Recent studies have postulated that distinct regulatory cascades control myogenic differentiation in the head and the trunk. However, although the tissues and signaling molecules that induce skeletal myogenesis in the trunk have been identified, the source of the signals that trigger skeletal muscle formation in the head remain obscure. Here we show that although myogenesis in the trunk paraxial mesoderm is induced by Wnt signals from the dorsal neural tube, myogenesis in the cranial paraxial mesoderm is blocked by these same signals. In addition, BMP family members that are expressed in both the dorsal neural tube and surface ectoderm are also potent inhibitors of myogenesis in the cranial paraxial mesoderm. We provide evidence suggesting that skeletal myogenesis in the head is induced by the BMP inhibitors, Noggin and Gremlin, and the Wnt inhibitor, Frzb. These molecules are secreted by both cranial neural crest cells and by other tissues surrounding the cranial muscle anlagen. Our findings demonstrate that head muscle formation is locally repressed by Wnt and BMP signals and induced by antagonists of these signaling pathways secreted by adjacent tissues. Vertebrate locomotion crucially depends on trunk skeletal muscles, which all derive from the segmented paraxial mesoderm termed somites (for review, see Christ and Ordahl 1995). During the past decade, the tissues and signaling molecules that induce the formation of skeletal muscle from somites have been intensively studied. These studies have indicated that somitic myogenesis in the trunk is affected by signals emanating from the axial tissues, the surface ectoderm, and the lateral plate mesoderm. Wnt family members expressed in the dorsal neural tube work together with Sonic hedgehog (Shh) expressed in the notochord to activate myogenic bHLH gene expression (that is, Myf-5 and MyoD) in the epaxial component of the myotome (Munsterberg et al. 1995;Stern et al. 1995;Tajbakhsh et al. 1998;Borycki et al. 2000;Gustafsson et al. 2002). In addition, Wnt signals from the dorsal ectoderm have been demonstrated to up-regulate the expression of MyoD in the hypaxial component of the myotome (Tajbakhsh et al. 1998). Furthermore, BMP signals from the lateral plate have been shown to delay the activation of myogenic bHLH gene expression in the hypaxial muscle progenitors relative to those that form the epaxial musculature (Pourquié et al. 1996). In contrast to our understanding of how skeletal muscle is induced in the trunk, the tissues and signaling pathways that induce the formation of skeletal muscle in the head have not yet been elucidated.In the vertebrate head, ∼40 skeletal muscles are present, which, instead of serving for locomotion, rather move the eye, control the cranial openings, or facilitate food uptake and, in humans, speech (for review, see Wachtler and Jacob 1986). Although the hypobranchial muscles, the tongue muscles, and the muscles of the posterior branchial arches (BAs), develop from the somites, the remainder of the head muscles (that is, the "genui...

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Parathyroid hormone-related protein (PTHrP) is essential to maintain a pool of dividing, immature chondrocytes in the growth plate of long bones. In chick and mouse, expression of Nkx3.2/Bapx1 in the growth plate is restricted to the proliferative zone and is downregulated as chondrocyte maturation begins. Nkx3.2/Bapx1 expression is lost in the growth plates of mice engineered to lack PTHrP signaling and, conversely, is maintained by ectopic expression of PTHrP in developing bones. Lettice et al., 1999;Tribioli and Lufkin, 1999). To explore the possibility that Nkx3.2/Bapx1 might negatively regulate chondrocyte maturation, but that this role may not have been revealed in Nkx3.2/Bapx1 deficient mice due to redundant/compensatory mechanisms that may modulate chondrocyte maturation in the growth plate, we have employed a gain-of-function approach in chick embryos to elucidate a potential role for Nkx3.2 in the regulation of chondrocyte maturation. MATERIALS AND METHODS Materials In situ hybridization (ISH)Details of the probes employed for ISH are available upon request. Frozen sections of embryonic chick tissue, and paraffin sections of mouse, were prepared exactly as described previously (Murtaugh et al., 1999). Nonradioactive section ISH, with digoxigenin (DIG)-labeled probes, was performed as described previously (Murtaugh et al., 2001). ISH with 35 Slabeled riboprobes and Hematoxylin and Eosin staining were performed as described previously (Chung et al., 1998). Retroviral misexpressionVirus preparation was as described elsewhere (Morgan and Fekete, 1996). Concentrated viral supernatant was injected into the nascent wing buds of late E3 [Hamburger-Hamilton (HH) stage 17-19] chick embryos. To ensure thorough infection, each wing bud was injected in several anteroposterior regions along its distal margin. Following injection, eggs were reincubated up to E8-E10, and processed for Alcian Blue and Alizarin Red staining (Murtaugh et al., 1999), or ISH as described above. Chick embryo explant culture, retroviral infection, and RT-PCR analysisDissection and culture of somitic or pre-somitic mesoderm (psm) explants was previously described (Munsterberg et al., 1995). Retrovirus infection was performed essentially as described by Zeng et al. (Zeng et al., 2002), with slight modifications (available upon request). Reverse transcriptase (RT) reactions and polymerase chain reaction (PCR) analysis were carried out as previously described (Munsterberg et al., 1995;Zeng et al., 2002). PCR conditions and primer sequences have either been previously published or are available on request. RESULTS Nkx3.2 expression is restricted to immature proliferative chondrocytes during endochondral ossification Although Nkx3.2 transcripts are initially expressed in all cartilaginous cells in the limb bud soon after mesenchymal condensation (Murtaugh et al., 2001), as chondrocyte maturation begins, expression of these transcripts becomes restricted to the distal portion of the developing cartilage elements (Fig. 1). AtHamburger-Hamilton (HH...

The physicochemical deposition of calcium-phosphate in the arterial wall is prevented by calcification inhibitors. Studies in cohorts of patients with rare genetic diseases have shed light on the consequences of loss-of-function mutations for different calcification inhibitors, and genetic targeting of these pathways in mice have generated a clearer picture on the mechanisms involved. For example, generalized arterial calcification of infancy (GACI) is caused by mutations in the enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (eNPP1), preventing the hydrolysis of ATP into pyrophosphate (PPi). The importance of PPi for inhibiting arterial calcification has been reinforced by the protective effects of PPi in various mouse models displaying ectopic calcifications. Besides PPi, Matrix Gla Protein (MGP) has been shown to be another potent calcification inhibitor as Keutel patients carrying a mutation in the encoding gene or Mgp-deficient mice develop spontaneous calcification of the arterial media. Whereas PPi and MGP represent locally produced calcification inhibitors, also systemic factors contribute to protection against arterial calcification. One such example is Fetuin-A, which is mainly produced in the liver and which forms calciprotein particles (CPPs), inhibiting growth of calcium-phosphate crystals in the blood and thereby preventing their soft tissue deposition. Other calcification inhibitors with potential importance for arterial calcification include osteoprotegerin, osteopontin, and klotho. The aim of the present review is to outline the latest insights into how different calcification inhibitors prevent arterial calcification both under physiological conditions and in the case of disturbed calcium-phosphate balance, and to provide a consensus statement on their potential therapeutic role for arterial calcification.