MOLECULAR AND FUNCTIONAL CHARACTERIZATION OF TWO POU HOMEODOMAIN GENES IN DROSOPHILA MELANOGASTER

Abstract

The formation of the Drosophila embryonic nervous system is dependent on a complex network of interacting developmental regulators. Such developmental regulators include different families of transcription factors which may account for the enormous diversity of neuronal phenotypes and functions. A new class of transcription factors, the POU domain proteins, is found to be expressed and function in neuronal tissues. Using PCR, two novel POU genes, namely c/POU19 and c/POU28, were isolated from Drosophila. dPOU19 and dPOU28 are transcribed as 3.1 kb and 2.2 kb mRNAs which encode 601 and 475 amino acid deduced proteins, respectively. Both deduced proteins have their POU domains near their C-termini. Their POU domains are most related to those of the class II POU proteins, for example, the human OCT1 and OCT2 proteins. Both POU domains are very homologous to each other, 99% identical in their POU-specific domains and 82% identical in their POU-homeodomains. Both genes are preferentially expressed during the first 12 hours of embryonic development. Their embryonic RNA in situ expression patterns reflect their protein distributions. Both dPOU 19 and dPOU28 gene products, namely DPOU 19 and DPOU28 respectively, are expressed as early as in the blastoderm stage and later they are coexpressed in the neuroblasts and ganglion mother cells during early neurogenesis. They are also expressed in the precursors, and a subset of the support cells and neurons of the peripheral nervous system. Their coexpression pattern is suggestive of a possible physiological interaction during neurogenesis and neurospecification. Both dPOU19 and dPOU28 are coexpressed to high levels in GMC4-2a, the first GMC produced by NB4-2, which gives rise to the RP2 neuron. However, only the ectopic expression of dPOU28 (and not dPOU19) in the progeny of GMC4-2a causes both of these cells to adopt their parental GMC4-2a fate and thus generate a duplication of the RP2 neurons. This result suggests that dPOU28 normally plays a role in GMC4- 2a cell fate specification. The overlapping functional roles of dPOU19 and dPOU28 in the specification of GMC4-2a cell identity was definitively demonstrated with the isolation and analysis of a collection of mutants in dPOU19 and dPOU28. These genes are not required for the birth of GMC4-2a but they act to specify the identity of GMC4-2a, with dPOU28 as the more dominant player. Both of these genes together contribute towards the development of a full complement of mature RP2 neurons. In some mutant combinations where no mature RP2 neurons are formed, some EVE positive GMC4-2a's can nevertheless divide, implying that the failure of the POU mutants to produce mature RP2 neurons is not clue to a block in GMC4-2a cell division per se but rather because the GMC4-2a's fail to acquire their correct cellular identity. Furthermore, within the hierachy of genes which are expressed in GMC4-2a, the POU genes are found to lie downstream of pros and ftz and upstream of eve. The data on the isolation, molecular, genetic and phenotypic characterisation of dPOU19 and dPOU28 are presented.