BIOLOGICAL OVERVIEW

The unplugged (unpg) gene was identified by systematic screening of P element enhancer detector lines for beta-galactosidase expression patterns, suggestive of regulation by the homeotic gene Ultrabithorax (Ubx). Line f85, which carries a P element insertion in the unpg locus, expresses beta-galactosidase in a pattern restricted to the lateral ectoderm of the first thoracic segment, suggesting that Ubx may negatively regulate expression in more posterior segments. The f85 line also displays a segmentally repeated pattern of expression in neuroblasts and neurons. unplugged is required for formation of specific tracheal branches, including some segment-specific branches. The segmentally reiterated ganglionic branches fail to penetrate the CNS in the absence of unpg function. In addition, unpg function is specifically required for development of the cerebral branch, a tracheal branch uniquely derived from the first thoracic segment (Chiang, 1995).

The expression of unpg in founder cells of the cerebral branch within the first tracheal placode suggests an early role during branch development. unpg function, however, is most likely not involved in the initial commitment to founder cell fates, since expression of the lacZ reporter gene by the enhancer trap is maintained in unpg mutant embryos. Instead, unpg appears to be involved in branching morphogenesis by regulating cell migration or extension; in the absence of such function, the founder cells either die or adopt other branch patterns. This is consistent with the observation that in unpg mutant embryos the absence of the cerebral branch is occasionally accompanied by the presence of an ectopic branch in the first tracheal metamere. The appearance of this ectopic branch resembles that of the dorsal branch, as well as the dorsal cephalic branch, both of which, like the cerebral branch, originate from the first tracheal placode. In addition to the cerebral branch, unpg is also expressed in cells of the ganglionic branches, but here expression occurs much later, suggesting that unpg may have secondary functions during ganglionic branch development. Consistent with this view is the observation that the ganglionic branches develop but fail to extend consistently to the CNS in unpg mutant embryos. This phenotype is reminiscent of the hypormorphic alleles of pointed and breathless mutants. breathless encodes a Drosophila homolog of the fibroblast growth factor (FGF) receptor, and its expression in the developing tracheal system is required for the migration of tracheal cells. Thus, the observed unpg phenotype appears to be consistent with the role of unpg in the specification of tracheal cell migration or extension (Chiang, 1995).

Restricted expression of unpg in the cerebral branch founder cells requires normal function of genes in the Bithorax complex (BX-C). In the absence of these homeotic genes, the expression of unplugged expands more posteriorly to the abdominal segments. This is consistent with the notion that Ultrabithorax controls tracheal development by regulating the expression of target genes. Since unpg encodes a transcription factor and is required for cerebral branch development, it is suggested that normal restriction of cerebral branch development to T1 is mediated by Ubx repression of unpg. This repression is mediated by the 2.7 kb fragment located downstream of the unpg transcription unit (Chiang, 1995).

GENE STRUCTURE

Direct comparison between the unpg cDNA and genomic sequences reveals that the transcription unit of unpg is organized in 3 exons (each 3.5 kb) and separated by two introns (each 62 bp). Sequence analysis of the first intron of unpg also reveals that it contains a transposable element; this 1360 bp element is moderately repetitive and has copy numbers ranging from 25 to 30 within the Drosophila genome. Whether presence of the 1360 bp element in the unpg locus has any functional significance is unknown (Chiang, 1995).

Bases in 5' UTR - 125

Exons - 3

Bases in 3' UTR - 196

PROTEIN STRUCTURE

Amino Acids - 486

Structural Domains

The Unpg protein contains a homeodomain (residues 319-378) belonging to a family that includes several vertebrate homeobox genes. The homeodomain shares amino acid identities ranging between 90% and 93% for homeodomains of the CHox7 gene in chicken, the HOX7Q and GBX2 genes in human, partial sequence of the MMoxA gene in mouse, the XlHox7a and XlHox7b gene in Xenopus, the G9 gene in goldfish, and the Hrox7 gene in abalone. Little is known about the expression and function of these vertebrate homologs; however, the MMoxA and G9 genes were initially isolated from brain libraries and thus may be involved in brain development or function. One of two introns in the unpg transcription unit interrupts homeodomain coding sequences at a location first noted in labial-class homeobox genes. This location, between Gln 44 and Val 45, is conserved for introns of many other homeodomain genes of different species, but is distinct from the location of the intron that interrupts homeodomain coding sequences in engrailed and related genes. Outside of the homeodomain, unpg shares no significant homology with other known proteins in the data base. However, Pro/Gln-rich regions are found amino terminal to the homeodomain, with one particular region from residues 111 to 142 comprising 52% proline and glutamine residues. Pro/Gln-rich regions are found in many proteins that are capable of transcriptional activation (Chiang, 1995).

date revised: 8 April 98

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