My doctoral thesis dealt mainly with a developmental biology project concentrating on the genetic characterization of a Drosophila gene teashirt (tsh)
, which was a relatively newly discovered gene, with its primary role in regulating trunk development.
Developmental expression of transduced mini-white (w) gene of Drosophila (fruitfly) is sensitive to its flanking genomic enhancers. Taking advantage of this phenomenon, a PlacW transposon was mobilized and screened for new transposant lines that showed patterned expression of the mini-w gene in the adult eyes. From a screen of about 1000 independent PlacW lines on the second chromosome, we identified seven lines, which showed patterned w expression in the adult eyes (Bhojwani et al., 1991). In this study, evidence was presented to show that four of these insertion lines located at the cytogenetic interval 40A ( in situ ) were found to be allelic to teashirt (tsh) homeotic gene (Fasano et al., 1991). The allelism of these insertions at tsh locus was established on the basis of their chromosomal locations, developmental expression of the lacZ reporter gene, lethal phenotypes and, finally, their failure to complement the known lethal alleles of tsh . These results showed that although only a small fraction of the total transposant lines displayed patterned w expression (anterior and posterior sectors of the eye), the genetic loci thus identified are those which play essential roles in pattern formation.
In addition to this, results of the role of tsh homeotic gene in patterning the adult fly were presented. These aspects were approached by two methods; firstly, by the study of lacZ reporter expression in imaginal discs and adult cuticle and, secondly, on the basis of the phenotype of dominant gain-of-function mutation of Antennapedia (Antp) in tsh mutant animals. The lacZ reporter expression of all the imaginal discs excepting eye-antennal disc displayed lacZ reporter in presumptive proximal region of the future adult appendages. In the eye-antennal discs, initially a weak lacZ reporter gene expression was seen in isolated patches corresponding to the third antennal segment. During subsequent stages of development, this expression became robust and eventually translated into strong tsh specific lacZ reporter expression in posterior compartments of the antennal structures in adult head. tsh expression was also seen in the posterior compartments of the maxillary palps. This posterior compartment specific expression in the adult structures could be further confirmed by comparing domains of the lacZ reporter gene expression with that of engrailed (en). Compartment specific activity of tsh in these adult structures strongly suggested its role as a selector gene as in case of en and other homeotic genes, which express in domains defined by anterior/posterior compartment boundary. Besides, tsh specific expression was also registered in most of the appendages/derivatives of the adult head.
Closer examination of adults homozygous for various PlacW insertions led to the identification of a hypomorphic allele. These mutant homozygotes (tsh75) revealed reduction in the size of maxillary palps reminiscent of the phenotype seen in flies heterozygous for dominant gain-of-function alleles of Antp homeotic gene.
This phenotype was taken as a clue to imply misexpression of Antp or other related genes in tsh mutant background. Based on this evidence, it was argued that in tsh mutant or heterozygous animals, the mutant phenotype of Antp dominant mutations would be further enhanced. True to this prediction, animals homo- or heterozygous for different tsh alleles identtified in this study and those reported earlier, displayed a stronger antenna-to-leg transformation due to dominant Antp mutations. Genetic interaction experiments between various allelic combinations of tsh and Antp and monitoring the effects of reducing the doses of tsh in Antp-dominant (AntpD) mutations (in which the ANTP protein levels are known to be elevated per se ), proved to be the key experiments in this study.
Enhancement of antenna-to-leg transformation apart, a novel homeotic transformation of the eye-to-notum was seen in animals transheterozygous for dominant Antp alleles and various tsh mutations. These results showed that cell fate determination in both eye and antennal discs requires normal tsh expression. A combination of Antp misexpression and absence or aberrant expression of tsh creates a situation where cell fate of eye disc is disposed toward that of notum, which is derived from the wing disc. tsh is presumably involved in stable overexpression of Antp and other as yet unidentified genes in the eye disc to maintain its determination to the ommatidial cell fate. In this respect, tsh behaves as a transregulator of Antp in the eye-antennal discs. Quite interestingly, in the eye-antennal imaginal discs of these double mutant larvae, the ANTP protein levels were found to dramatically elevate with decreasing doses of tsh , suggesting tsh to be a crucial negative regulator (effector) of Antp in defining head versus thoracic identity in Drosophila . Thus, tsh not only regulates trunk (thorax+abdomen) development in Drosophila but is also recruited towards a novel function in controlling head development. tsh acts as an “effector” that tips this balance by negatively regulating a master control gene, Antennapedia (Antp ), which is known to be a classic homeotic selector switch acting in the differentiation of head and thoracic segments in the fly (Bhojwani et al. , 1997). This study therefore revealed a role of tsh during adult development and opened prospects of further identification of its role in segmental fate determination of the adult fly, as evidenced by subsequent studies on the gene.