Elimination of the developing female reproductive tract in male fetuses is an essential step in mammalian sexual differentiation. In males, the fetal testis produces the transforming growth factor beta (TGF-beta) family member anti-Müllerian hormone (Amh, also known as Müllerian-inhibiting substance (Mis)), which causes regression of the Müllerian ducts, the primordia of the oviducts, uterus and upper vagina. Amh induces regression by binding to a specific type II receptor (Amhr2) expressed in the mesenchyme surrounding the ductal epithelium. Mutations in AMH or AMHR2 in humans and mice disrupt signaling, producing male pseudohermaphrodites that possess oviducts and uteri. The type I receptor and Smad proteins that are required in vivo for Müllerian duct regression have not yet been identified. Here we show that targeted disruption of the widely expressed type I bone morphogenetic protein (BMP) receptor Bmpr1a (also known as Alk3) in the mesenchymal cells of the Müllerian ducts leads to retention of oviducts and uteri in males. These results identify Bmpr1a as a type I receptor for Amh-induced regression of Müllerian ducts. Because Bmpr1a is evolutionarily conserved, these findings indicate that a component of the BMP signaling pathway has been co-opted during evolution for male sexual development in amniotes.

Abstract-Despite the advantages of reversibly altering cardiac transgene expression, the number of successful studies with inducible cardiac-specific transgene expression remains limited. The utility of the current system is hampered by the large number of lines needed before a nonleaky inducible line is isolated and by the use of a heterologous virus-based minimal promoter in the responder line. We developed an efficient, experimentally flexible system that enables us to reversibly affect both abundant and nonabundant cardiomyocyte proteins. The use of bacterial-codon-based transactivators led to aberrant splicing, whereas other more efficient transactivators, by themselves, caused disease when expressed in the heart. The redesign of the system focused on developing stable transactivator-expressing lines in which expression was driven by the mouse ␣-myosin heavy chain promoter. A minimal responder locus was derived from the same promoter, in which the GATA sites and thyroid responsive elements responsible for robust cardiac specific expression were ablated, leading to an attenuated promoter that could be inducibly controlled. In all cases, whether activated or not, expression mimicked that of the parental promoter. By use of this system, an inducible expression of an abundant contractile protein, the atrial isoform of essential myosin light chain 1, and a powerful biological effector, glycogen synthase kinase-3␤ (GSK-3␤), were obtained. Subsequently, we tested the hypothesis that GSK-3␤ expression could reverse a preexisting hypertrophy. Inducible expression of GSK-3␤ could both attenuate a hypertrophic response and partially reverse a pressure-overload-induced hypertrophy. The system appears to be robust and can be used to temporally control high levels of cardiac-specific transgene expression.

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Receptors for bone morphogenetic proteins (BMPs), members of the transforming growth factor-␤ (TGF␤) superfamily, are persistently expressed during cardiac development, yet mice lacking type II or type IA BMP receptors die at gastrulation and cannot be used to assess potential later roles in creation of the heart. Here, we used a Cre͞lox system for cardiac myocyte-specific deletion of the type IA BMP receptor, ALK3. ALK3 was specifically required at mid-gestation for normal development of the trabeculae, compact myocardium, interventricular septum, and endocardial cushion. Cardiac muscle lacking ALK3 was specifically deficient in expressing TGF␤2, an established paracrine mediator of cushion morphogenesis. Hence, ALK3 is essential, beyond just the egg cylinder stage, for myocyte-dependent functions and signals in cardiac organogenesis. Bone morphogenetic proteins (BMPs), named for their firstdiscovered role in bone differentiation, mediate a diverse spectrum of developmental events, such as dorsal-ventral patterning, mesoderm specification, and the spacing of embryo implantation (1, 2). BMPs are postulated to mediate cardiac development in mammals, by extrapolation from the role of decapentaplegic, a related factor in Drosophila (3), their function in cardiac looping in fish (4) or frogs (5), and, especially, studies of cardiac myogenesis in avians (6, 7). Avian explant studies also implicate BMPs as an essential paracrine signal from myocardium adjacent to the endocardial cushion during the epithelialmesenchymal transformation and later events that ultimately give rise to the atrio-ventricular (AV) valves (8). This process is known to involve TGF␤2 and TGF␤3 as well (9-12), but the molecular connection between BMPs and TGF␤ is uncertain.Despite inferences from the distribution of BMPs during heart formation (13), and BMP effects on cardiac differentiation in teratocarcinoma cells (14), evidence analogous to that for other species is lacking in mammals themselves. One barrier to using conventional germline deletions to resolve these issues in mice is the greater potential for redundancy among BMP family members than in flies. In the mid-gestation heart, BMP2 and -4 are found, respectively, in myocardial layers of the AV canal and in the AV cushion itself, BMP5, -6, and -7 are initially homogeneous throughout the myocardium, and BMP10, which is heart-specific, is found exclusively in trabeculae (14,15). This diversity, with overlapping and nonoverlapping programs of expression, suggests the utility of addressing the question via BMP receptors, instead, which are smaller in number.BMPs bind two serine͞threonine kinase receptors, type II (BMPRII) and type I (ALK3͞BMPR-IA and ALK6͞BMPR-IB), which form a heteromeric signaling complex acting in series, as for other TGF␤ family receptors (16). In the presence of ligand, the type II receptor phosphorylates the type I receptors, which activate signaling by intracellular effectors including Smad transcription factors (16). ALK3 is ubiquitous throughout development, wherea...

During vertebrate limb development, positional information must be specified along three distinct axes. Although much progress has been made in our understanding of the molecular interactions involved in anterior-posterior and proximal-distal limb patterning, less is known about dorsal-ventral patterning. The genes Wnt-7a and Lmx-1, which are expressed in dorsal limb ectoderm and mesoderm, respectively, are thought to be important regulators of dorsal limb differentiation. Whether a complementary set of molecules controls ventral limb development has not been clear. Here we report that Engrailed-1, a homeodomain-containing transcription factor expressed in embryonic ventral limb ectoderm, is essential for ventral limb patterning. Loss of Engrailed-1 function in mice results in dorsal transformations of ventral paw structures, and in subtle alterations along the proximal-distal limb axis. Engrailed-1 seems to act in part by repressing dorsal differentiation induced by Wnt-7a, and is essential for proper formation of the apical ectodermal ridge.