Dickkopf-1 (DKK1) Reveals That Fibronectin Is a Major Target of Wnt Signaling in Branching Morphogenesis of the Mouse Embryonic Lung
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CORE Metadata, citation and similar papers at core.ac.uk Provided by Elsevier - Publisher Connector Developmental Biology 277 (2005) 316–331 www.elsevier.com/locate/ydbio Dickkopf-1 (DKK1) reveals that fibronectin is a major target of Wnt signaling in branching morphogenesis of the mouse embryonic lung Stijn P. De Langhea, Fre´de´ric G. Salaa, Pierre-Marie Del Morala, Timothy J. Fairbanksa, Kenneth M. Yamadab, David Warburtona, Robert C. Burnsa, Saverio Belluscia,* aDevelopmental Biology Program, Department of Surgery, USC Keck School of Medicine and the Saban Research Institute of Children’s Hospital Los Angeles, Los Angeles, CA 90027, USA bCraniofacial Developmental Biology and Regeneration Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892, USA Received for publication 6 April 2004, revised 16 September 2004, accepted 20 September 2004 Available online 22 October 2004 Abstract Members of the Dickkopf (Dkk) family of secreted proteins are potent inhibitors of Wnt/h-catenin signaling. In this study we show that Dkk1, -2, and -3 are expressed distally in the epithelium, while Kremen1, the needed co-receptor, is expressed throughout the epithelium of the developing lung. Using TOPGAL mice [DasGupta, R., Fuchs, E., 1999. Multiple roles for activated LEF/TCF transcription complexes during hair follicle development and differentiation. Development 126, 4557–4568] to monitor the Wnt pathway, we show that canonical Wnt signaling is dynamic in the developing lung and is active throughout the epithelium and in the proximal smooth muscle cells (SMC) until E12.5. However, from E13.5 onwards, TOPGAL activity is absent in the SMC and is markedly reduced in the distal epithelium coinciding with the onset of Dkk-1 expression in the distal epithelium. To determine the role of Wnt signaling in early lung development, E11.5 organ cultures were treated with recombinant DKK1. Treated lungs display impaired branching, characterized by failed cleft formation and enlarged terminal buds, and show decreased a-smooth muscle actin (a-SMA) expression as well as defects in the formation of the pulmonary vasculature. These defects coincide with a pattern of decreased fibronectin (FN) deposition. DKK1-induced morphogenetic defects can be mimicked by inhibition of FN and overcome by addition of exogenous FN, suggesting an involvement of FN in Wnt-regulated morphogenetic processes. D 2004 Elsevier Inc. All rights reserved. Keywords: Lung; Dickkopf-1; Wnt signaling; Fibronectin; Branching morphogenesis; Smooth muscle; Vascular development Introduction and branch laterally and distally in a highly reproducible pattern. They develop asymmetrically, so that in the mouse Early embryonic lung development is characterized by a the right bud gives rise to four lobes, whereas the left bud succession of cycles of bud initiation, bud outgrowth, and gives rise to only one. bud arrest. Lung formation starts at E9.5, with an out- The differentiation process of the epithelium and pocketing of the endoderm from the ventral foregut. mesenchyme is tightly coordinated with the branching Initially, the two primary buds undergo repetitive outgrowth process. The most distal epithelial and mesenchymal cells are essentially undifferentiated and as they leave the distal domain, they differentiate progressively (Hogan, 1999). The * Corresponding author. Developmental Biology Program, Department differentiation process is dependent upon the combination of of Surgery, USC Keck School of Medicine and the Saban Research Institute of Children’s Hospital Los Angeles, 4650 W Sunset Boulevard, 804 SRT, signals received. Experimental evidence is rapidly emerging MS 35, Los Angeles, CA 90027. Fax: +1 323 906 8096. that the balance between activating and inhibitory signals is E-mail address: [email protected] (S. Bellusci). a mechanism of fine regulation in development. For 0012-1606/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ydbio.2004.09.023 S.P. De Langhe et al. / Developmental Biology 277 (2005) 316–331 317 example, Sonic hedgehog activity is modulated by Hedge- expression of pro-SP-C protein and Vascular endothelial hog Interacting Protein (HIP), which binds to SHH and growth factor-A (Vegf-A), the latter correlating with a prevents its binding to its receptor, Patched (PTC) (Chuang reduction in alveolar capillaries. So far, inactivation of only et al., 2003). Similar inhibitory mechanisms exist between two WNT ligands has resulted in lung defects. Wnt7b,a Bone Morphogenetic Protein 4 and extracellular modulators canonical Wnt, is expressed in epithelial cells of the like Noggin, Gremlin, or Chordin (reviewed by Balemans peripheral lung. Wnt7bÀ/À mice exhibit perinatal death and Van Hul, 2002). In the Wnt pathway, secreted Frizzled- due to respiratory failure. Defects were observed in related proteins (sFRP) and more recently Dickkopf (DKK) proliferation of the lung mesenchyme resulting in lung are secreted molecules shown to inhibit Wnt signaling hypoplasia. In addition, Wnt7bÀ/À embryos and newborn (reviewed by Kawano and Kypta, 2003). mice exhibit severe defects in the smooth muscle compo- This work focuses on the role of DKK1 during lung nent of the major pulmonary vessels, with increased development. Through our study of DKK1, our goal is also to apoptosis of the vascular smooth muscle cells (VSMCs), investigate the role of the Wnt pathway during this process. resulting in rupture of the major blood vessels and The Wnt growth factor family in the mouse is comprised of hemorrhages in the lungs after birth (Shu et al., 2002). 19 different secreted ligands that interact with 10 known Wnt5a, a noncanonical Wnt, which is able to inhibit seven-transmembrane receptors of the Frizzled (Fz) gene canonical Wnt signaling (Topol et al., 2003; Westfall et family and either one of two single-span transmembrane al., 2003), is expressed at high levels in the distal lung proteins, low-density-lipoprotein-receptor-related proteins mesenchyme. Wnt5aÀ/À mice die perinatally from lung (LRP-5 and LRP-6) (Pinson et al., 2000; Tamai et al., 2000; defects including truncation of the trachea, overexpansion of Wehrli et al., 2000). Historically, Wnt proteins have been the peripheral airways, and delayed lung maturation. grouped into two classes, canonical and noncanonical. Absence of Wnt5a activity in mutant lungs leads to Canonical Wnts bind to frizzled receptors, inhibiting increased cell proliferation and up-regulation of the expres- glycogen-synthase kinase-3h (GSK-3h)-mediated phosphor- sion of Fgf10, Bmp4, Shh, and Ptc (Li et al., 2002). ylation of h-catenin. Hypophosphorylated h-catenin accu- To determine the role of canonical Wnt signaling in early mulates in the cytoplasm, after which it translocates to the lung development, we treated E11.5 lung explants with nucleus, where it heterodimerizes with members of the TCF/ recombinant Dickkopf-1 (DKK1), a potent and specific LEF transcription factor family to activate the transcription of diffusible inhibitor of Wnt action that is also endogenously TCF/LEF target genes (reviewed by Seto and Bellen, 2004). secreted by the distal lung epithelium. We show that organ Noncanonical Wnts activate other Wnt signaling pathways, cultures treated with DKK1 display impaired branching, such as the planar-cell-polarity (PCP)-like pathway that characterized by failed cleft formation and enlarged terminal guides cell movements during gastrulation (Heisenberg et buds. DKK1-treated lung explants further show reduced a- al., 2000). Activation of the PCP pathway can antagonize the smooth muscle actin (a-SMA) expression and defects in the canonical pathway intracellularly (Ishitani et al., 2003; Kuhl formation of the pulmonary vascular network. These defects et al., 2001; Torres et al., 1996). Secreted Wnt antagonists can coincide with a pattern of decreased fibronectin (FN) be divided into two functional classes, the secreted Frizzled- deposition and Platelet-derived growth factor-A (Pdgf-a) related protein (sFRP) class and the Dickkopf class. Members expression. DKK1-induced morphogenetic defects can be of the sFRP class bind directly to Wnts, thereby altering their mimicked by inhibition of FN and overcome by addition of ability to bind to the Wnt receptor complex, while members exogenous FN. Our findings suggest an intersection of the Dickkopf class inhibit Wnt signaling by binding to the between the Wnt and FN-integrin pathways and an involve- LRP5/LRP6 component of the Wnt receptor complex. In ment of FN in Wnt-regulated morphogenetic processes. addition to LRP5/6, DKK1 interacts with the high-affinity DKK1 co-receptors Kremen1 (Krm1) or Kremen2 (Krm2), which functionally cooperate with Dkk1 to block Wnt/beta- Materials and methods catenin signaling (Mao et al., 2002 and reviewed by Kawano and Kypta, 2003). DKK1, DKK3, and DKK4 act as inhibitors Transgenic embryos of canonical Wnt signaling, while DKK2 can function both as an inhibitor in the presence of Kremen2 or as an activator in TOPGAL and Sp-C-GFP mice were a generous gift of its absence (Hoang et al., 2004; Mao and Niehrs, 2003). Dr. Elaine Fuchs and Dr. Jonathan Heath, respectively. Wnt signaling plays an important role in lung develop- Flk1LacZ/+ mice were obtained from Jackson Laboratories. ment. Conditional inactivation of the b-catenin gene in the All mice were bred in the C57BL6 background. Lungs were epithelium of the developing mouse lung leads to neonatal isolated from embryonic day 11.5 (E11.5)