Identification of ubiquitin ligases required for skeletal muscle atrophy.

Skeletal muscle adapts to decreases in activity and load by undergoing atrophy. To identify candidate molecular mediators of muscle atrophy, we performed transcript profiling. Although many genes were up-regulated in a single rat model of atrophy, only a small subset was universal in all atrophy models. Two of these genes encode ubiquitin ligases: Muscle RING Finger 1 (MuRF1), and a gene we designate Muscle Atrophy F-box (MAFbx), the latter being a member of the SCF family of E3 ubiquitin ligases. Overexpression of MAFbx in myotubes produced atrophy, whereas mice deficient in either MAFbx or MuRF1 were found to be resistant to atrophy. These proteins are potential drug targets for the treatment of muscle atrophy.

Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development.

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.

The receptor tyrosine kinase MuSK is required for neuromuscular junction formation in vivo.

Formation of neuromuscular synapses requires a series of inductive interactions between growing motor axons and differentiating muscle cells, culminating in the precise juxtaposition of a highly specialized nerve terminal with a complex molecular structure on the postsynaptic muscle surface. The receptors and signaling pathways mediating these inductive interactions are not known. We have generated mice with a targeted disruption of the gene encoding MuSK, a receptor tyrosine kinase selectively localized to the postsynaptic muscle surface. Neuromuscular synapses do not form in these mice, suggesting a failure in the induction of synapse formation. Together with the results of an accompanying manuscript, our findings indicate that MuSK responds to a critical nerve-derived signal (agrin), and in turn activates signaling cascades responsible for all aspects of synapse formation, including organization of the postsynaptic membrane, synapse-specific transcription, and presynaptic differentiation.

Mice lacking the CNTF receptor, unlike mice lacking CNTF, exhibit profound motor neuron deficits at birth.

Ciliary neurotrophic factor (CNTF) supports motor neuron survival in vitro and in mouse models of motor neuron degeneration and was considered a candidate for the muscle-derived neurotrophic activity that regulates motor neuron survival during development. However, CNTF expression is very low in the embryo, and CNTF gene mutations in mice or human do not result in notable abnormalities of the developing nervous system. We have generated and directly compared mice containing null mutations in the genes encoding CNTF or its receptor (CNTFR alpha). Unlike mice lacking CNTF, mice lacking CNTFR alpha die perinatally and display severe motor neuron deficits. Thus, CNTFR alpha is critical for the developing nervous system, most likely by serving as a receptor for a second, developmentally important, CNTF-like ligand.

Neuronal deficits, not involving motor neurons, in mice lacking BDNF and/or NT4.

Nerve growth factor and other neurotrophins signal to neurons through the Trk family of receptor tyrosine kinases. TrkB is relatively promiscuous in vitro, acting as a receptor for brain-derived neurotrophic factor (BDNF), neurotrophin-4 (NT4) and, to a lesser extent, NT3 (refs 3-5). Mice lacking TrkB show a more severe phenotype than mice lacking BDNF, suggesting that TrkB may act as a receptor for additional ligands in vivo. To explore this possibility, we generated mice lacking NT4 or BDNF as well as mice lacking both neurotrophins. Unlike mice lacking other Trks or neurotrophins, NT4-deficient mice are long-lived and show no obvious neurological defects. Analysis of mutant phenotypes revealed distinct neuronal populations with different neurotrophin requirements. Thus vestibular and trigeminal sensory neurons require BDNF but not NT4, whereas nodose-petrosal sensory neurons require both BDNF and NT4. Motor neurons, whose numbers are drastically reduced in mice lacking TrkB, are not affected even in mice lacking both BDNF and NT4. These results suggest that another ligand, perhaps NT3, does indeed act on TrkB in vivo.

Ciliary neurotrophic factor maintains the pluripotentiality of embryonic stem cells.

Ciliary neurotrophic factor was discovered based on its ability to support the survival of ciliary neurons, and is now known to act on a variety of neuronal and glial populations. Two distant relatives of ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M, mimic ciliary neurotrophic factor with respect to its actions on cells of the nervous system. In contrast to ciliary neurotrophic factor, leukemia inhibitory factor and oncostatin M also display a broad array of actions on cells outside of the nervous system. The overlapping activities of leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor can be attributed to shared receptor components. The specificity of ciliary neurotrophic factor for cells of the nervous system results from the restricted expression of the alpha component of the ciliary neurotrophic factor receptor complex, which is required to convert a functional leukemia inhibitory factor/oncostatin M receptor complex into a ciliary neurotrophic factor receptor complex. The recent observation that the alpha component of the ciliary neurotrophic factor receptor complex is expressed by very early neuronal precursors suggested that ciliary neurotrophic factor may act on even earlier precursors, particularly on cells previously thought to be targets for leukemia inhibitory factor action. Here we show the first example of ciliary neurotrophic factor responsiveness in cells residing outside of the nervous system by demonstrating that embryonic stem cells express a functional ciliary neurotrophic factor receptor complex, and that ciliary neurotrophic factor is similar to leukemia inhibitory factor in its ability to maintain the pluripotentiality of these cells.

Regulation of mouse preimplantation development: inhibitory effect of the calmodulin antagonist W-7 on the first cleavage.

The calmodulin antagonist W-7 inhibits cleavage of 1-cell mouse embryos in a concentration-dependent manner. This inhibition is likely to be specific for a calmodulin-mediated process, since the less active congener W-5 does not inhibit cleavage when used at concentrations of W-7 that do. Concentrations of W-7 that inhibit cleavage and do not inhibit either the uptake or incorporation of [35S]methionine do inhibit [3H]thymidine incorporation; similar concentrations of W-5 do not inhibit [3H]thymidine incorporation. Consistent with W-7's ability to inhibit cleavage by inhibiting DNA synthesis is that addition of W-7 at later times that correspond with exit from S phase results in cleavage to the 2-cell stage. Although W-7 does inhibit cleavage of 1-cell embryos, it does not inhibit transcriptional activation, which occurs in the 2-cell embryo and is characterized by the synthesis of a group of proteins of Mr = 70,000. Results of these experiments suggest a role for calmodulin in the first cell cycle of the mouse embryo and provide another example in which zygotic gene activation is not dependent on progression through the first cell cycle.

Regulation of mouse preimplantation development: differential effects of CZB medium and Whitten's medium on rates and patterns of protein synthesis in 2-cell embryos.

In contrast to Whitten's medium, CZB medium, which lacks glucose but is supplemented with glutamine, supports development of 1-cell embryos beyond the 2-cell stage for embryos obtained from mice that exhibit the 2-cell block. The molecular basis for this effect, however, is not known. We report that correlated with the ability of CZB medium to support development beyond the 2-cell stage is an enhanced rate of total protein synthesis and higher level of synthesis of proteins that reflect activation of transcription of the embryonic genome when compared to embryos cultured in Whitten's medium +/- ethylenediaminetetraacetate (EDTA). The overall patterns of protein synthesis of embryos cultured in CZB or Whitten's medium +/- EDTA are similar, although the synthesis of the transcription-dependent proteins is markedly delayed in embryos cultured in Whitten's-EDTA. Last, there are no significant differences in the adenosine 5'-triphosphate (ATP) levels in 2-cell embryos cultured from the 1-cell stage in each of the media.

Regulation of mouse preimplantation development: inhibition of synthesis of proteins in the two-cell embryo that require transcription by inhibitors of cAMP-dependent protein kinase.

Perturbing the changes in protein phosphorylation accompanying the first cleavage can inhibit the appearance of a set of proteins whose synthesis is inhibited by alpha-amanitin (transcription-requiring proteins, TRPs) (W. T. Poueymirou and R. M. Schultz, 1987, Dev. Biol. 121, 489-498); synthesis of the TRPs is likely to represent activation of transcription of the embryonic genome that occurs at the 2-cell stage during mouse development. In the present study, we report the effects of three different inhibitors of the cAMP-dependent protein kinase, N-[2-(methylamino)ethyl]-5-isoquinoline-sulfonamide (H8), (Rp)-cAMPs, and protein kinase inhibitor (PKI), each of which inhibits the kinase by a different mechanism, on cleavage of 2-cell embryos and synthesis of the TRPs. Two-cell embryos possess PK-A activity, which is inhibited by each of these inhibitors. Both H8 and (Rp)-cAMPs inhibit cleavage of 2-cell embryos in a concentration-dependent manner; similar concentrations of H7, which is a less potent inhibitor of PK-A, do not inhibit cleavage. H8 and (Rp)-cAMPS inhibit in a concentration-dependent manner TRP synthesis, whereas higher concentrations of H7 are required to inhibit TRP synthesis. Microinjected PKI also inhibits synthesis of the TRPs. In addition, H8 inhibits the accumulation of translatable messenger RNAs that are likely to encode for the TRPs. Last, H8, but not H7, inhibits the phosphorylation of a phosphoprotein in 2-cell embryos. Results of these studies suggest a role for protein phosphorylation catalyzed by cAMP-dependent protein kinase in regulating transcription in the early mouse embryo.

Differential effects of activators of cAMP-dependent protein kinase and protein kinase C on cleavage of one-cell mouse embryos and protein synthesis and phosphorylation in one- and two-cell embryos.

Membrane-permeable cAMP analogs or elevation of intracellular cAMP by cyclic nucleotide phosphodiesterase (PDE) inhibitors activates cAMP-dependent protein kinase. Biologically active phorbol esters or diacylglycerol activate the calcium-, phospholipid-dependent protein kinase, protein kinase C (PK-C). We report that membrane-permeable cAMP analogs, PDE inhibitors, biologically active phorbol esters, or a synthetic diacylglycerol inhibited cleavage of 1-cell mouse embryos to the 2-cell stage. The cAMP analogs and PDE inhibitors were effective only when added prior to S of the first cell cycle, whereas PK-C activators inhibited cleavage when added up until late G2/M. The PDE inhibitor Ro 20 1724/1 inhibited both DNA and protein synthesis in 1-cell embryos, whereas the phorbol ester, 12-O-tetradecanoyl-phorbol-13 acetate, or alpha-amanitin did not. In addition, 1-cell embryos prevented from cleaving by PDE inhibitors did not show specific changes in the pattern of protein phosphorylation associated with the 2-cell embryo, whereas such changes occurred in 1-cell embryos inhibited from cleaving with PK-C activators. Transcription in the 2-cell embryo results in the synthesis of a specific set of proteins, which is inhibited by alpha-amanitin. Although treatment of 1-cell embryos with aphidicolin or PK-C activators during G1 did not inhibit the synthesis of these proteins, treatment with cAMP analogs or PDE inhibitors during G1 inhibited the appearance of these proteins. These results are discussed in terms of how the synthesis of transcription-dependent proteins in the 2-cell embryo may be regulated by protein phosphorylation.

Effects of protein kinase C activators on germinal vesicle breakdown and polar body emission of mouse oocytes.

Protein phosphorylation mediated by cAMP-dependent protein kinase is instrumental in maintaining meiotic arrest of mouse oocytes. To assess whether protein phosphorylation mediated by calcium/phospholipid-dependent protein kinase (protein kinase C) might also inhibit the resumption of meiosis, we treated oocytes with activators of this enzyme. The active phorbol esters 12-O-tetra-decanoyl phorbol-13-acetate (TPA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD) inhibited germinal vesicle breakdown (GVBD), as did a more natural activator of protein kinase, C, sn-1,2-dioctanoylglycerol (diC8). An inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD), did not inhibit GVBD. We then examined whether protein kinase C activators inhibit a step in the cAMP-modulated pathway that regulates resumption of meiosis. TPA did not inhibit the maturation-associated decrease in oocyte cAMP. Microinjected heat-stable protein inhibitor of cAMP-dependent protein kinase failed to induce GVBD in the presence of TPA. Both TPA and diC8 partially inhibited specific changes in oocyte phosphoprotein metabolism that are tightly correlated with resumption of meiosis; these agents also induced the apparent phosphorylation of specific oocyte proteins. These results suggest that protein kinase C activators may inhibit resumption of meiosis by acting distal to a decrease in cAMP-dependent protein kinase activity, but prior to changes in oocyte phosphoprotein metabolism that are presumably required for resumption of meiosis. Finally, we compared the effects of db-cAMP and protein kinase C activators on polar body emission following GVBD. TPA, 4 beta-PDD or diC8, but not 4 alpha-PDD or db-cAMP, inhibited polar body emission in a dose-dependent manner. The morphology and cytology of oocytes in which polar body emission was inhibited by TPA or 4 beta-PDD differed from that of oocytes treated with diC8. Thirty to 60% of the former were round in shape and exhibited a clump of chromosomes but no spindle; the remainder were distended in shape and exhibited a metaphase I spindle. All oocytes treated with diC8, however, were round, had dispersed chromosomes, and no spindle. These results suggest that, in contrast to resumption of meiosis, polar body emission is inhibited by activation of protein kinase C but not cAMP-dependent protein kinase.