Blocking extracellular activation of myostatin as a strategy for treating muscle wasting.

Many growth factors are intimately bound to the extracellular matrix, with regulated processing and release leading to cellular stimulation. Myostatin and GDF11 are closely related members of the TGFß family whose activation requires two proteolytic cleavages to release the growth factor from the prodomain. Specific modulation of myostatin and GDF11 activity by targeting growth factor-receptor interactions has traditionally been challenging. Here we demonstrate that a novel strategy for blocking myostatin and GDF11, inhibition of growth factor release, specifically and potently inhibits signaling both in vitro and in vivo. We developed human monoclonal antibodies that selectively bind the myostatin and GDF11 precursor forms, including a subset that inhibit myostatin proteolytic activation and prevent muscle atrophy in vivo. The most potent myostatin activation-blocking antibodies promoted robust muscle growth and resulted in significant gains in muscle performance in healthy mice. Altogether, we show that blocking the extracellular activation of growth factors is a potent method for preventing signaling, serving as proof of concept for a novel therapeutic strategy that can be applied to other members of the TGFß family of growth factors.

Vertebrate eye development as modeled in Drosophila.

Pax6, a member of the paired-box family of transcription factors, is critical for oculogenesis in both vertebrates and insects. Identification of potential vertebrate Pax6 targets has been guided by studies in Drosophila, where the Pax6 homologs eyeless ( ey ) and twin of eyeless ( toy ) function within a network of genes that synergistically pattern the developing fly eye. These targets, which share homology with the fly genes sine oculis, eyes absent and dachshund, exist in mice and humans as the Six, Eya and Dach gene families. Members of these gene families are present in the developing vertebrate eye, and preliminary studies suggest that they may function in a network analogous to that in the fly. Thus, despite radically different architecture, a similar molecular scaffold underlies both vertebrate and fly eye patterning, suggesting that the considerable power of Drosophila genetics can be harnessed to study mammalian ocular development.

BMP7 acts in murine lens placode development.

Targeted inactivation of the Bmp7 gene in mouse leads to eye defects with late onset and variable penetrance (A. T. Dudley et al., 1995, Genes Dev. 9, 2795-2807; G. Luo et al., 1995, Genes Dev. 9, 2808-2820). Here we report that the expressivity of the Bmp7 mutant phenotype markedly increases in a C3H/He genetic background and that the phenotype implicates Bmp7 in the early stages of lens development. Immunolocalization experiments show that BMP7 protein is present in the head ectoderm at the time of lens placode induction. Using an in vitro culture system, we demonstrate that addition of BMP7 antagonists during the period of lens placode induction inhibits lens formation, indicating a role for BMP7 in lens placode development. Next, to integrate Bmp7 into a developmental pathway controlling formation of the lens placode, we examined the expression of several early lens placode-specific markers in Bmp7 mutant embryos. In these embryos, Pax6 head ectoderm expression is lost just prior to the time when the lens placode should appear, while in Pax6-deficient (Sey/Sey) embryos, Bmp7 expression is maintained. These results could suggest a simple linear pathway in placode induction in which Bmp7 functions upstream of Pax6 and regulates lens placode induction. At odds with this interpretation, however, is the finding that expression of secreted Frizzled Related Protein-2 (sFRP-2), a component of the Wnt signaling pathway which is expressed in prospective lens placode, is absent in Sey/Sey embryos but initially present in Bmp7 mutants. This suggests a different model in which Bmp7 function is required to maintain Pax6 expression after induction, during a preplacodal stage of lens development. We conclude that Bmp7 is a critical component of the genetic mechanism(s) controlling lens placode formation.

Expression of the Kallmann syndrome gene in human fetal brain and in the manipulated chick embryo.

Kallmann syndrome is an inherited disorder characterized by an abnormality in olfactory system development. The gene for the X-linked form of this disorder (KAL) maps to Xp22.3 and encodes a protein sharing homologies with molecules involved in neuronal migration and axonal pathfinding. Here we report the expression pattern of the KAL gene in various parts of the human fetal brain. We found KAL transcripts in granule cells of the olfactory bulb and the cerebellum, in the dorsomedial thalamus and in the developing cerebral cortex. To determine whether or not signals from the olfactory nerve are required for KAL expression in the olfactory bulb, we analyzed chick embryos in which the olfactory placode was surgically removed. Those embryos lacking an olfactory nerve had a histologically abnormal bulb which nevertheless expressed the KAL gene at high levels. These findings indicate that, while the development of the proper cytoarchitecture of the olfactory bulb requires the innervation by olfactory axons, the expression of KAL is independent of such developmental processes.

The expression pattern of the chick homeobox gene gMHox suggests a role in patterning of the limbs and face and in compartmentalization of somites.

MHox is a homeodomain protein that binds an essential element in the core of the muscle creatine kinase enhancer. In the mouse embryo, MHox expression is restricted to mesenchymal cells; in adult mice the gene is highly expressed in skeletal and cardiac muscle. To further define the functions of MHox during embryogenesis, we have cloned its chicken homolog, termed gMHox, and analyzed its properties and detailed expression patterns. Our studies show that the amino acid sequence and DNA-binding properties of the avian and murine gene products are very similar. Furthermore, the sites of expression are alike with high levels of expression in the splanchnic mesoderm, in the somatic mesoderm, in the limb bud mesoderm, in the dermatome and in the dermis, and in the ectomesenchyme of the face. gMHox became downregulated as chondrogenesis proceeded, whereas its expression was maintained in perichondrium and undifferentiated mesenchymal cells beneath the surface ectoderm. Such a pattern of expression suggests that gMHox may participate in maintenance of mesenchymal cell lineages derived from both mesoderm and the neural crest and in patterning of the limbs and the face. Removal of the surface ectoderm overlying the somites has no visible effect on the architecture of somites but results in the failure of gMHox to be expressed in the underlying dermatome, suggesting that regulation of gMHox expression in these cells is dependent on cues emanating from the surface ectoderm.

Developmental regulation of the orphan receptor COUP-TF II gene in spinal motor neurons.

Members of the steroid/thyroid hormone receptor superfamily are involved in the control of cell identity and of pattern formation during embryonic development. Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) can act as regulators of various steroid/thyroid hormone receptor pathways. To begin to study the role of COUP-TFs during embryogenesis, we cloned a chicken COUP-TF (cCOUP-TF II) which is highly homologous to human COUP-TF II. Northern analysis revealed high levels of cCOUP-TF II transcripts during organogenesis. Nuclear extracts from whole embryos and from embryonic spinal cords were used in electrophoretic mobility shift assays. These assays showed that COUP-TF protein is present in these tissues and is capable of binding to a COUP element (a direct repeat of AGGTCA with one base pair spacing). Analysis of cCOUP-TF expression by in situ hybridization revealed high levels of cCOUP-TF II mRNA in the developing spinal motor neurons. Since the ventral properties of the spinal cord, including the development of motor neurons, is in part established by inductive signals from the notochord, we transplanted an additional notochord next to the dorsal region of the neural tube in order to induce ectopic motor neurons. We observed that an ectopic notochord induced cCOUP-TF II gene expression in the dorsal spinal cord in a region coextensive with ectopic domains of SC1 and Islet-1, two previously identified motor neuron markers. Collectively, our studies raise the possibility that cCOUP-TF II is involved in motor neuron development.

Expression pattern of the Kallmann syndrome gene in the olfactory system suggests a role in neuronal targeting.

Kallmann syndrome is a genetic disorder characterized by a defect in olfactory system development, which appears to be due to an abnormality in the migration of olfactory axons and gonadotropin releasing hormone (Gn-RH) producing neurons. The X-linked Kallmann syndrome gene shares significant similarities with molecules involved in neural development. We have now isolated the evolutionarily conserved chicken homologue of the Kallmann gene. In the developing and adult chicken, high levels of expression were found in the mitral cells of the olfactory bulb (the target of olfactory axons) and in the Purkinje cells of the cerebellar cortex, both areas affected in patients with Kallmann syndrome. We propose a model in which the Kallmann syndrome gene product is a signal molecule required for neuronal targeting throughout life.