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To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.05.030 Byline: Marek Dudas (a)(d), Jieun Kim (a), Wai-Yee Li (a), Andre Nagy (a), Jonas Larsson (b), Stefan Karlsson (b), Yang Chai (c), Vesa Kaartinen (a) Keywords: Alk5; Cleft face; Cleft palate; Cranial neural crest; Craniofacial malformation; Mandible; Palatal fusion Abstract: Transforming growth factor beta (TGF-[beta]) proteins play important roles in morphogenesis of many craniofacial tissues; however, detailed biological mechanisms of TGF-[beta] action, particularly in vivo, are still poorly understood. Here, we deleted the TGF-[beta] type I receptor gene Alk5 specifically in the embryonic ectodermal and neural crest cell lineages. Failure in signaling via this receptor, either in the epithelium or in the mesenchyme, caused severe craniofacial defects including cleft palate. Moreover, the facial phenotypes of neural crest-specific Alk5 mutants included devastating facial cleft and appeared significantly more severe than the defects seen in corresponding mutants lacking the TGF-[beta] type II receptor (TGF[beta]RII), a prototypical binding partner of ALK5. Our data indicate that ALK5 plays unique, non-redundant cell-autonomous roles during facial development. Remarkable divergence between Tgfbr2 and Alk5 phenotypes, together with our biochemical in vitro data, imply that (1) ALK5 mediates signaling of a diverse set of ligands not limited to the three isoforms of TGF-[beta], and (2) ALK5 acts also in conjunction with type II receptors other than TGF[beta]RII. Author Affiliation: (a) Developmental Biology Program, The Saban Research Institute of Children's Hospital Los Angeles, Departments of Pathology and Surgery, Keck School of Medicine, University of Southern California, Los Angeles, CA 90027, USA (b) Molecular Medicine and Gene Therapy, Institute of Laboratory Medicine and Department of Medicine, Lund University Hospital, Sweden (c) Center for Craniofacial Molecular Biology, School of Dentistry University of Southern California, CA 90033, USA (d) Institute of Biology and Ecology, P. J. Safarik University in Kosice, Slovakia Article History: Received 30 January 2006; Revised 28 April 2006; Accepted 23 May 2006

The role of beta (TGF- ?) proteins in development of craniofacial tissues during morphogenesis is discussed.

Developmental abnormalities in endocardial cushions frequently contribute to congenital heart malformations including septal and valvular defects. While compelling evidence has been presented to demonstrate that members of the TGF-[beta] superfamily are capable of inducing endothelial-to-mesenchymal transdifferentiation in the atrioventricular canal, and thus play a key role in formation of endocardial cushions, the detailed signaling mechanisms of this important developmental process, especially in vivo, are still poorly known. Several type I receptors (ALKs) for members of the TGF-[beta] superfamily are expressed in the myocardium and endocardium of the developing heart, including the atrioventricular canal. However, analysis of their functional role during mammalian development has been significantly complicated by the fact that deletion of the type I receptors in mouse embryos often leads to early embryonal lethality. Here, we used the Cre/loxP system for endothelial-specific deletion of the type I receptor Alk2 in mouse embryos. The endothelial-specific Alk2 mutant mice display defects in atrioventricular septa and valves, which result from a failure of endocardial cells to appropriately transdifferentiate into the mesenchyme in the AV canal. Endocardial cells deficient in Alk2 demonstrate decreased expression of Msx1 and Snail, and reduced phosphorylation of BMP and TGF-[beta] Smads. Moreover, we show that endocardial cells lacking Alk2 fail to delaminate from AV canal explants. Collectively, these results indicate that the BMP type I receptor ALK2 in endothelial cells plays a critical non-redundant role in early phases of endocardial cushion formation during cardiac morphogenesis. Keywords: Atrioventricular cushion; ALK2; BMP; TGF-[beta]; Transformation; Cardiac development

Cleft palate is among the most common birth defects in humans, caused by a failure in the complex multistep developmental process of palatogenesis. It has been recently shown that transforming growth factor [beta]3 (Tgf-[beta]3) is an absolute requirement for successful palatal fusion, both in mice and humans. However, very little is known about the mechanisms of Tgf-[beta]3 signaling during this process. Here we show that putative Tgf-[beta] type I receptors, Alk-1, Alk-2, and Alk-5, are all endogenously expressed in the palatal epithelium. Activation of Alk-5 in the Tgf-[beta]3 (- / -) palatal epithelium is able to rescue palatal fusion, whereas inactivation of Alk-5 in the wild-type palatal epithelium prevents palatal fusion. The effect of Alk-2 is similar, but less pronounced. The induction of fusion by activation of Alk-5 or Alk-2 is stronger in the posterior parts of the palates at the embryonic day 14 (E14), while their activation at E13.5 also restores anterior fusion, reflecting the natural anterior-posterior direction of palate maturation in vivo. We also show that Smad2 is endogenously activated in the palatal midline epithelial seam (MES) during the fusion process. By using a mutant Alk-5 receptor that is an active kinase but is unable to activate Smads, we show that activation of Smad-independent Tgf-[beta] responses is not sufficient to induce fusion of shelves deficient in Tgf-[beta]3. Based on these observations, we conclude that the Smad2-dependent Alk-5 signaling pathway is dominant in palatal fusion driven by Tgf-[beta]3. Keywords: Cleft palate; Palate fusion; Tgf-[beta]3 knockout mouse; Tgf-[beta] receptor; Alk-5; Smad2; Smad7; Recombinant adenovirus