Dr. DiNardo proposes to continue his investigation of inductive events that establish the body pattern during Drosophila embryogenesis. He and other investigators have shown that certain aspects of Drosophila pattern formation are determined by organizer regions analogous to the classic organizers described in vertebrate system. The molecular components of the Drosophila system responsible for organizing pattern across each segment include to hedgehog and wingless, for which vertebrate homologues function in the organizers in vertebrates. Wingless and hedgehog are expressed in adjacent rows of cells flanking the parasegmental boundary. Reciprocal signaling occurs between the WG and HH expressing cells. For example engrailed, a homeoprotein expressed in the HH cells is maintained by inductive effects of WG secreted from the adjacent row of cells. Engrailed, in turn, maintains the expression of HH. In addition a signal from the en/HH cells helps to maintain expression WG in the WG cells. DiNardo has recently shown that WG and HH organize much of the pattern within the epidermis by specifying distinct cell types. WG and HH are also crucial to the patterning within imaginal disks. DiNardo raises three major questions: Are WG HH the only signals in this patterning system? do WG and HH act to specify cell fates over a distance, or do other signals mediate the patterning by WG and HH? What are the components of the transduction pathways? The focus of the current proposal is on the patterning of the larval parasegments and the imaginal discs as related to the function of the lines gene. There are four distinguishable cell types along the anterior-posterior axis of a parasegment. WG specifies the 4th cell type whereas HH specifies the 1st, 2nd and 3rd cell types. In the absence of HH only the 4th cell type is specified. HH also plays a role in blocking the activity of patched in the 1st and 2nd cell types. (In patched-HH double mutants the 1st and 2nd cell types are specified, demonstrating the blocking function of HH on patched.) Moreover, this indicates that that the 3rd cell type requires HH but not through its antagonism of patched. In lines mutants, 1-3 celltypes are present and the fourth cell type is replaced by the 3rd celltype. In lines-HH double mutants the 3rd cell type is restored but the 4th celltypes are missing. Thus it appears that HH normally antagonizes the activity of lines. In ventral patterning lines does not alter the expression of WG, HH, or en but apparently regulates dpp expression. Mosaic analysis of lines during imaginal development of the leg indicates a compression along the proximo-distal axis and outgrowth. The first specific aim is to complete the phenotypic analysis of lines in epidermal patterning. This will include determining if maternally contributed lines is essential for lines function during embryogenesis and if it is, is the maternal function related D/V pattern or segmental polarity. Once the lines gene has been cloned genetic interaction experiments will be performed to order lines relative to other genes thought to be in the HH pathway including fused, PKA, patched, and ciD. The second specific aim is to the determine the primary effect of removing lines activity during imaginal disc development. Preliminary mosaic analysis indicated that lines mutant cells either died or were incorporated into outgrowths that did not differentiate. Consequently it could not be determined if lines activity acted autonomously. The FLP-FRT system will be used to induce lines clones and the expression of WG, HH ptc, Dll and Dpp will be examined. The third specific aim is to clone the lines gene. So far attempts to isolate P-element induced lines mutant which could be readily used for cloning have failed. However, the region containing lines has been narrowed to a segment containing four complementation groups (including lines) and a chromosome walk is underway to clone this region. Several approaches to identifying the lines gene will used including mapping gamma ray alleles and RFLPs associated with lines mutant alleles, analysis of the expression of candidate transcription units, and by transgene rescue experiments. Once the lines gene has been isolated and appropriate molecular tools are constructed, a number of experiments are proposed to further probe lines function. The fourth specific aim is to identify other mutants affecting epidermal pattern. 890 P-element induced lethal lines will be screened or cuticle defects that represent a switch in cell fates. A number of other secondary screens are proposed to reduce the number of lethal lines to those of particular interest.
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