castor

Targets of Activity

CAS/Ming is required for the correct CNS expression of engrailed (Cui, 1992). Ganglion mother cells generated early in development normally express en. Interstripe neurons usually expressing en are absent in cas/ming mutants, and a reduction of en expression by a factor of two is found in later cells (Mellerick, 1992).

To determine if Cas is a pdm repressor, Pdm-1 and Pdm-2 expression was analyzed in cas null embryos. In stage 9 and in younger embryos, no differences were detected between the cas- and wild-type expression patterns of Pdm-1 or -2. However, starting at stage 10, NBs fail to terminate expression of both Pdms. Ectopic Pdm expression is observed in most, if not all, late developing sublineages in all CNS ganglia. The sustained Pdm expression is most likely due to transcriptional derepression, since PDM-1 mRNA in situ hybridizations also reveal that its message persists in cas- late NBs. In vivo analysis of pdm-1 genomic DNA has identified the main cis-regulatory elements controlling its embryonic expression. These control elements lie within a 6.3 kb DNA fragment, flanking the 5' side of its transcribed sequence. The pdm-1 regulatory DNA contains enhancer(s) that can drive the expression of reporter genes in the same cells in which pdm-1 is normally expressed. In a cas- background, transgenes are ectopically expressed in NBs during late sublineage development. This result demonstrates that the enhancer(s) within the 6.3 kb regulatory DNA are negatively regulated by Cas. To explore the possibility that Cas may play a direct role in silencing pdm gene expression, Cas-DNA immunoprecipitation was carried out with pdm-1 promoter fragments to test for potential Cas DNA-binding sites. DNA sequence analysis of bound fragments reveals 32 potential DNA-binding sites, all sharing at least 8 out of the 10 bp with the Hb consensus sites. Cas binds to these sites. Base pair substitutions in one of the sites demonstrate that the A/T rich core sequence is essential for Cas binding. All together, the results suggest that Hb and Cas regulate pdm expression by interacting directly with their cis-regulators to deactivate controlling enhancer(s), with Hb repressing the pdm genes early and Cas silencing late in CNS development. To test if Cas can silence Pdm-1 expression outside of Cas's endogenous sublineage boundaries, the effects of misexpressing Cas were studied early in CNS development. Indicating that Cas can act as a pdm repressor outside of its normal late expression boundaries, the temporally misexpressed Cas significantly reduces Pdm-1 expression when compared to wild-type embryos stained under identical conditions (Kambadur, 1998).

The role of Castor in regulating pdm genes raises the possibility that it may regulate expressions of other POU genes. To test this, the expression domains of Cas and Drifter/Ventral veins lacking were examined. Drf expression was examined in cas- embryos. In addition to its established role in midline glia and tracheal development, Drf is also expressed in a subset of NB progeny in both the developing brain and ventral cord. Many Cas-expressing NB sublineages also express Drf. Thus, it appears that Cas does not repress drf expression: to the contrary, a marked reduction in late-lineage Drf expression is observed in cas- embryos, suggesting Cas either directly or indirectly plays a role in activating and/or sustaining drf expression in these sublineages. Ectopically activated Cas has no effect on Drf expression. In the absence of castor function, I-POU expression is lost in a subset of ventral cord cells, but ectopic Cas has no effect on the I-POU wild-type expression pattern. It is not known if Cas is a direct activator of drf and/or I-POU. However, the data indicate that if Cas is playing a direct activator role, it most likely requires co-factors that are not expressed outside of its normal domain (Kambadur, 1998).

The mammalian NAB proteins have been identified previously as potent co-repressors of the EGR family of zinc finger transcription factors. Drosophila NAB (dNAB: CG15000), like its mammalian counterparts, binds EGR1 and represses EGR1-mediated transcriptional activation from a synthetic promoter. In contrast, dNAB does not bind the Drosophila EGR-related protein Klumpfuss. dnab RNA is expressed exclusively in a subset of neuroblasts in the embryonic and larval central nervous system (CNS), as well as in several larval imaginal disc tissues. Targeted deletion mutations were created in the dnab gene and the identification is described of additional, EMS-induced dnab mutations by genetic complementation analysis. Null alleles in dnab cause larval locomotion defects and early larval lethality (L1-L2). A putative hypomorphic allele in dnab instead causes early adult lethality due to severe locomotion defects. In the dnab -/- CNS, axon outgrowth/guidance and glial development appear normal; however, a subset of Eve+ neurons forms in reduced numbers. In addition, mosaic analysis in the eye reveals that dnab -/- clones are either very small or absent. Similarly, dNAB overexpression in the eye causes eyes to be very small with few ommatidia. These dramatic eye-specific phenotypes will prove useful for enhancer/suppressor screens to identify dnab-interacting genes (Clements, 2002).

To identify regulators of dnab gene expression, mutants of several NB-expressing transcription factors were screened for loss of dnab RNA expression. Castor is a zinc finger transcription factor expressed in Drosophila NBs and GMCs; castor loss of function causes reductions in CNS axonal density and reductions in the number of neurons expressing the homeodomain protein Engrailed. Furthermore, Castor positively regulates expression of the POU domain transcription factors Drifter and Acj6 and negatively regulates expression of the POU domain transcription factors Pdm-1 and Pdm-2 in the embryonic CNS. dnab expression was examined in cas minus embryos, which contain deletions removing the entire castor gene; dnab expression is affected in these mutants. dnab expression in midline cells at stage 11 appears normally; however, expression fails to spread to NBs during stages 12/13. In the cephalic lobes, only a few cells express dnab. This finding is in contrast to wild-type, where dnab is robustly expressed in many cephalic NB. These results indicate that dnab is either a direct or indirect target gene of Castor. The temporal expression patterns of castor and dnab support these conclusions. Castor expression first appears in NBs at stage 10 and spreads to 9-10 NBs per hemisegment by late stage 11, the stage at which dnab expression is first observed. The Castor protein has been shown to bind the consensus DNA sequence (G/C)C(C/T)(C/T)AAAAA(A/T). A genomic region containing the entire dnab transcription unit, as well as 10 kb upstream and 10 kb downstream of dnab was scanned for Castor binding sites. This analysis revealed that no consensus Castor binding sites occur in the putative dnab promoter region or in dnab introns, although at least two closely related sites occur in the putative promoter region. It may be possible that Castor directly regulates dnab expression through these sites, or through sites in a distal enhancer element. Alternatively, dnab expression might be directly regulated by the transcription factors encoded by the Castor target genes drifter, Acj6, pdm-1, or pdm-2. In these scenarios, drifter and Acj6 might normally function as positive regulators of dnab expression (Clements, 2002).

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.