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• W2044638670 abstract "Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, we have studied the functional role of Cdc42 in pancreatic tubulogenesis. We present evidence that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later for maintaining apical cell polarity. Finally, we show that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors.For a video summary of this article, see the PaperFlick file with the Supplemental Data available online. Understanding how cells polarize and coordinate tubulogenesis during organ formation is a central question in biology. Tubulogenesis often coincides with cell-lineage specification during organ development. Hence, an elementary question is whether these two processes are independently controlled, or whether proper cell specification depends on formation of tubes. To address these fundamental questions, we have studied the functional role of Cdc42 in pancreatic tubulogenesis. We present evidence that Cdc42 is essential for tube formation, specifically for initiating microlumen formation and later for maintaining apical cell polarity. Finally, we show that Cdc42 controls cell specification non-cell-autonomously by providing the correct microenvironment for proper control of cell-fate choices of multipotent progenitors. For a video summary of this article, see the PaperFlick file with the Supplemental Data available online. Organs such as the lung, kidney, pancreas, salivary gland, and mammary gland are primarily made up of tubes that act as biological pipes for transporting vital fluids and gases. Two principle mechanisms for mammalian tubulogenesis have been described. One mechanism involves reiterative sprouting and stereotypical branching of a tubular anlagen consisting of fully polarized epithelial cells. This process has for example been described in the lung (Metzger et al., 2008Metzger R.J. Klein O.D. Martin G.R. Krasnow M.A. The branching programme of mouse lung development.Nature. 2008; 453: 745-750Crossref PubMed Scopus (501) Google Scholar). The other mechanism is represented in glandular organs, e.g., the pancreas, mammary gland, prostate, and salivary glands, where tubulogenesis arises when groups of unpolarized epithelial cells form microlumens, which subsequently participate in the formation of tubes where branching is not entirely stereotypical (Hogan and Kolodziej, 2002Hogan B.L. Kolodziej P.A. Organogenesis: molecular mechanisms of tubulogenesis.Nat. Rev. Genet. 2002; 3: 513-523Crossref PubMed Scopus (254) Google Scholar). Whereas the first process is relatively well understood, the latter is not. Tubulogenesis involves a series of dynamic and interdependent cellular processes, including cytoskeletal reorganization, assembly of intercellular junctional complexes, and cell polarization. Rho-GTPases are molecular switches that control such complex processes. For example, Cdc42, which is one of the most studied Rho-GTPase family members, is a master regulator of cytoskeletal dynamics and cell polarity—a function which is evolutionarily conserved from yeast to mammals (Etienne-Manneville, 2004Etienne-Manneville S. Cdc42–the centre of polarity.J. Cell Sci. 2004; 117: 1291-1300Crossref PubMed Scopus (515) Google Scholar). Recently, Cdc42 was demonstrated to control lumen formation in three-dimensional (3D) organotypic cultures of MDCK and Caco-2 cells by controlling apical segregation of phosphoinositides and spindle orientation during cell division, respectively (Jaffe et al., 2008Jaffe A.B. Kaji N. Durgan J. Hall A. Cdc42 controls spindle orientation to position the apical surface during epithelial morphogenesis.J. Cell Biol. 2008; 183: 625-633Crossref PubMed Scopus (246) Google Scholar, Martin-Belmonte et al., 2007Martin-Belmonte F. Gassama A. Datta A. Yu W. Rescher U. Gerke V. Mostov K. PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42.Cell. 2007; 128: 383-397Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). Whether Cdc42 controls lumen formation in vivo in a similar manner remains unclear. The pancreas is a glandular organ where the tubular network interconnects the acinar cells and enables coordinated transport of digestive enzymes into the duodenum. Organogenesis of the pancreas begins with the evagination of the dorsal and ventral anlagen from the foregut endoderm. In mice, this event starts at embryonic day (E) 8.5. The pancreatic epithelium is in contact with various sources of mesodermally derived tissues whose signals are crucial for pancreatic growth and differentiation (Gittes, 2008Gittes G.K. Developmental biology of the pancreas: A comprehensive review.Dev. Biol. 2008; 326: 4-35Crossref PubMed Scopus (318) Google Scholar). Pancreatic and duodenal homeobox 1(Pdx1) expressing multipotent pancreatic progenitors give rise to all epithelial cells of the adult pancreas, including duct, acinar, and endocrine cells (Gu et al., 2002Gu G. Dubauskaite J. Melton D.A. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.Development. 2002; 129: 2447-2457Crossref PubMed Google Scholar). Here, we have used the mouse pancreas to address two fundamentally important questions in developmental biology, namely the molecular control of mammalian organ asymmetry and tubulogenesis in vivo and the interplay between tube formation and cell-fate decisions during organogenesis. First, we show that tube formation starts at E11.5 by the initiation of scattered microlumens throughout the epithelium. These lumens expand by spreading of cell polarization, and not by sprouting, followed by fusion of lumens and their rearrangement into an interconnected tubular system. Second, by ablating Cdc42 at different time points during pancreas development, we demonstrate that Cdc42 is required for microlumen formation and subsequently for maintenance of a polarized tubular phenotype. Third, the failure to organize pancreatic epithelial progenitors into tubes causes a dramatic upregulation of acinar cell differentiation at the expense of duct and endocrine cell differentiation. Finally, we show that this is non-cell-autonomously caused by changes in epithelial cell-extracellular matrix (ECM) interactions. To understand the basis for pancreatic tubulogenesis, we first characterized the establishment of epithelial cell polarity and tube formation during pancreas development. Mucin1 (Muc1) is an O-glycosylated transmembrane protein expressed on the apical surface of many epithelia, including the pancreas (Cano et al., 2004Cano D.A. Murcia N.S. Pazour G.J. Hebrok M. Orpk mouse model of polycystic kidney disease reveals essential role of primary cilia in pancreatic tissue organization.Development. 2004; 131: 3457-3467Crossref PubMed Scopus (138) Google Scholar). Thus, Muc1 was used as an apical marker, whereas the cell-cell adhesion protein E-cadherin (Ecad) and the basement membrane protein laminin (Lam) were used as lateral and basal markers, respectively (Figures 1A–1E). To image the lumenal system three-dimensionally, whole-mount Muc1 immunofluorescence analysis was carried out (Figures 2A–2F). Except for a stunted opening into the duodenum, the E10.5 dorsal pancreatic bud is multilayered, consisting of epithelial cells that lack apicobasal cell polarity. Furthermore, the epithelium lacks lumenal structures and is surrounded by laminin (Figure 1A). The first sign of lumen formation occurs at E11.5 with the stochastic appearance of microlumens scattered throughout the epithelium (Figures 1B, 2A, and 2D; arrowhead indicates microlumen). The microlumens are made up of clusters of epithelial cells with a common apical surface facing the lumen. Subsequently, the microlumens expand by inducing apical cell polarity in neighboring epithelial cells resulting in a complex network of independently organized lumenal structures (Figures S1A and S1B available online). At E12.5 the lumens coalesce into a complex continuous lumenal network within the multilayered epithelium (Figures 1C, 2B, and 2E). Notably, at this point no tubes have formed. However, between E13.5 and E15.5 the lumenal network remodels and matures into a tubular network, i.e., this is the stage when the first tubes consisting of a monolayered fully polarized epithelium surrounded by a basal lamina forms (Figures 1D, 1E, 2C, 2F, and S2 available online). This process can be visualized by changes in the organization of basal lamina components, including laminin. Whereas laminin is distributed mainly on the periphery of the early pancreatic bud, it covers the entire tubular monolayered epithelium at later time points (Figure S2). Moreover, ultrastructural analysis at E15.5 confirmed the hallmarks of a fully polarized tubular epithelium, including cell-cell junctions and protruding microvilli on the apical surface (Figure 1F).Figure 2Cdc42 Is Essential for Tubulogenesis in the Developing PancreasShow full captionTo characterize tubulogenesis in 3D, pancreases from E11.5– E13.5 were analyzed by whole-mount immunostaining with antibodies against mucin1 (Muc1; green), followed by 3D reconstructions of confocal images.(A–C) In the WT, microlumens appeared stochastically at E11.5 (A). At E12.5 (B), microlumens expanded and coalesced into a continuous lumenal network. At E13.5 (C), the initiated lumenal network remodeled into tubular structures.(A′–C′) In the Cdc42 KO, aberrant lumenal structures appeared at E11.5 (A′). The subsequent steps to generate a lumenal network at E12.5 (B′) and E13.5 (C′) were blocked. The subcellular distribution of Muc1 was altered as well.(D–F) To visualize tube formation in 3D at higher magnification, WT pancreas sections (40 μm) were immunostained with antibodies against Muc1 (green). Microlumens (indicated with arrowheads in D) were specifically observed at E11.5. Generation of a continuous lumenal network (E12.5; E) and the first tubular structures (E13.5; F) define characteristic developmental stages during pancreatic tubulogenesis.Scale bars, 50 μm (A–C and A′–C′), 20 μm (D–F).View Large Image Figure ViewerDownload Hi-res image Download (PPT) To characterize tubulogenesis in 3D, pancreases from E11.5– E13.5 were analyzed by whole-mount immunostaining with antibodies against mucin1 (Muc1; green), followed by 3D reconstructions of confocal images. (A–C) In the WT, microlumens appeared stochastically at E11.5 (A). At E12.5 (B), microlumens expanded and coalesced into a continuous lumenal network. At E13.5 (C), the initiated lumenal network remodeled into tubular structures. (A′–C′) In the Cdc42 KO, aberrant lumenal structures appeared at E11.5 (A′). The subsequent steps to generate a lumenal network at E12.5 (B′) and E13.5 (C′) were blocked. The subcellular distribution of Muc1 was altered as well. (D–F) To visualize tube formation in 3D at higher magnification, WT pancreas sections (40 μm) were immunostained with antibodies against Muc1 (green). Microlumens (indicated with arrowheads in D) were specifically observed at E11.5. Generation of a continuous lumenal network (E12.5; E) and the first tubular structures (E13.5; F) define characteristic developmental stages during pancreatic tubulogenesis. Scale bars, 50 μm (A–C and A′–C′), 20 μm (D–F). The Par-aPKC complex is regulated by Cdc42 and plays a critical role in cell polarity initiation and maintenance. Binding of Cdc42-GTP activates the Par-aPKC complex and the phosphorylated form of aPKC (p-aPKC) was used as a marker to identify the activated form of aPKC (Wu et al., 2007Wu X. Li S. Chrostek-Grashoff A. Czuchra A. Meyer H. Yurchenco P.D. Brakebusch C. Cdc42 is crucial for the establishment of epithelial polarity during early mammalian development.Dev. Dyn. 2007; 236: 2767-2778Crossref PubMed Scopus (60) Google Scholar). At E11.5, p-aPKC was specifically distributed along the apical surface of microlumens together with characteristic apical markers, including ZO-1, Par6, Muc1, and crumbs3 (Figures 3A, 3B, and S3A). In situ hybridization analysis demonstrated that Cdc42 mRNA is ubiquitously expressed in the entire pancreas at all development stages analyzed (Figure S3B). To specifically ablate Cdc42 during pancreas development, floxed Cdc42 mice were intercrossed with Pdx1-cre mice (Gu et al., 2002Gu G. Dubauskaite J. Melton D.A. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.Development. 2002; 129: 2447-2457Crossref PubMed Google Scholar, Wu et al., 2006Wu X. Quondamatteo F. Lefever T. Czuchra A. Meyer H. Chrostek A. Paus R. Langbein L. Brakebusch C. Cdc42 controls progenitor cell differentiation and beta-catenin turnover in skin.Genes Dev. 2006; 20: 571-585Crossref PubMed Scopus (129) Google Scholar). From here on, Cdc42fl/fl or Cdc42fl/+ mice are referred to as wild-type (WT) mice, whereas Cdc42fl/+; Pdx1-cre and Cdc42fl/fl; Pdx1-cre mice are referred to as Cdc42 het and Cdc42 knockout (KO) mice, respectively. Cdc42 het were indistinguishable from WT controls (data not shown). Analysis of the recombination efficiency of the Pdx1-cre line using the R26R LacZ reporter line demonstrated undetectable recombination at E10.5, whereas approximately 90%–95% of the epithelial cells underwent cre-mediated recombination at E11.5. Consistently, cre efficiently ablated the Cdc42 protein (Figure S3C and S3D). As a consequence the first phenotype was apparent at E11.5. In contrast to the WT epithelium where apical polarity is induced concomitant with microlumen formation, apical membrane proteins, e.g., Muc1, remained intracellular within the Cdc42 KO epithelium (compare Figure 1B with 1B′ and S4A with S4A′). Furthermore, quantitative polymerase chain reaction (QPCR) analysis revealed a 2-fold upregulation of Muc1 mRNA expression, which most likely is due to an increased number of Muc1+ cells (Figure S4D). In contrast, E-cadherin and laminin showed normal distribution (Figures 1B′–1E′), suggesting that Cdc42 ablation did not affect the formation of the lateral and basal surfaces. The unpolarized Cdc42 KO epithelia failed to retain its integrity and became fragmented into epithelial cords. Cell proliferation gradually transformed these structures into large cellular aggregates lacking tubular structures (Figures 1C′–E′). Whole-mount immunofluorescence analysis of Muc1 in Cdc42 KO E11.5 to E13.5 pancreas confirmed the lack of tubes as well as the change in Muc1 distribution (Figures 2A′–2C′). Transmission electron microscopy (TEM) analysis at E15.5 corroborated the failure to establish tubes (Figure 1F′). Thus, Cdc42 is essential for tubulogenesis in the developing pancreas. Careful examination of microlumen formation revealed that induction of apical cell polarity appears to initially involve only one cell. At E11.5, Muc1 is confined to a distinct vesicular compartment close to the plasma membrane (Figure S4A). This intracellular vesicular compartment may represent the same secretory vesicles delivered to the de novo apical membrane concomitant with formation of primitive cell-cell junctions (Figures S4B and S4C). Importantly, these events are only seen at E11.5. Altogether, these results suggest that microlumens are initiated by one cell that upon a given signal induces targeting of secretory vesicles containing apical membrane proteins to the presumptive apical domain. Shortly thereafter neighboring cells undergo apical polarization resulting in a shared apical domain facing a lumen. In the absence of Cdc42, characteristic apical membrane proteins and membrane-associated proteins, including Muc1, ZO-1, Par6, crumbs3, and cortical F-actin, are primarily confined to lumenal structures within cells (Figures 3A–3C). Careful TEM analysis revealed that these lumens (from here on referred to as “autocellular” lumens) are in fact in direct contact with the cell surface via ZO-1+ and claudin3+, “autocellular” tight junction-like junctions (Figures S1C, S1D, and S5). Intercellular lumens between two cells also form, but less frequently (Figures 3C, and S1E, and S1F). The apical membrane phenotype of both the autocellular and intercellular lumenal surfaces is reinforced by the appearance of microvilli-like structures (Figures S1C–S1F and S1H). Finally, Cdc42 ablation does not affect the intracellular distribution of proteins involved in the general vesicular targeting machinery, such as VAP-A (VAMP-associated protein A) (Figure S4E). Based on these findings, Cdc42 is not required for apical membrane biogenesis or for tight junction formation. However, whether the apical domain and tight junctions in the absence of Cdc42 are fully mature and functional remains to be determined. In summary, Cdc42 is necessary for maintaining an apical surface facing the outside of cells and for establishing multicellular (>2 cells) microlumens with shared apical surfaces. Ablating Cdc42 during the earliest stages of pancreas development failed to address whether Cdc42, in addition to its role in microlumen formation, plays a role in maintaining cell polarity in the tubular epithelium. To address this question, we used a tamoxifen (TM) inducible model (Pdx1-cre ER™ mice) for timed ablation of Cdc42 within the pancreatic epithelium (Gu et al., 2002Gu G. Dubauskaite J. Melton D.A. Direct evidence for the pancreatic lineage: NGN3+ cells are islet progenitors and are distinct from duct progenitors.Development. 2002; 129: 2447-2457Crossref PubMed Google Scholar). By intercrossing the R26R LacZ reporter line (Soriano, 1999Soriano P. Generalized lacZ expression with the ROSA26 Cre reporter strain.Nat. Genet. 1999; 21: 70-71Crossref PubMed Scopus (4029) Google Scholar) with Pdx1-cre ER™ mice, recombined cells became traceable by their expression of beta-galactosidase (βGal). To address the role of Cdc42 in maintenance of tubes, Cdc42fl/+; Pdx1-cre ER™; R26R and Cdc42fl/fl; Pdx1-cre ER™; R26R mice were generated. Cdc42 was ablated by a single tamoxifen pulse at E12.5 followed by analysis at E15.5. Thus, at the time of tamoxifen induction these cells were polarized and confined to the primitive tubular network. This mosaic system (Cdc42fl/fl; Pdx1-cre ER™) resulted in a recombination efficiency of 16% (data not shown), which is not sufficient to block tubulogenesis and alter the overall tissue architecture (Figure S6). In control samples, all βGal+ cells were randomly distributed within tubes as fully polarized epithelial cells with an apical surface enriched with Muc1, claudin3, Par6, and cortical F-actin (Figures 4A–4F). In the mosaic Cdc42 KO model βgal+ cells were found both within and outside the tubular epithelium. However, the few βGal+ Cdc42 KO cells that were found interspersed between WT cells within tubes failed to maintain apical cell polarity, as demonstrated by failure to maintain Muc1, Par6, and cortical F-actin on the apical side (Figures 4A′, 4C′, and 4E′). Furthermore, less claudin3 was found at tight junctions of βGal+ cells (Figures 4F and 4F′). Interestingly, basal polarity as detected by laminin deposition was unaffected (Figures 4G and 4G′). Delaminating βGal+ Cdc42 KO cells clustered into epithelial aggregates without an apical surface (Figures 4B′ and 4D′). Intracellular accumulation of apical markers, such as Muc1, and loss of cortical F-actin were observed (Figures 4B′ and 4D′). Loss of apical polarity resulted in replacement of the apical membrane by lateral membrane, as evidenced by expression of the lateral surface marker, E-cadherin (Figure S7). In conclusion, Cdc42 plays a cell-autonomous role not only in microlumen formation but also in maintenance of apical polarity in fully polarized tubular epithelial cells. QPCR analysis showed that both the aPKCζ and aPKCι isoforms are expressed in the developing pancreas (Figure S4D). The activated form of aPKC, p-aPKC, is expressed on the apical membrane throughout the pancreatic lumens and tubes until E13.5 (Figures S8A–S8D). Ablation of Cdc42 resulted in undetectable levels of p-aPKC within the epithelium from E11.5 onward, whereas expression of p-aPKC within blood vessels was unaffected (Figures S8A′–S8D′). To test the hypothesis that Cdc42 controls microlumen formation through aPKC activation we examined whether blocking aPKC activation would mimic the Cdc42 KO phenotype. For this purpose, we used a two-dimensional (2D) explants culture system that provides a simple and effective way to analyze tubulogenesis in vitro (Percival and Slack, 1999Percival A.C. Slack J.M. Analysis of pancreatic development using a cell lineage label.Exp. Cell Res. 1999; 247: 123-132Crossref PubMed Scopus (72) Google Scholar). Treatment of the explants with the myristoylated substrate of aPKCζ (aPKC-PS) effectively inhibited the activation of aPKC, whereas expression of the protein remained unaffected (Figures S9A and S9B) (Nunbhakdi-Craig et al., 2002Nunbhakdi-Craig V. Machleidt T. Ogris E. Bellotto D. White 3rd, C.L. Sontag E. Protein phosphatase 2A associates with and regulates atypical PKC and the epithelial tight junction complex.J. Cell Biol. 2002; 158: 967-978Crossref PubMed Scopus (212) Google Scholar). Untreated controls formed a continuous tubular network, whereas Cdc42 KO explants failed to form tubes due to their lack of apical cell polarity (Figure S9C). aPKC-PS-treated WT explants failed to generate a continuous tubular network. Instead, the epithelium remained compact with large aggregates of epithelial cells (Figure S9C). However, in contrast to the intracellular accumulation of Muc1 in the Cdc42 KO epithelial cells, apical cell polarity appeared unaffected in aPKC-PS-treated epithelial aggregates (Figure S9C). These results show that aPKC plays a crucial role in lumen coalescence into a continuous tubular network in vitro. This observation is consistent with the multiple lumen phenotype in the intestine of aPKC mutants in Zebrafish (Horne-Badovinac et al., 2001Horne-Badovinac S. Lin D. Waldron S. Schwarz M. Mbamalu G. Pawson T. Jan Y. Stainier D.Y. Abdelilah-Seyfried S. Positional cloning of heart and soul reveals multiple roles for PKC lambda in zebrafish organogenesis.Curr. Biol. 2001; 11: 1492-1502Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Altogether, these results suggest that Cdc42 controls tubulogenesis in vivo at several levels, and that aPKC activation through Par6 represents one of several Cdc42-controlled pathways involved in tubulogenesis. Cdc42 acts in several ways to establish a functional and mature apical surface, e.g., by interacting with the master polarity complex proteins Par3, Par6, and aPKC (Bryant and Mostov, 2008Bryant D.M. Mostov K.E. From cells to organs: building polarized tissue.Nat. Rev. Mol. Cell Biol. 2008; 9: 887-901Crossref PubMed Scopus (547) Google Scholar). Par3 interaction with the Par6-aPKC complex is indispensable for apical domain development (Horikoshi et al., 2009Horikoshi Y. Suzuki A. Yamanaka T. Sasaki K. Mizuno K. Sawada H. Yonemura S. Ohno S. Interaction between PAR-3 and the aPKC-PAR-6 complex is indispensable for apical domain development of epithelial cells.J. Cell Sci. 2009; 122: 1595-1606Crossref PubMed Scopus (112) Google Scholar). Therefore, failure to establish a common apical domain in the absence of Cdc42 may be attributed to disturbed interaction of Par3 with the Par6-aPKC complex. Recently, it was demonstrated that Rho kinase (ROCK) also controls formation of the Par3-Par6-aPKC complex by phosphorylation of Par3, which prevents its interaction with Par6 and aPKC (Nakayama et al., 2008Nakayama M. Goto T.M. Sugimoto M. Nishimura T. Shinagawa T. Ohno S. Amano M. Kaibuchi K. Rho-kinase phosphorylates PAR-3 and disrupts PAR complex formation.Dev. Cell. 2008; 14: 205-215Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To test if blocking ROCK activity could restore apical polarity and tubulogenesis in the Cdc42 KO epithelium, WT and Cdc42 KO explants were incubated with the ROCK inhibitor, Y27632. Pharmacological inhibition of Rho kinase activity in vitro restored tube formation in the Cdc42 KO epithelium (Figure S10). Further studies are required to fully understand this intriguing result. Between E13.5 and E15.5, lineage commitment of multipotent Pdx1+ pancreatic progenitors toward exocrine and endocrine lineages is active (Gittes, 2008Gittes G.K. Developmental biology of the pancreas: A comprehensive review.Dev. Biol. 2008; 326: 4-35Crossref PubMed Scopus (318) Google Scholar, Jorgensen et al., 2007Jorgensen M.C. Ahnfelt-Ronne J. Hald J. Madsen O.D. Serup P. Hecksher-Sorensen J. An illustrated review of early pancreas development in the mouse.Endocr. Rev. 2007; 28: 685-705Crossref PubMed Scopus (261) Google Scholar). Notably, these lineages appear at distinct anatomical positions within the developing tubular network. All peripheral epithelial cells, including “tip cells” and acinar progenitors (Zhou et al., 2007Zhou Q. Law A.C. Rajagopal J. Anderson W.J. Gray P.A. Melton D.A. A multipotent progenitor domain guides pancreatic organogenesis.Dev. Cell. 2007; 13: 103-114Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar), are exposed to ECM proteins, including laminin, and mesenchymal cells throughout development (Figures S2A and S11A). In contrast, before formation of a monolayered tubular epithelium, the central parts (trunk) of the epithelium consisting of endocrine and ductal progenitors are sparsely exposed to ECM and mesenchymal cells. The failure to organize Cdc42 KO multipotent pancreatic progenitors into tubes provides a model for addressing the importance of tissue/microenvironment asymmetry in cell specification. Ablation of Cdc42 had no impact on the expression of Pdx1, Nkx6.1, and Sox9 in Pdx1+ multipotent progenitors up until E13.5 (Figure S11B). A subpopulation of the multipotent progenitors confined to the tips of the tubular network, “tip cells,” express carboxypeptidase A1 (Cpa1), along with Pdx1, Ptf1a, and c-Myc until E14 (Zhou et al., 2007Zhou Q. Law A.C. Rajagopal J. Anderson W.J. Gray P.A. Melton D.A. A multipotent progenitor domain guides pancreatic organogenesis.Dev. Cell. 2007; 13: 103-114Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). The fact that the Cdc42 KO pancreatic epithelium lacked distinct “tip-trunk” structures (Figures 5A, 5B, 5A′, and 5B′) led to a randomized distribution of “tip cell” markers, such as Cpa1 and Ptf1a. Although the total number of epithelial cells was unaffected, the relative number of Cpa1+ and Ptf1a+ progenitors increased at E13.5 (Figures 5C and 5D; data not shown). In addition, cleaved Caspase3 stainings showed no significant difference in the frequency of apoptotic cells between the WT and Cdc42 KO epithelium (0.56% ± 0.04% [WT] and 0.41% ± 0.11% [KO]). Thus, Cdc42-controlled tissue/microenvironment asymmetry is required for regulating the distribution and number of Cpa1+ and Ptf1a+ pancreatic progenitors. Quantification of Ngn3 mRNA expression demonstrated a significant reduction in Cdc42 KO samples at E14.5 (Figure 5G). Consistently, immunofluorescence staining of Ngn3 at E15.5 showed significantly fewer Ngn3-positive cells in the KO epithelium (Figures 5E and 5E′). In line with this observation, a dramatic reduction of insulin- and glucagon-expressing cells was observed at E15.5 (Figures 5F, 5F′, and 5H). These results show that differentiation toward endocrine cell lineages is severely compromised upon Cdc42 deficiency. In contrast, expression of Ptf1a, elastase, and amylase mRNAs and amylase and Cpa1 proteins increased, suggesting that acinar cell differentiation increased in the absence of Cdc42 (Figures 6A, 6B, 6A′, 6B′, and 6D–6F). In the WT pancreas Sox9 was distributed throughout the branching tubular tree, except for within the terminal acinar structures (Seymour et al., 2007Seymour P.A. Freude K.K. Tran M.N. Mayes E.E. Jensen J. Kist R. Scherer G. Sander M. SOX9 is required for maintenance of the pancreatic progenitor cell pool.Proc. Natl. Acad. Sci. USA. 2007; 104: 1865-1870Crossref PubMed Scopus (407) Google Scholar). In the E15.5 Cdc42 KO epithelium very few Sox9+ duct cells were found and they were randomly distributed within the epithelial aggregates (Figures 6C and 6C′). At E17.5, the phenotype was comparable to E15.5 with many acinar cells but few endocrine and duct cells (Figure S12B). Postnatally, the Cdc42 KO animals were growth retarded and developed cysts in the stomach and a distended duodenum (Figure S12A). Moreover, acinar cysts developed within the pancreas. The majority of these cysts were multicellular consisting of polarized cells with apical junctions. However, large cysts from single cells were also observed (Figures S12B and S12C and data not shown). In summary, Cdc42 ablation results in increased acinar cell differentiation at the expense of endocrine and duct cell differentiation. This indicates that Cdc42 is required for proper specification of multipotent pancreatic progenitors into acinar, duct, and endocrine cells. In vitro culture of the embryonic pancreas in the absence of mesenchyme suppresses cell proliferation and acinar differentiation but enhances endocrine specification. Therefore, it was proposed that multipotent progenitors choose i" @default.
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• W2044638670 date "2009-11-01" @default.
• W2044638670 modified "2023-10-04" @default.
• W2044638670 title "Cdc42-Mediated Tubulogenesis Controls Cell Specification" @default.
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• W2044638670 doi "https://doi.org/10.1016/j.cell.2009.08.049" @default.
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