Chick pulmonary Wnt5a directs airway and vascular tubulogenesis

Maria Loscertales 2 Amanda J. Mikels 1 Jimmy Kuang-Hsein Hu 0 Patricia K. Donahoe 2 Drucilla J. Roberts ) 2 3 0 Department of Genetics, Harvard Medical School , 77 Avenue Louis Pasteur, Boston, MA 02115 , USA 1 Department of Developmental Biology, Stanford University School of Medicine , Stanford, CA 94305 , USA 2 Pediatric Surgical Research Laboratories, Department of Surgery, Massachusetts General Hospital, Harvard Medical School , 185 Cambridge Street, Boston, MA 02114 , USA 3 Department of Pathology, Massachusetts General Hospital, Harvard Medical School , Boston, MA 02114 , USA Wnt5a is an important factor patterning many aspects of early development, including the lung. We find pulmonary non-canonical Wnt5a uses Ror2 to control patterning of both distal air and vascular tubulogenesis (alveolarization). Lungs with mis/overexpressed Wnt5a develop with severe pulmonary hypoplasia associated with altered expression patterns of Shh, L-CAM, fibronectin, VEGF and Flk1. This hypoplastic phenotype is rescued by either replacement of the Shh protein or inhibition of fibronectin function. We find that the effect of Wnt5a on vascular patterning is likely to be through fibronectin-mediated VEGF signaling. These results demonstrate the pivotal role of Wnt5a in directing the essential coordinated development of pulmonary airway and vasculature, by affecting fibronectin levels directly, and by affecting the fibronectin pattern of expression through its regulation of Shh. Data herein suggest that Wnt5a functions in mid-pulmonary patterning (during alveolarization), and is distinct from the Wnt canonical pathway which is more important in earlier lung development. INTRODUCTION Pulmonary development involves coordinated patterning of both the airway and vascular systems (Cardoso and Lu, 2006). Their synchronization is necessary for effective gas transfer, and maldevelopment of either is frequently lethal (Berrocal et al., 2004; Cullinane et al., 1992; deMello, 2004; Galambos and deMello, 2007). Although there have been many studies of airway development (for reviews, see Cardoso, 2001; Costa et al., 2001; Kimura and Deutsch, 2007), relatively little is known about pulmonary vascular pattern formation or the coordinated development of the two. Although the anatomy of the avian lung differs from that of the mammalian lung, both develop similarly and have anatomical functional equivalents. The avian lung forms by a series of closed circular buds arising from the main airway branches, which differs from the dichotomous branching morphogenesis in mammalian lung development. In contrast to the mammalian lung, which terminates in alveoli, the avian lung forms a looping anastomotic network of air-vascular surfaces (parabronchi) that end in terminal air buds and air capillaries. For all vertebrates, pulmonary vascular patterning continues in coordination with airway development. In the chick embryo, vasculogenesis is the main process by which the pulmonary vasculature initially forms, being guided by the budding airways (Anderson-Berry et al., 2005; Hislop, 2005). This interstitial microvasculature connects the large pulmonary vessels to the terminal buds of the airways and the capillaries via two mechanisms, sprouting and intussusceptive angiogenesis (Makanya et al., 2007). Sprouting angiogenesis predominates from early to mid-gestation (E15), and is the major mechanism for setting up the basic hexagonal interstitial vascular pattern seen in avians. Intussusceptive angiogenesis, a novel process involving endothelial cell extension into the lumen of a vessel (splitting the vessel), predominates from ~E15, producing the massive expansion of the vasculature that is necessary for vital gas exchange (alveologenesis). Intussusceptive angiogenesis occurs simultaneously with the rapid growth of the airway epithelium into the mesenchyme to create the huge surface area of the mature lung. After E18, the air capillaries and the blood capillaries are in close proximity, forming the functional equivalent of the mammalian alveolus (for a review, see Maina, 2006). The chick embryo is fully air breathing by E18, 2-3 days before hatching. Airway and vascular channels, epithelial and endothelial tubules, are the fundamental structural units of the lung (Cardoso and Lu, 2006), and develop in response to morphogenetic growth factors, transcription factors, extracellular matrix proteins and their receptors (Hogan and Kolodziej, 2002). Although much is known about the factors regulating airway development, those controlling this coordinated air and vascular patterning are unknown. The Wnt family of secreted signaling molecules functions in numerous key developmental events (Wodarz and Nusse, 1998). Wnts are broadly categorized into two groups based on their signal transduction pathway. Canonical Wnts transduce their signals through receptors of the frizzled (Fz) family by a -catenindependent pathway (Wodarz and Nusse, 1998). Non-canonical Wnts signal via either the planar cell polarity (PCP) pathway or the Wnt/Ca2+ pathway (Veeman et al., 2003; Widelitz, 2005), and may use Fz receptors or other receptors, including the orphan tyrosine kinase Ror2 (Keeble et al., 2006; Oishi et al., 2003). The Wnt signaling pathway has been well described and includes numerous regulators (Pandur et al., 2002; Widelitz, 2005). Most described Wnt antagonists interfere with the canonical pathway, such as the Dickkopf (Dkk) proteins, of which Dkk1 specifically interrupts only the canonical-Wnt function (Glinka et al., 1998; Kawano and Kypta, 2003; Niehrs, 2006). Whereas canonical Wnt signaling is known to regulate lung development early in branching morphogenesis (Dean et al., 2005; Mucenski et al., 2005; Okubo and Hogan, 2004), the non-canonical Wnts (such as Wnt5a) appear to act later (Li et al., 2005). Wnt5a moderates many cellular events, including cell adhesion (Jonsson and Andersson, 2001; Torres et al., 1996; Toyofuku et al., 2000; Weeraratna et al., 2002), migration (Jonsson and Andersson, 2001), proliferation (Liang et al., 2003; Yamaguchi et al., 1999) and differentiation (He et al, 1997; Kuhl et al., 2001; Kuhl et al., 2000; Sheldahl et al., 1999). These findings suggest that Wnt5a is a good candidate for coordinating pulmonary air and vascular pattern formation. One mechanism by which Wnt5a might regulate these cellular events is by affecting the extracellular matrix, a component of which is fibronectin. Fibronectin has been implicated in branching morphogenesis (Roman and McDonald, 1992; Sakai et al., 2003), vasculogenesis (Bull et al., 1993; Hall et al., 2000; Jozaki et al., 1990) and intussusceptive angiogenesis (Makanya et al., 2007), and it interacts with VEGF (Vascular Endothelial Growth Factor) (Goerges and Nugent, 2004), which is also implicated in lung vascular and airway branching morphogenesis (Warburton et al., 2005). Although it is known that canonical Wnt signaling decreases fibronectin expression i (...truncated)