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Wnt Ligand/Frizzled 2 Receptor Signaling Regulates Tube Shape and Branch-Point Formation in the Lung Through Control of Epithelial Cell Shape

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Wnt Ligand/Frizzled 2 Receptor Signaling Regulates Tube Shape and Branch-Point Formation in the Lung Through Control of Epithelial Cell Shape Wnt ligand/Frizzled 2 receptor signaling regulates tube shape and branch-point formation in the lung through control of epithelial cell shape Rachel S. Kadzika, Ethan David Cohenb, Michael P. Morleyc, Kathleen M. Stewartc,d, Min Min Luc, and Edward E. Morriseya,c,d,e,1 Departments of aCell and Developmental Biology and cMedicine, dCardiovascular Institute, and eInstitute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA 19104; and bDivision of Endocrinology, Department of Medicine, University of Rochester, Rochester, NY 14642 Edited* by Brigid L. M. Hogan, Duke University Medical Center, Durham, NC, and approved July 16, 2014 (received for review April 11, 2014) Changing the morphology of a simple epithelial tube to form a apical expression of phospho-myosin light chain 2 (pMLC2) in- highly ramified branching network requires changes in cell behavior dicative of the decreased Rho signaling that is required for that lead to tissue-wide changes in organ shape. How epithelial cells thickening of the lung epithelium before new branch formation. in branched organs modulate their shape and behavior to promote Importantly, activation of Rho signaling can rescue the loss of bending and sculpting of the epithelial sheet is not well understood, Fzd2 signaling during lung branching morphogenesis. Together, and the mechanisms underlying this process remain obscure. We our data highlight a previously unappreciated mechanism in the show that the Wnt receptor Frizzled 2 (Fzd2) is required for domain formation of branched networks by the control of epithelial cell branch formation during the initial establishment of the respiratory shape through Wnt signaling. tree. Live imaging and transcriptome analysis of lung-branching morphogenesis demonstrate that Fzd2 promotes changes in epi- Results thelial cell length and shape. These changes in cell morphology Loss of Fzd2 in the Lung Epithelium Causes the Formation of Distal deform the developing epithelial tube to generate and maintain new Cysts in the Lung. Fzd2 is a Wnt receptor expressed at high levels domain branches. Fzd2 controls branch formation and the shape in the developing lung epithelium and has been implicated in of the epithelial tube by regulating Rho signaling and by the regulating epithelial differentiation downstream of Gata6 (6). To localization of phospho-myosin light chain 2, in turn controlling assess further the role of Fzd2 in the developing lung, we gen- flox/flox the changes in the shape of epithelial cells during morphogen- erated mice carrying the Fzd2 allele and crossed these mice cre into the Shh allele (7) to delete Fzd2 specifically in the lung esis. This study demonstrates the importance of Wnt/Fzd2 cre flox/flox signaling in promoting and maintaining changes in epithelial cell epithelium (Fig. S1). Fzd2 expression in the Shh :Fzd2 shape that affect development of a branching network. mutants is reduced efficiently in the lung epithelium by embryonic day (E) 12.5, as observed by in situ hybridization and quantitative real-time PCR (qPCR) (Fig. 1 A and B and Fig. S1B). Although evelopment of many epithelial-derived organs requires a pro- cre flox/flox initial lung formation occurs in Shh :Fzd2 mutants, by cess of bending, folding, and reorganization of a primitive D E14.5 mutant lungs contain multiple large cysts in the distal epithelial sheet or tube to generate a functional 3D organ. The regions of the lung (Fig. 1 C and D) that persist into adulthood. mammalian lung is derived from a simple endoderm tube through In normal lung development, new lateral branches are add- a complex series of morphological changes that generates the ed along a main founder branch in a stereotyped pattern in highly arborized airways required for postnatal respiration. In humans, the first 16 generations of branching are thought to be genetically hard-wired; this notion is supported by work on mouse Significance lungs showing that the branching pattern across multiple mouse strains is highly reproducible (1, 2). Despite such insight, little is We generated a conditional mouse allele for the Wnt receptor understood about the genetic control of the molecular and cellular Fzd2 and used it to assess the role of Fzd2-mediated Wnt sig- mechanisms underlying branching morphogenesis in the lung. naling in the lung. Loss of Fzd2 specifically in the developing The epithelial cells that line tubular branching networks can lung epithelium results in defects in domain branch-point for- be thought of as a large planar epithelial surface that must undergo mation which alter the primary branching program of the lung. changes in cell morphology in specific subregions for proper branch We show that Fzd2 is required to sculpt the developing epi- formation to occur. Several pathways, including the Wnt signaling thelium in the lung through activation of the small GTPase RhoA pathway, have been implicated in regulating epithelial cell behavior and control of epithelial cell shape. These results reveal the in a plane. Although the canonical Wnt signaling pathway regulates importance of Wnt/RhoA signaling in altering the shape of the gene expression through nuclear translocation of β-catenin and its developing epithelium of branched organs such as the lung. subsequent coactivation of LEF/TCF transcription factors, non- Such studies highlight the interconnectedness of signaling canonical Wnt signaling involves a less well-defined signaling pathways during the formation of a branched network. network that leads to alterations in epithelial cell shape and cyto- skeletal structure. Noncanonical Wnt signaling is known to regulate Author contributions: R.S.K., M.P.M., and E.E.M. designed research; R.S.K., E.D.C., K.M.S., changes in epithelial cell shape in convergent–extension movements M.M.L., and E.E.M. performed research; R.S.K., E.D.C., and M.M.L. contributed new reagents/ (3, 4) and bending of the neural plate (5), but whether this pathway analytic tools; R.S.K., M.P.M., K.M.S., and E.E.M. analyzed data; and R.S.K. and E.E.M. wrote regulates the development of branched organs is unknown. the paper. In the current study we show that the Wnt receptor Frizzled 2 The authors declare no conflict of interest. (Fzd2) plays a key role in regulating the epithelial cell behavior *This Direct Submission article had a prearranged editor. and tube morphology necessary for formation of new branch Data deposition: The microarray data reported in this article have been deposited in the points during airway morphogenesis. Fzd2 is essential for regu- Gene Expression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. lating changes in epithelial cell shape and cell lengthening along GSE49370). the apical–basal axis which we show are critical for formation of 1To whom correspondence should be addressed. Email: [email protected]. new domain branch points and maintaining proper airway tube This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. shape in the developing lung. Loss of Fzd2 leads to decreased 1073/pnas.1406639111/-/DCSupplemental. 12444–12449 | PNAS | August 26, 2014 | vol. 111 | no. 34 www.pnas.org/cgi/doi/10.1073/pnas.1406639111 Downloaded by guest on September 29, 2021 E12.5 E14.5 branch formation during the establishment of the respiratory tree, we also observe defects in later branching modalities, including cre fl/fl Fzd2 In Situ Control Shh ; Fzd2 planar bifurcation, which likely contribute to cyst formation. A C Loss of Fzd2 Does Not Change Wnt/β-Catenin Signaling Significantly in Lung Epithelium. Fzd2 activates both β-catenin–dependent and –independent Wnt signaling, depending on cellular context (8–11). Therefore we evaluated the effect of loss of Fzd2 expression Control on canonical Wnt signaling in vivo using two Wnt reporter mouse cre B D lines and qPCR for endogenous Wnt target genes (12, 13). Shh : flox/flox Fzd2 :TOPGAL mutant lungs did not exhibit an appreciable change in LacZ histochemical staining compared with control lungs (Fig. S3 A–F). qPCR for known Wnt targets in the developing lung did not reveal a significant change in canonical Wnt signaling ac- Shhcre; Fzd2fl/fl tivity (Fig. S3G). These findings are consistent with a previously 36 somites 44 somites 48 somites 54 somites 60 somites reported global Fzd2-null allele that demonstrated that the loss of Fzd2 does not significantly affect canonical Wnt/β-catenin signaling EFGHI (8). Previous work established a role for Wnt/β-catenin signaling in regulating cell-fate decisions in the lung during development (14– control 16). However, we do not observe any change in proximal-to- E-cadherin Vimentin distal patterning or cell differentiation with the loss of Fzd2 (Fig. fl/fl J KMNL S4). These data suggest that Wnt/Fzd2 signaling has a previously unappreciated role in regulating morphological changes inde- :Fzd2 cre pendent of Wnt/β-catenin signaling or cell specification. E-cadherin Shh Vimentin O i. 42-44 somites ii. 46-48 somites iii. 52-54 somites iv. 57-60 somites Loss of Fzd2 Disrupts the Molecular Branching Program of the Lung. 1 To assess further the molecular changes that underlie the br- 1 1 1 anching defects described above, we determined the changes in cre flox/flox 2 2 the transcriptome of Shh :Fzd2 mutant lungs at E12.5. control 3 2 3 These data revealed changes in signaling factors known to reg- 4 ulate lung branching morphogenesis, including Fgf10, Bmp4, Fgfr2, and Shh (Fig. 2 A and B and Dataset S1). Previous work fl/fl 1 1 1 has proposed a model for lung branch formation that includes 1 2 2 C :Fzd2 roles for Fgf10 signaling as well as Bmp4 and Shh (Fig. 2 ) (17, Left Lobe Domain Branches cre 2 18). This model describes a role for Fgf10 in establishing the site 3 Shh E-cadherin of new bud formation, although recent work has questioned Vimentin whether focal Fgf10 expression is required for proper branching Fig.
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