Rac function in epithelial tube morphogenesis

Abstract

Epithelial cell migration and morphogenesis require dynamic remodeling of the actin cytoskeleton and cell-cell adhesion complexes. Numerous studies in cell culture and in model organisms have demonstrated the small GTPase Rac to be a critical regulator of these processes; however, little is known about Rac function in the morphogenic movements that drive epithelial tube formation. Here, we use the embryonic salivary glands of Drosophila to understand the role of Rac in epithelial tube morphogenesis. We show that inhibition of Rac function, either through loss of function mutations or dominant-negative mutations, disrupts salivary gland invagination and posterior migration. In contrast, constitutive activation of Rac induces motile behavior and subsequent cell death. We further show that Rac regulation of salivary gland morphogenesis occurs through modulation of cell-cell adhesion mediated by the E-cadherin/beta-catenin complex and that shibire, the Drosophila homolog of dynamin, functions downstream of Rac in regulating beta-catenin localization during gland morphogenesis. Our results demonstrate that regulation of cadherin-based adherens junctions by Rac is critical for salivary gland morphogenesis and that this regulation occurs through dynamin-mediated endocytosis.

Pak1 control of E-cadherin endocytosis regulates salivary gland lumen size and shape

“…Cdc42 DN expression had no effect on lumen length, as with loss of Pak1 function; however, Rac1 DN expression led to formation of a shorter lumen that is likely to be a result of the role of Rac1 in gland migration that we reported previously (see Fig. S4F in the supplementary material) (Pirraglia et al, 2006). E-cad failed to localize to the basolateral membrane of gland cells expressing Cdc42 DN , as observed also in Pak1 14 mutant gland cells (see Fig.…”

“…Embryo fixation and staining were performed as described previously (Pirraglia et al, 2006). Primary antibodies used were: anti-Phalloidin (AF488; Invitrogen; 1:200), rabbit anti-Avalanche (Syntaxin 7 -FlyBase; obtained from H. Kramer, UT Southwestern Medical Center, Dallas, TX, USA; 1:1000), chicken anti-Avalanche (obtained from D. Bilder, UC Berkeley, Berkeley, CA, USA; 1:500), mouse anti--galactosidase (Promega; 1:10,000 for whole-mount, 1:500 for immunofluorescence), rabbit anti-Bazooka (obtained from J. Zallen, Sloan Kettering Institute, New York, NY, USA; 1:1000), rat anti-CrebA (generated in the Myat Lab, New York, NY, USA; 1:10,000 for whole-mount, 1:5000 for immunofluorescence) (Andrew et al, 1997), rabbit anti-Croquemort (obtained from N. Franc, The Scripps Research Institute, La Jolla, CA, USA; 1:500), mouse anti-Crumbs [Developmental Studies Hybridoma Bank (DSHB); 1:100 for whole-mount, 1:10 for immunofluorescence], rat anti-E-cadherin (DSHB; 1:20), mouse anti-Discs large (DSHB; 1:500), rabbit anti-Engrailed (obtained from P. O'Farrell, UC San Francisco, San Francisco, CA, USA; 1:1000), rabbit anti-Fork head (obtained from M. Stern and S. Beckendorf, UC Berkeley, Berkeley, CA, USA; 1:1000), mouse anti-GFP (Roche Diagnostics; 1:500), mouse anti-Myc (Invitrogen; 1:500), mouse anti-Neurotactin (DSHB; 1:10), rabbit anti-Pak (obtained from N. Harden, Simon Fraser University, Burnaby, B.C.…”

“…Recombinant lines carrying different UAS-transgene insertions were confirmed by PCR analysis as described previously (Pirraglia et al, 2006).…”

Cdc42- and Rac1-mediated endothelial lumen formation requires Pak2, Pak4 and Par3, and PKC-dependent signaling

“…Previous studies have revealed an important role for Rho GTPases in EC morphogenesis Davis and Senger, 2005;Hoang et al, 2004) as well as important functions such as the control of vascular permeability (Stockton et al, 2004). Furthermore, Rho GTPases such as Cdc42 and Rac1 are known to play a crucial role in epithelial lumen formation (Martin-Belmonte et al, 2007;Pirraglia et al, 2006), similar to that observed for EC lumen formation Davis and Bayless, 2003). Our previous studies utilized dominant-negative mutants as well as GFP-Rho GTPase fusion proteins to analyze EC lumen formation .…”

A Targeted Gain-of-Function Screen Identifies Genes Affecting Salivary Gland Morphogenesis/Tubulogenesis in Drosophila

“…The actin cytoskeleton is modified through proteins such as Btk29/Tec29 in conjunction with Chickadee (Chandrasekaran and Beckendorf 2005). Small GTPases such as Rac and Rho affect the invagination of the glands (Pirraglia et al 2006;Xu et al 2008). Crumbs and Klarsicht affect the delivery of apical membrane and thus cell shape at late stages of morphogenesis (Myat and Andrew 2002).…”