RhoGAP RGA-8 supports morphogenesis in C. elegans by polarizing epithelia.

CDC-42 regulation of non-muscle myosin/NMY-2 is required for polarity maintenance in the one-cell embryo of Caenorhabditis elegans CDC-42 and NMY-2 regulate polarity throughout embryogenesis, but their contribution to later events of morphogenesis are less understood. We have shown that epidermal enclosure requires the GTPase CED-10/Rac1 and WAVE/Scar complex, its effector, to promote protrusions that drive enclosure through the branch actin regulator Arp2/3. Our analysis here of RGA-8, a homolog of SH3BP1/Rich1/ARHGAP17/Nadrin, with BAR and RhoGAP motifs, suggests it regulates CDC-42, so that actin and myosin/NMY-2 promote ventral enclosure during embryonic morphogenesis. Genetic and molecular data suggest RGA-8 regulates CDC-42, and phenocopies the CDC-42 pathway regulators WASP-1/WSP-1 and the F-BAR proteins TOCA-1 and TOCA-2. Live imaging shows RGA-8 and WSP-1 enrich myosin and regulate F-actin in migrating epidermal cells during ventral enclosure. Loss of RGA-8 alters membrane recruitment of active CDC-42. We propose TOCA proteins and RGA-8 use BAR domains to localize and regenerate CDC-42 activity, thus regulating F-actin levels, through the branched actin regulator WSP-1, and myosin enrichment. RhoGAP RGA-8 thus polarizes epithelia, to promote cell migrations and cell shape changes of embryonic morphogenesis.

The RhoGAP HUM-7/Myo9 integrates signals to modulate RHO-1/RhoA during embryonic morphogenesis in Caenorhabditiselegans.

During embryonic morphogenesis, cells and tissues undergo dramatic movements under the control of F-actin regulators. Our studies of epidermal cell migrations in developing Caenorhabditiselegans embryos have identified multiple plasma membrane signals that regulate the Rac GTPase, thus regulating WAVE and Arp2/3 complexes, to promote branched F-actin formation and polarized enrichment. Here, we describe a pathway that acts in parallel to Rac to transduce membrane signals to control epidermal F-actin through the GTPase RHO-1/RhoA. RHO-1 contributes to epidermal migration through effects on underlying neuroblasts. We identify signals to regulate RHO-1-dependent events in the epidermis. HUM-7, the C. elegans homolog of human MYO9A and MYO9B, regulates F-actin dynamics during epidermal migration. Genetics and biochemistry support that HUM-7 behaves as a GTPase-activating protein (GAP) for the RHO-1/RhoA and CDC-42 GTPases. Loss of HUM-7 enhances RHO-1-dependent epidermal cell behaviors. We identify SAX-3/ROBO as an upstream signal that contributes to attenuated RHO-1 activation through its regulation of HUM-7/Myo9. These studies identify a new role for RHO-1 during epidermal cell migration, and suggest that RHO-1 activity is regulated by SAX-3/ROBO acting on the RhoGAP HUM-7.

Molecular population genetics of inversion breakpoint regions in Drosophila pseudoobscura.

Paracentric inversions in populations can have a profound effect on the pattern and organization of nucleotide variability along a chromosome. Regions near inversion breakpoints are expected to have greater levels of differentiation because of reduced genetic exchange between different gene arrangements whereas central regions in the inverted segments are predicted to have lower levels of nucleotide differentiation due to greater levels of genetic flux among different karyotypes. We used the inversion polymorphism on the third chromosome of Drosophila pseudoobscura to test these predictions with an analysis of nucleotide diversity of 18 genetic markers near and away from inversion breakpoints. We tested hypotheses about how the presence of different chromosomal arrangements affects the pattern and organization of nucleotide variation. Overall, markers in the distal segment of the chromosome had greater levels of nucleotide heterozygosity than markers within the proximal segment of the chromosome. In addition, our results rejected the hypothesis that the breakpoints of derived inversions will have lower levels of nucleotide variability than breakpoints of ancestral inversions, even when strains with gene conversion events were removed. High levels of linkage disequilibrium were observed within all 11 breakpoint regions as well as between the ends of most proximal and distal breakpoints. The central region of the chromosome had the greatest levels of linkage disequilibrium compared with the proximal and distal regions because this is the region that experiences the highest level of recombination suppression. These data do not fully support the idea that genetic exchange is the sole force that influences genetic variation on inverted chromosomes.

Evolutionary history of the third chromosome gene arrangements of Drosophila pseudoobscura inferred from inversion breakpoints.

The third chromosome of Drosophila pseudoobscura is polymorphic for numerous gene arrangements that form classical clines in North America. The polytene salivary chromosomes isolated from natural populations revealed changes in gene order that allowed the different gene arrangements to be linked together by paracentric inversions representing one of the first cases where genetic data were used to construct a phylogeny. Although the inversion phylogeny can be used to determine the relationships among the gene arrangements, the cytogenetic data are unable to infer the ancestral arrangement or the age of the different chromosome types. These are both important properties if one is to infer the evolutionary forces responsible for the spread and maintenance of the chromosomes. Here, we employ the nucleotide sequences of 18 regions distributed across the third chromosome in 80-100 D. pseudoobscura strains to test whether five gene arrangements are of unique or multiple origin, what the ancestral arrangement was, and what are the ages of the different arrangements. Each strain carried one of six commonly found gene arrangements and the sequences were used to infer their evolutionary relationships. Breakpoint regions in the center of the chromosome supported monophyly of the gene arrangements, whereas regions at the ends of the chromosome gave phylogenies that provided less support for monophyly of the chromosomes either because the individual markers did not have enough phylogenetically informative sites or genetic exchange scrambled information among the gene arrangements. A data set where the genetic markers were concatenated strongly supported a unique origin of the different gene arrangements. The inversion polymorphism of D. pseudoobscura is estimated to be about a million years old. We have also shown that the generated phylogeny is consistent with the cytological phylogeny of this species. In addition, the data presented here support hypothetical as the ancestral arrangement. One of the youngest arrangements, Arrowhead, has one of the highest population frequencies suggesting that selection has been responsible for its rapid increase.