozygous for the ciD mutation, however, transcription of Hh target genes is regulated by Wingless (Wg) signaling rather than by Hh signaling. We show that ciD encodes a chimeric protein composed of the regulatory domain of dTCF/Pangolin (Pan) and the DNA binding domain of Ci. Pan is a Wg-regulated transcription factor that is activated by binding of Armadillo (Arm) to its regulatory domain. Arm is thought to activate Pan by contributing a transactivation domain. We find that a constitutively active form of Arm potentiates activity of a CiD transgene and coimmunoprecipitates with CiD protein. The Wg-responsive activity of CiD could be explained by recruitment of the Arm transactivation function to the promoters of Hh-target genes. We suggest that wild-type Ci also recruits a protein with a transactivation domain as part of its normal mechanism of activation.

Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity.

One of the first steps in neurogenesis is the diversification of cells along the dorsoventral axis. In Drosophila the central nervous system develops from three longitudinal columns of cells: ventral cells that express the vnd/nk2 homeobox gene, intermediate cells, and dorsal cells that express the msh homeobox gene. Here we describe a new Drosophila homeobox gene, intermediate neuroblasts defective (ind), which is expressed specifically in the intermediate column cells. ind is essential for intermediate column development: Null mutants have a transformation of intermediate to dorsal column neuroectoderm fate, and only 10% of the intermediate column neuroblasts develop. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, whereas ind represses msh in the intermediate column. 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.

Hedgehog signaling regulates transcription through cubitus interruptus, a sequence-specific DNA binding protein.

Hedgehog (Hh) is a member of a family of secreted proteins that direct patterning at multiple stages in both Drosophila and vertebrate development. During Drosophila embryogenesis, Hh protein is secreted by the cells of the posterior compartment of each segment. hh activates transcription of wingless (wg), gooseberry (gsb), and patched (ptc) in the cells immediately adjacent to Hh-secreting cells. Hh signaling is thought to involve the segment polarity gene cubitus interruptus (ci). ci encodes a zinc finger protein of the Gli family of sequence-specific DNA binding proteins. ci mRNA is expressed in all non-Hh expressing cells. Here we demonstrate ci activity is both necessary and sufficient to drive expression of Hh-responsive genes in the Drosophila embryos. We show that Ci is a sequence-specific DNA binding protein that drives transcription from the wg promoter in transiently transfected cells. We demonstrate that Ci binding sites in the wg promoter are necessary for this transcriptional activation. These data taken together provide strong evidence that Ci is a transcriptional effector of Hh signaling.

Hedgehog signaling regulates transcription through Gli/Ci binding sites in the wingless enhancer.

The segment polarity gene cubitus interruptus (ci) encodes a transcriptional effector of Hedgehog (Hh) signaling in Drosophila. The Ci gene product is a zinc finger protein belonging to the Gli family of sequence-specific DNA binding proteins. After gastrulation, segmental expression of the segment polarity gene wingless (wg) is maintained by Hh signaling in a pathway requiring Ci activity. In the absence of Hh or Ci activity, wg expression is initiated normally and then fades in the ectoderm after stage 10. We have previously identified a wingless enhancer region whose Ci binding sites mediate Ci-dependent transcriptional activation in transiently transfected cells. Here we demonstrate that Hh and Patched (Ptc) act through those Ci binding sites to modulate the level of Ci-dependent transcriptional activation in S2 cells. We demonstrate that this same wg enhancer region is Hh responsive in vivo and that its Ci binding sites are necessary for its activity. This provides strong evidence that Hh affects wg transcription through post-translational activation of Ci.

The Drosophila smoothened gene encodes a seven-pass membrane protein, a putative receptor for the hedgehog signal.

Smoothened (smo) is a segment polarity gene required for correct patterning of every segment in Drosophila. The earliest defect in smo mutant embryos is loss of expression of the Hedgehog-responsive gene wingless between 1 and 2 hr after gastrulation. Since smo mutant embryos cannot respond to exogenous Hedgehog (Hh) but can respond to exogenous Wingless, the smo product functions in Hh signaling. Smo acts downstream of or in parallel to Patched, an antagonist of the Hh signal. The smo gene encodes an integral membrane protein with characteristics of G protein-coupled receptors and shows homology to the Drosophila Frizzled protein. Based on its predicted physical characteristics and on its position in the Hh signaling pathway, we suggest that smo encodes a receptor for the Hh signal.