In this application a molecular and phenotypic analysis of the gene oro is proposed. oro was identified by the investigator as an enhancer of Bar in eye development. oro interacts with the segment polarity gene hedgehog (hh) in the eye and produces an embryonic segment polarity phenotype by itself. oro is uncovered by the deficiency Df(2l)al. A P-element inserted near, but not in, the oro locus has been identified. Thus, a small deficiency generated by imprecise excision of P l(2)06751, called oroDP1, has been identified which does not complement oro1.
The first aim i s to clone the oro gene. Several P1 and lambda clones which cover part of the interval to which oro has been mapped are available. It will be first investigated by Southern analysis whether one of the three lambda phages uncovers the breakpoint. If this turns out to be the case, fragments of the corresponding phage will be subcloned and further analyzed to identify a transcript. In case of a negative result, subclone libraries of the 80kB P1 plasmid will be generated to identify fragments which uncover the oro breakpoint. Genomic fragments falling out of this search will be used for Northern analysis and to screen for cDNA clones. Sequence analysis of cDNA clones and transformation rescue of the oro phenotype is further proposed. In the second aim it is proposed to induce further oro alleles, in particular nulls. The strategy is the same employed for the identification of oro1 itself, i.e., screening for EMS and g-ray induced dominant enhancers of Bar. The reason to prefer this scheme is that it represents an F1 screen which is less work intensive than an F2 screen for embryonic lethals; the rationale that it should be successful is that one copy of oro1, itself a strong hypomorphic allele, leads to an enhancement of the Bar phenotype in the eye; null alleles of oro should lead to an even stronger enhancement.
The third aim focusses on the function of oro in eye development. Preliminary data had placed oro between hh and decapentaplegic (dpp). First, the expression pattern of oro in the eye disc will be studied in detail by in situ hybridization and antibody staining. The latter requires the production of oro antibody, which will be achieved by injecting oro-fusion protein. Next, homozygous oro clones will be generated, using the FLP/FRT technique. Initially, oro1 will be used; the data will be complemented by using a null allele, as soon as this becomes available. Clones will be analyzed in the adult (defects in mature ommatidial cells) and in larval discs (defects in morphogenetic furrow progression, neural differentiation and expression of the genes dpp, atonal (ato), string (stg) and hairy (h). A hs-oro construct will be generated and transformed into flies to analyze the effect of overexpression of oro on eye development. Finally, clones doubly mutant for oro and hh, oro and patched, and oro and PKA will be analyzed to establish how oro relates to the function of these genes.
Aim 4 addresses the interaction between oro and known segment polarity genes during embryogenesis. Expression of oro in the wild-type embryo and in mutations of other segment polarity genes (wingless, engrailed) will be analyzed. Expression of wingless, engrailed, hedgehog in the background of oro loss of function and oro overexpression will be analyzed.
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