In vivo Time Lapse Confocal Analysis of the RhoA Head Involution Defect and Molecular and Genetic Characterization of Five Extant RhoA Mutant Alleles. Melissa Maloof¹, Rachel Stottlar¹, Pria Chang¹, Laura Johansen¹, Katherine Sinclair¹, Maureen Filak¹, Fafa H. Koudoro¹, Rahul Warrior², Susan R. Halsell¹. 1) Biology, James Madison University, Harrisonburg, VA; 2) Developmental and Cell Biology, University of California, Irvine, CA.

   RhoA signal transduction functions in myriad morphogenetic processes throughout the Drosophila life cycle. Characterized RhoA mutant alleles are homozygous embryonic lethal with a characteristic defect in head involution. This work presents analysis of the head involution defect using time-lapse confocal microscopy. Cells and their actin cytoskeleton were visualized using an actin-binding GFP-moesin fusion protein driven by the spaghetti-squash promoter (SGMCA; Edwards et. al. 1997. Dev. Biol. 191, 103). Wild type and null RhoA mutants were analyzed. To date, data indicates the dorsal ridge forms normally and begins its anterior-ward movement, but is ultimately impeded on the dorsal side of the embryo when the procephalon and clypeolabrum fail to retract; this is consistent with the dorsal anterior hole observed in cuticle preparations of RhoA mutant embryos. Phenotypic and molecular characterization of five EMS induced RhoA mutations (233 and 246, Ward, Evans and Thummel, Genetics. 165:1397; 3.5.1, 4.4.2 and 7.23.1) will also be described. Four of the five alleles exhibit apparent complete loss of function phenotypes; they are 100% embryonic lethal and all show consistent anterior dorsal holes in the cuticle. In addition, three of these alleles have been sequenced and each shows a mutation consistent with a null phenotype. All are single G to A transitions and alter either the start codon, a splice donor site or introduce a premature stop codon. The fifth allele, however, may be a hypomorphic mutation. This is based on the observation that the mutation is not 100% lethal embryonically and the dead embryo cuticle phenotype is variable, with the majority of embryos exhibiting defects in the head skeleton by no dorsal anterior hole. Genetic and molecular characterization of this allele continues.