DEVELOPMENT 1933 RESEARCH ARTICLE INTRODUCTION Bone morphogenetic proteins (BMPs) are a large subclass of the TGF superfamily that are involved in many aspects of development (Chang et al., 2001; Hogan, 1996; Kishigami and Mishina, 2005). Bmp4 is widely expressed throughout embryogenesis and its earliest known function is during gastrulation. Most mice homozygous for a null allele of Bmp4 (Bmp4 –/– mice) die at embryonic day (E) 6.5, with little or no mesoderm (Winnier et al., 1995). In the rare mutants that survive past this time, primordial germ cells (PGCs) are not specified (Lawson et al., 1999) and lens induction does not occur (Furuta and Hogan, 1998). Chimeric analysis has shown that BMP4 is required for chorioallantoic fusion, vascularization of the allantois, migration and survival of PGCs, and establishment of left-right asymmetry (Fujiwara et al., 2001; Fujiwara, 2002). Tissue-specific inactivation of Bmp4 has revealed additional roles for BMP4 in development of the heart, limbs and craniofacial structures (Jiao et al., 2003; Liu et al., 2005; Liu et al., 2004; Selever et al., 2004). Strict regulation of BMP4 dosage is essential for normal development, as evidenced by patterning defects observed in mice with reduced or elevated levels of BMP4 activity. Bmp4 null heterozygotes (Bmp4 +/– mice) on a C57BL/6J background display several phenotypes of variable penetrance, including reduced numbers of PGCs, polydactyly, failure to maintain spermatogenesis and defects in the kidneys, eyes and craniofacial structures (Dunn et al., 1997; Katagiri et al., 1998; Lawson et al., 1999; Miyazaki et al., 2000). By contrast, mice that lack the BMP antagonists chordin and/or noggin are stillborn, show loss of ventral cell fates in the spinal cord and defects in the development of the forebrain, somites and skeleton (Bachiller et al., 2000; Brunet et al., 1998; McMahon et al., 1998). BMP4 dosage is regulated at multiple levels, including at the level of proteolytic activation (Miyazono et al., 2005; Nakayama et al., 2000; Yanagita, 2005). BMP4 is synthesized as an inactive precursor that is cleaved by FURIN and/or other members of the proprotein convertase (PC) family (Cui et al., 1998) at two evolutionarily conserved sites within the inactive prodomain. An initial cleavage occurs at an optimal FURIN consensus motif adjacent to the mature ligand domain (-RSKR-, denoted the S1 site) and this allows for subsequent cleavage at an upstream minimal FURIN motif (-RISR-, the S2 site) within the prodomain (Cui et al., 2001). In Xenopus embryos, BMP4 synthesized from exogenous precursor in which the S2 site is non-cleavable is less active, signals over a shorter range and accumulates at lower levels than does BMP4 cleaved from native precursor (Cui et al., 2001). Biochemical analysis of BMP4 cleavage in Xenopus oocytes revealed that mature BMP4 remains noncovalently attached to the prodomain following cleavage at the S1 site (Degnin et al., 2004). If cleavage at the S2 site does not occur, this complex is targeted to the lysosome for degradation, either within the biosynthetic pathway, or within the endocytic pathway following receptor activation and internalization. As a result, mature BMP4 in complex with the prodomain signals only at short range, to nearby cells. Cleavage at the S2 site occurs when the mature/prodomain complex traffics to a more acidic environment, which unmasks and facilitates cleavage of the S2 site. This leads to dissociation of the prodomain fragments from mature BMP4, and the free ligand is stable and able to signal over long range. Cleavage at the S2 site might therefore determine how much BMP4 is available for signaling. The ability of cleavages within the prodomain to regulate the signaling range of mature BMP4 is of particular interest because BMP4 and its Drosophila ortholog, decapentaplegic (DPP) can function as either short- or long-range signaling molecules depending on the tissue in which they are expressed. Xenopus BMP4, for example, acts over multiple cells within the embryonic Mutation of an upstream cleavage site in the BMP4 prodomain leads to tissue-specific loss of activity Devorah C. Goldman 1 , Renee Hackenmiller 1 , Takuya Nakayama 1, *, Shailaja Sopory 1 , Crispin Wong 1 , Holger Kulessa 2,† and Jan L. Christian 1,§ ProBMP4 is initially cleaved at a site adjacent to the mature ligand (the S1 site) allowing for subsequent cleavage at an upstream (S2) site. Mature BMP4 synthesized from a precursor in which the S2 site cannot be cleaved remains in a complex with the prodomain that is targeted for lysosomal degradation, and is thus less active when overexpressed in Xenopus. Here we report that mice carrying a point mutation that prevents S2 processing show severe loss of BMP4 activity in some tissues, such as testes and germ cells, whereas other tissues that are sensitive to Bmp4 dosage, such as the limb, dorsal vertebrae and kidney, develop normally. In a haploinsufficient background, inability to cleave the S2 site leads to embryonic and postnatal lethality due to defects in multiple organ systems including the allantois, placental vasculature, ventral body wall, eye and heart. These data demonstrate that cleavage of the S2 site is essential for normal development and, more importantly, suggest that this site might be selectively cleaved in a tissue-specific fashion. In addition, these studies provide the first genetic evidence that BMP4 is required for dorsal vertebral fusion and closure of the ventral body wall. KEY WORDS: Bone morphogenetic protein, Proprotein convertase, Proteolytic activation, Embryonic patterning, Cleavage mutant mouse Development 133, 1933-1942 (2006) doi:10.1242/dev.02368 1 Department of Cell and Developmental Biology, Oregon Health and Sciences University, School of Medicine, 3181 SW Sam Jackson Park Road, Portland, OR 97239-3098, USA. 2 Vanderbilt University Medical Center, Division of Gastroenterology, D4108 Medical Center North, Nashville, TN 37232-2279, USA. *Present address: Department of Biology, Gilmer Hall Rm 253, University of Virginia, PO Box 400328, Charlottesville, VA 22904-4328, USA † Deceased § Author for correspondence (e-mail: christia@ohsu.edu) Accepted 16 March 2006