Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Afadin and RhoA control pancreatic endocrine mass via lumen morphogenesis D. Berfin Azizoglu,1 Caitlin Braitsch,1 Denise K. Marciano,2,3 and Ondine Cleaver1,3 1Department of Molecular Biology, Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA; 2Department of Medicine, Division of Nephrology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA Proper lumen morphogenesis during pancreas development is critical to endocrine and exocrine cell fate. Recent studies showed that a central network of lumens (termed core), but not the surrounding terminal branches (termed periphery), produces most islet endocrine cells. To date, it remains unclear how pancreatic lumens form and remodel and which aspects of lumen morphogenesis influence cell fate. Importantly, models testing the function of the central lumen network as an endocrine niche are lacking. Here, we identify mechanisms underlying lumen for- mation and remodeling and show that central lumen network morphogenesis impacts pancreatic endocrine mass. We show that loss of the scaffolding protein Afadin disrupts de novo lumenogenesis and lumen continuity in the tip epithelium. Codepletion of the actomyosin regulator RhoA and Afadin results in defects in the central lumens and arrests lumen remodeling. This arrest leads to prolonged perdurance of the central lumen network over develop- mental time and expansion of the endocrine progenitor population and, eventually, endocrine mass. Our study uncovers essential roles of Afadin and RhoA in pancreatic central lumen morphogenesis, which subsequently de- termines endocrine cell mass. [Keywords: apical polarity; Rab GTPases; actomyosin; vesicular trafficking; islet; progenitor niche] Supplemental material is available for this article. Received September 26, 2017; revised version accepted December 4, 2017. Extensive efforts have been directed toward developing β- delaminate from the epithelium and differentiate into en- cell replacement therapies for diabetic patients. Although docrine cells, including glucagon-producing α and insulin- β-cell (or endocrine cell) generation has been accom- producing β cells (Pan and Wright 2011). These differenti- plished by recapitulating in vivo developmental steps, ation events coincide with major morphological changes these protocols have yet to be optimized and become ther- of the developing pancreatic progenitor epithelium, in- apeutically viable (Pagliuca et al. 2014; Massumi et al. cluding lumen morphogenesis. 2016). Several studies suggest that a three-dimensional Close spatiotemporal association between pancreatic microenvironment enhances endocrine differentiation (Ji- lumen formation and cell fate determination brings up ang et al. 2007; Kesavan et al. 2009; Kelly et al. 2011; Greg- the question of whether lumen morphogenesis impacts gio et al. 2013; Bankaitis et al. 2015). To date, what this pancreatic fate. Indeed, loss of Cdc42 in the murine pan- environment entails and how it forms remain unknown. creas leads to failure to initiate pancreatic lumens and a Therefore, a deeper understanding of the endocrine differ- concomitant increase in acinar cells at the expense of en- entiation “niche” will likely propel development of novel docrine differentiation (Kesavan et al. 2009). The investi- treatments for diabetes. gators noted that the resulting abnormal epithelial Differentiation of pancreatic progenitors into the endo- morphology increased exposure to laminin, which in crine lineage has been thoroughly characterized (Pan and turn promoted acinar fate. This work suggests that epithe- Wright 2011). Endodermal progenitors first transition lial morphology and lumen formation profoundly influ- through a ducto–endocrine bipotential state, identified ence pancreatic fate determination. by expression of the transcription factor Sox9. A fraction We and others previously characterized pancreatic lu- of Sox9+ progenitors transiently expresses Neurogenin3 men morphogenesis in the mouse embryo (Hick et al. (Neurog3), and a subset of these becomes endocrine pro- 2009; Kesavan et al. 2009; Villasenor et al. 2010). This genitors (Pan and Wright 2011). These progenitors then © 2018 Azizoglu et al. This article is distributed exclusively by Cold 3Cosenior authors. Spring Harbor Laboratory Press for the first six months after the full-issue Corresponding authors: [email protected], denise. publication date (see http://genesdev.cshlp.org/site/misc/terms.xhtml). [email protected] After six months, it is available under a Creative Commons License (At- Article published online ahead of print. Article and publication date are tribution-NonCommercial 4.0 International), as described at http://creati- online at http://www.genesdev.org/cgi/doi/10.1101/gad.307637.117. vecommons.org/licenses/by-nc/4.0/. GENES & DEVELOPMENT 31:1–15 Published by Cold Spring Harbor Laboratory Press; ISSN 0890-9369/17; www.genesdev.org 1 Downloaded from genesdev.cshlp.org on September 29, 2021 - Published by Cold Spring Harbor Laboratory Press Azizoglu et al. process can be categorized into four steps. First, microlu- endocrine mass by birth. With these findings, we demon- mens initiate between epithelial cells as isolated foci in strate that Afadin and RhoA functionally interact to direct the embryonic day 10.5 (E10.5) stratified bud. Next, morphogenesis of the pancreatic core, which determines lumens progressively fuse into longer channels through- endocrine mass. out the epithelium by E12.5. These channels take on a three-dimensional (3D) net-like morphology, called a plexus, that undergoes remodeling and extension between Results E13 and E18.5. By birth, the lumenal plexus resolves into a Afadin is required to form a continuous lumenal network hierarchical tree. Recent work by Bankaitis et al. (2015) in the developing pancreas characterized two distinct compartments in the pancreas at E13.5–E18.5 based on their lumenal/tubular morpholo- Given the requirement of Afadin in renal vesicle lumen gy. Regions at the center of the pancreas, which contain a formation, we asked whether it plays a similar role in lumenal plexus in the process of remodeling, were termed pancreatic lumenogenesis. Afadin is expressed in many the core, while regions surrounding the core, which dis- mouse tissues and localizes to the apical membrane in ep- play ramifying branches, were termed the periphery. ithelial cells of the renal vesicle (Yang et al. 2013). Howev- The core plexus progressively resolves into a ramifying er, its expression has not been reported in the pancreas. tree at perinatal stages (Bankaitis et al. 2015). Further- We examined Afadin in the embryonic pancreatic bud more, the investigators identified the core region as and found that it localized to cell–cell boundaries, marked the site of endocrine differentiation. Hence, the core is a by the adherens junction protein E-cadherin, throughout transiently formed region distinguished by lumen mor- development (Fig. 1A–D′). At E10.5, Afadin localized phology and differs from the periphery in its cellular along the primary central lumen (data not shown) and composition. showed enrichment at scattered membrane domains Although the core and periphery are morphologically within the stratified epithelium prior to formation of lu- distinct, it is unknown how these two regions form during mens (Fig. 1A,A′). Afadin was also observed adjacent to pancreas development or how their morphogenesis is in- foci expressing the lumenal glycoprotein Mucin1 fluenced by initial lumen formation or subsequent remod- (Muc1), suggesting that it marked nascent lumens (Fig. eling. The functional relevance of morphogenesis of either 1B,B′). region in endocrine differentiation also remains to be de- At E12.5, Afadin became enriched at junctions flanking termined, as a model that alters either the core or the pe- the apical membrane (Fig. 1C,C′) and restricted to subap- riphery has not yet been reported. Elucidation of these ical junctions at lumenal boundaries at later stages (Fig. questions requires a better understanding of pancreatic lu- 1D,D′). Superresolution microscopy revealed localization men development. of Afadin at sites with progressive enrichment of E-cad- We took a genetic approach to study pancreatic lumen herin or the tight junction (TJ) protein ZO-1, pointing to development and its influence on endocrine cell fate. Giv- a function in junction formation in the pancreas, as de- en the function of the scaffolding molecule Afadin in kid- scribed previously in other systems (Supplemental Fig. ney tubulogenesis, we hypothesized that Afadin may play S1A–B′; Takahashi et al. 1998; Ikeda et al. 1999; Zhadanov a similar role during pancreas development (Takai et al. et al. 1999). Interestingly, a subset of Afadin foci did not 2008; Yang et al. 2013; Marciano 2017). We further hy- colocalize with either junctional protein, suggesting po- pothesized that the Afadin mutant model could be used tential junction-independent functions prior to lumen for- as a tool to manipulate pancreatic lumens to assess impact mation (Supplemental Fig. S1A–B′, arrows). Thus, Afadin on cell fate, including endocrine cells. In this study, we localizes to scattered membrane domains and non- found that pancreas-specific ablation of Afadin results in junctional foci prior to lumen formation and becomes re- delayed and discontinuous