castor

Transcriptional Regulation

The ventral nerve cord (VNC) of Drosophila exhibits significant segmental-specific characteristics during embryonic development. Homeotic genes are expressed over long periods of time and confer identity to the different segments. castor (cas) is one of the genes which are expressed in neuroblasts along the VNC. However, at late embryonic stages, cas transcripts are found only in head and thoracic segments and terminal abdominal segments, while Cas protein lasts longer in all segments. This study investigated the regulation of temporal and spatial expression of cas by the bithorax complex genes. In the loss-of-function mutants of Ultrabithorax (Ubx) and abdominal-A (abdA), cas transcripts were ectopically expressed in abdominal segments at late embryonic stage. However, unlike in Ubx and abdA mutants, in Abdominal-B (AbdB) loss-of-function mutant embryos, cas disappeared in the terminal region. Ectopic Ubx and abdA suppressed cas expression, but ectopic AbdB activated cas expression in most abdominal segments. Moreover, cas was co-expressed in the cells in which AbdB was normally expressed, and overexpressed in the ectopically expressed AbdB embryos. These results suggest that the expression of cas is segment-specifically regulated negatively by Ubx and abdA genes, but positively by the AbdB gene (Ahn, 2010).

cas is transiently expressed in a subset of neuroblasts in their cell lineage. Its transcripts are present with homologous patterns in thorax and abdomen at early embryonic stages. At late embryonic stages, cas transcripts are found in only a few cells per hemisegment in thoracic and posterior abdominal segments, but not in other abdominal segments. This indicates that cas is expressed in segment-specific mode during late embryonic stages. This study investigated how this regional diversity was produced (Ahn, 2010).

The segment-specific expression of cas was regulated by the homeotic genes. Ubx or abdA mutation caused the homeotic transformation of abdominal cuticle belts to the more anterior ones. These transformation patterns were also observed in cas expression. Mutations in Ubx or abdA genes caused ectopic cas expression in abdominal segments, which was normally observed in the thoracic segments at stage 15 of wild-type embryo, suggesting transformation of the thoracic pattern to an abdominal pattern at that stage. This transformation was synergistically enhanced in the Ubx and abdA double mutants (Ahn, 2010).

cas was ectopically expressed in A1 segment in Ubx mutant embryos. This result is coincident with the function of Ubx to specify the posterior thorax and a portion of A1 segment. cas was also ectopically expressed in A1 to A4 segments in abdA mutant embryos, which coincide with the function of abdA. In Ubx abdA double mutant embryos, cas was expressed in virtually all abdominal segments and in more cells than in each single mutant embryo. The roles of Ubx and abdA on cas expression in the abdominal segments were confirmed in the ectopically expressed Ubx and abdA mutant embryos. For this experiment, proper embryonic stages were very important because cas expression changed dramatically in the abdominal segments between stages 14 and 15. Whether ectopic Ubx or abdA repressed cas expression in the abdominal segments was tested at this stage. The GAL4-mediated induction of Ubx or abdA suppressed cas expression in the abdominal segments (Ahn, 2010).

However, in contrast to the Ubx and abdA mutations, AbdB mutation caused reverse effects on cas expression in the abdominal segments, which have never been reported. Loss-of AbdB function caused lack of cas expression, while ectopic ABDB activated cas expression in the abdominal segments. This phenotype was also observed in Polycomb mutant embryos. Although Polycomb mutation induced ectopic expression of Ubx, abdA and AbdB at the same time, cas was ectopically expressed in the abdominal segments of stage15 embryos, suggesting that ABDB dominated the effects of ectopic UBX or ABDA. The co-localization of cas and ABDB is found in a few cells in the posterior abdominal segments, supporting the positive regulation of cas by ABDB (Ahn, 2010).

This idea was further intensified by the appearance of the ectopic cas mRNA in the numerous abdominal cells with the ectopic AbdB expression. Real-time PCR experiment showed the overexpession of cas mRNA in the ectopically expressed AbdB embryos, also supporting the positive regulation of cas by ABDB. Furthermore, seven AbdB DNA binding sites were found within 5kb upstream from the cas transcription start site enhancing the possibility that ABDB directly regulates the cas expression. ABDB binds preferentially to a sequence with an unusual 5'-TTAT-3' core (Ahn, 2010).

One of questions was why all the cells with the AbdB expression does not show cas mRNA expression. In wild-type embryos, all AbdB-expressing cells does not show cas mRNA. Only a few cells among AbdB-expressing cells could maintain the expression of cas and the other cells lost it. This might be that the homoetic proteins carry out their function by interacting with other cofactors to regulate distinct sets of downstream genes (Ahn, 2010).

Accumulating evidence shows that the bithorax complex genes are involved at different steps in the segment-specific divergence of the CNS. Ubx or abdA activity is required for the abdominal pathway of the NB1-1 lineage. Both ectopic induction of Ubx- or abdA expression until several hours after gastrulation and homeotic de-repression in Polycomb mutants, override thoracic determination of NB1-1. The abdominal NB6-4 lineage is also specified by the abdA and AbdB. abdA is expressed in the NB6-4 lineage of abdominal segments A1-A6, whereas AbdB is expressed in the NB6-4 lineage of segments A7-A8. They specify the abdominal NB6-4 lineage by down-regulating levels of G1 Cyclin (CycE) (Ahn, 2010).

In summary, UBX and ABDA suppress cas expression in abdominal segments, so that mutation in both genes causes ectopic expression of cas in abdominal segments at late embryonic stage. However, ABDB activates cas expression, which is supported by co-localization of cas and ABDB in cells ectopically expressing AbdB, and real-time PCR in ectopically expressed AbdB embryos (Ahn, 2010).

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.