Apical constriction is an ancient developmental process that triggers key morphogenetic events such as neurulation in vertebrates and gastrulation in insects. However, the pathways involved are not fully understood. During pupariation, presumptive leg joints undergo apical constriction and epithelial invagination to initiate joint morphogenesis. We hypothesize that the process is mediated by an increase in actomyosin contractility and a decrease in cell-cell adhesion. The goal of this grant is to study the roles of the Rho GTPases Rho1, Rac1 and Cdc42 and their regulators, the RhoGEFs and RhoGAPs, in controlling the stability and turnover of adherence junctions (AJs), and the contribution of these genes to apical constriction and epithelial invagination during joint morphogenesis. We find that the inhibition of Rho1 or the activation of Rac1 or Cdc42 blocks joint morphogenesis. We also find that RhoGAP68F (GAP68F), a putative GAP for Cdc42, and RhoGAP5A (GAP5A), a putative GAP for Rac1, are expressed at presumptive joints and are required to initiate joint morphogenesis. We provide strong preliminary evidence to suggest that these regulators act through distinct pathways to control apical constriction. To test the role of the Rho GTPases and their regulators in apical constriction we propose the following specific AIMs:
AIM1 : We will test the hypothesis that Rho1, Cdc42 and Rac1 respectively regulate the influx, efflux and the stable pool of AJs at the ZA to promote apical constriction.
AIM2 : Large-scale interaction screens identified Rab4 and Sec3 as partners of GAP68F suggesting that GAP68F inhibits endocytic recycling from two distinct recycling routes to decrease the surface expression of AJs and thereby adhesive cell-cell contacts. In agreement, we find accumulation of GAP68F in the endocytic compartment. Therefore, we will test the hypothesis that GAP68F inhibits endocytic recycling and thereby adhesive cell-cell contacts to promote apical constriction.
AIM3 : We find that GAP5A is enriched at or near the zonula adherence (ZA). We will test the hypothesis that GAP5A acts at the ZA to decrease the stability of AJs and thereby adhesive cell-cell contacts to promote apical constriction. The successful completion of these studies will highlight the contribution of junctional stability and endocytic trafficking to apical constriction and epithelial invagination with implications to vertebrate systems.
The successful completion of the AIMs will help explain how the activities of the small RhoGTPases Rho1, Rac1 and Cdc42 are coordinated during development to control epithelial morphogenesis, and how the RhoGEFs and RhoGAPs regulate the activities of the RhoGTPases in topographically distinct subcellular sites to trigger changes in epithelial topology. Apical constriction is an evolutionarily conserved process and therefore these studies will have direct implications to vertebrate systems.
|Coravos, Jonathan S; Martin, Adam C (2016) Apical Sarcomere-like Actomyosin Contracts Nonmuscle Drosophila Epithelial Cells. Dev Cell 39:346-358|
|de Madrid, Beatriz Hernandez; Greenberg, Lina; Hatini, Victor (2015) RhoGAP68F controls transport of adhesion proteins in Rab4 endosomes to modulate epithelial morphogenesis of Drosophila leg discs. Dev Biol 399:283-95|
|Hatini, Victor; Kula-Eversole, Ela; Nusinow, David et al. (2013) Essential roles for stat92E in expanding and patterning the proximodistal axis of the Drosophila wing imaginal disc. Dev Biol 378:38-50|
|Del Signore, Steven J; Hayashi, Teru; Hatini, Victor (2012) odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms. Mech Dev 129:147-61|
|Greenberg, Lina; Hatini, Victor (2011) Systematic expression and loss-of-function analysis defines spatially restricted requirements for Drosophila RhoGEFs and RhoGAPs in leg morphogenesis. Mech Dev 128:5-17|
|Greenberg, Lina; Hatini, Victor (2009) Essential roles for lines in mediating leg and antennal proximodistal patterning and generating a stable Notch signaling interface at segment borders. Dev Biol 330:93-104|
|Nusinow, David; Greenberg, Lina; Hatini, Victor (2008) Reciprocal roles for bowl and lines in specifying the peripodial epithelium and the disc proper of the Drosophila wing primordium. Development 135:3031-41|
|Hatini, Victor; Green, Ryan B; Lengyel, Judith A et al. (2005) The Drumstick/Lines/Bowl regulatory pathway links antagonistic Hedgehog and Wingless signaling inputs to epidermal cell differentiation. Genes Dev 19:709-18|