Promoter Structure

To identify cis-regulatory regions that are responsible for thenormal unpg expression in embryos, six restriction fragmentsencompassing 20 kb of unpg genomic sequence were tested fortheir ability to direct expression of a lacZ reporter gene containing a minimal hsp70 promoter. The only region capable of driving a lacZ reporter geneexpression similar to the unpg protein distribution is located atthe 3' end of the unpg transcription unit. The 2.7 kb fragment givescharacteristic unpg-like expression in the CNS and the cerebraland ganglionic branches of the tracheal system during embryogenesis. This fragment also gives unpg-like expression in the cells around the first tracheal pit at thegermband extended stage. Thus a 2.7 kb fragment of unpg 3' flanking sequence containsmost of the cis-regulatory elements for the normal unpg expression in embryos (Chiang, 1995)

Transcriptional Regulation

unplugged expression occurs in portions of the trachealsystem that penetrate the CNS, including the cerebral branchspecific to T1. To test the possibility that genes inthe BX-C play a role in regulating unpg expression, the distribution of unpg transcript was examined in Ultrabithorax and abdominal-A mutants, and inUbx, abd-A, Abd-B triple mutants. In Ubx mutantembryos additional unpg expression is observed in cells surroundingthe tracheal pits of T2 and T3, indicative of a role forUbx in repression of unpg in the posterior segments and consistent with homeotic transformation in Ubx mutants ofposterior T2 and T3 toward a T1 identity. In abd-A mutantsembryos extra patches of unpg-expressing cells around thetracheal pits extend posteriorly to A7, indicating a role for Abd-Ain the repression of unpg expression in the abdominalsegments. The homeotic gene Abdominal-B probably contributes to the repression of unpg expression in A7, since slightly elevated expression in A7 is observed in the triplemutants (Chiang, 1995).

The specific branching defects observed in unplugged mutantembryos prompted an examination of the relationship between unpgand pointed (pnt), a gene encoding a member of the ets familyof transcription factors; pnt is expressed in tracheal placodesand in the developing tracheal branches. In the absence of pnt gene function, tracheal cells fail to migrate and branches do not extend to target tissues. In particular, stalling of the ganglionic branches at the ventral oblique musculature in hypomorphic pnt embryos is reminiscentof the most extreme phenotype observed in unpg embryos.To determine the regulatory relationship between pnt and unpg genes, pnt homozygous embryos were immunostained with Unplugged-specific antibody, in order to follow the fate of the ganglionicbranches. At stage 12.5, ganglionic branch precursor cells migrating toward the CNS are clearly visible in wild-type embryos. In pnt mutant embryos, no migratory cellsthat are expressing Unpg protein can be detected; however, a few precursor cells accumulating low levels of Unpg protein occasionally can be identified. By stage 14, the ganglionic branches of normal embryos are welldeveloped, as is evident from a group of 8 to 9 unpg-expressingcells along the ventrolateral region of each hemisegment. In pnt mutant embryos, only 3 to 4unpg-expressing cells can be detected; these unpg-expressingcells are clustered in a group suggesting that the precursor cells remain immobile and fail to extend from thetracheal pits. These results suggest that the ganglionic branch phenotype in pointed embryos may be in part due to a loss or reduction of unpg gene expression and failure of unpg-expressingcells to extend into the CNS (Chiang, 1995). It would be of interest to determine if unpg functions downstream of pnt in the ventral midline of the CNS.

unplugged: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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