Mice lacking alpha-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the Central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated alphaCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking alpha CGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction in morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in alphaCGRP-deficient animals. These results suggest that alphaCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Development of the neuromuscular junction: genetic analysis in mice.

Formation of the skeletal neuromuscular junction is a multi-step process that requires communication between the nerve and muscle. Studies in many laboratories have led to identification of factors that seem likely to mediate these interactions. 'Knock-out' mice have now been generated with mutations in several genes that encode candidate transsynaptic messengers and components of their effector mechanisms. Using these mice, it is possible to test hypotheses about the control of synaptogenesis. Here, we review our studies on neuromuscular development in mutant mice lacking agrin alpha CGRP, rapsyn, MuSK, dystrophin, dystrobrevin, utrophin, laminin alpha 5, laminin beta 2, collagen alpha 3 (IV), the acetylcholine receptor epsilon subunit, the collagenous tail of acetylcholinesterase, fibroblast growth factor-5, the neural cell adhesion molecule, and tenascin-C.

Organization and reorganization of neuromuscular junctions in mice lacking neural cell adhesion molecule, tenascin-C, or fibroblast growth factor-5.

Many proteins have been hypothesized to mediate intercellular interactions that regulate the formation, maturation, and maintenance of the skeletal neuromuscular junction. Three of the best characterized of these are a membrane-associated adhesion molecule, neural cell adhesion molecule (N-CAM), an extracellular matrix component, tenascin-C, and a soluble growth factor, fibroblast growth factor-5 (FGF-5). To assess the roles of these molecules in synaptogenesis in vivo, we examined neuromuscular junctions in homozygous mutant mice lacking N-CAM, tenascin-C, FGF-5, or both N-CAM and tenascin-C. End plates were 14% smaller in N-CAM-deficient mice than in controls, and formation of junctional folds was delayed in this mutant. In all other respects tested, however, the structure and molecular architecture of neuromuscular junctions were normal in all three single mutants and in the double mutant. We also tested the abilities of damaged motor axons to reinnervate mutant muscle after axotomy and of intact motor axons to sprout after partial denervation. Again, no significant differences among genotypes were observed. Together, these results demonstrate that N-CAM, tenascin-C, and FGF-5 are dispensable for major aspects of synaptic development and regeneration.

Rapsyn is required for MuSK signaling and recruits synaptic components to a MuSK-containing scaffold.

Agrin-induced clustering of acetylcholine receptors (AChRs) in the postsynaptic membrane is a key step in synaptogenesis at the neuromuscular junction. The receptor tyrosine kinase MuSK is a component of the agrin receptor, while the cytoplasmic protein rapsyn is necessary for the clustering of AChRs and all other postsynaptic membrane components studied to date. We show here that MuSK remains concentrated at synaptic sites in rapsyn-deficient mutant mice, suggesting that MuSK forms a primary structural scaffold to which rapsyn attaches other synaptic components. Using nonmuscle cells, we show that rapsyn-MuSK interactions are mediated by the ectodomain of MuSK, suggesting the existence of a transmembrane intermediate. In addition to rapsyn's structural role, we demonstrate that it is required for an early step in MuSK signaling, AChR phosphorylation. This signaling requires the kinase domain of MuSK, but not its ectodomain. Thus, MuSK may interact with rapsyn in multiple ways to play both structural and signaling roles in agrin-induced differentiation.

Defective neuromuscular synaptogenesis in agrin-deficient mutant mice.

During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.

Pre-Main-Sequence Star Candidates in the Bar of the Large Magellanic Cloud

Candidate pre-main-sequence stars were observed in the bar of the Large Magellanic Cloud during the search for dark matter in the galactic halo. Seven blue stars of apparent visual magnitude 15 to 17 had irregular photometric variations and hydrogen emission lines in their optical spectra, which suggested that these stars are pre-main-sequence stars of about 10 solar masses. These stars are slightly more massive and definitely more luminous than are Herbig AeBe pre-main-sequence stars in our own galaxy. Continued observations of these very young stars from another galaxy, which are probably at the pre-hydrogen-burning stage, should provide important clues about early stages of star formation.

Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, ErbB2 and ErbB3, in developing mammalian muscle.

Developing motor axons induce synaptic specializations in muscle fibers, including preferential transcription of acetylcholine receptor (AChR) subunit genes by subsynaptic nuclei. One candidate nerve-derived signaling molecule is AChR-inducing activity (ARIA)/heregulin, a ligand of the erbB family of receptor tyrosine kinases. Here, we asked whether ARIA and erbB kinases are expressed in patterns compatible with their proposed signaling roles. In developing muscle, ARIA was present not only at synaptic sites, but also in extrasynaptic regions of the muscle fiber. ARIA was synthesized, rather than merely taken up, by muscle cells, as indicated by the presence of ARIA mRNA in muscle and of ARIA protein in a clonal muscle cell line. ARIA-responsive myotubes expressed both erbB2 and erbB3, but little EGFR/erbB1 or erbB4. In adults, erbB2 and erbB3 were localized to the postsynaptic membrane. ErbB3 was restricted to the postsynaptic membrane perinatally, at a time when ARIA was still broadly distributed. Thus, our data are consistent with a model in which ARIA interacts with erbB kinases on the muscle cell surface to provide a local signal that induces synaptic expression of AChR genes. However, much of the ARIA is produced by muscle, not nerve, and the spatially restricted response may result from the localization of erbB kinases as well as of ARIA. Finally, we show that erbB3 is not concentrated at synaptic sites in mutant mice that lack rapsyn, a cytoskeletal protein required for AChR clustering, suggesting that pathways for synaptic AChR expression and clustering interact.

Regulation of the acetylcholine receptor epsilon subunit gene by recombinant ARIA: an in vitro model for transynaptic gene regulation.

Structural specialization of the postsynaptic skeletal muscle membrane is in part mediated by the motor neuron-induced transcriptional regulation of synaptic muscle nuclei. ARIA, a factor that stimulates production of acetylcholine receptors (AChRs), is a candidate signaling molecule for such regulation. Here we examine the transynaptic inducing potential of this polypeptide factor. ARIA immunoreactivity is detectable at synaptic sites in vivo. In vitro, recombinant heregulin beta 1 (rHRG beta 1), the human homolog of ARIA, induces expression of the AChR epsilon gene, the subunit most sensitive to synaptic input. The inducing property of rHRG beta 1 is demonstrated most dramatically in primary muscle cultures from transgenic mice bearing an epsilon promoter-nuclear lacZ reporter transgene. Transient transfection experiments using the Sol 8 muscle cell line indicate that sequences that confer responsiveness to ARIA are located within a 150 bp epsilon subunit promoter region and are E box-independent. These results suggest that ARIA performs a vital role by directing spatially restricted gene expression at the neuromuscular junction.

Expression of four immunoglobulin superfamily adhesion molecules (L1, Nr-CAM/Bravo, neurofascin/ABGP, and N-CAM) in the developing mouse spinal cord.

To identify cell adhesion molecules (CAMs) expressed by mammalian motoneurons, we applied the polymerase chain reaction to a murine motor neuron-like cell line, NSC-34. Using primers derived from a group of L1-related CAMs, we cloned two alternatively spliced forms of mouse L1, which differ by a 12-base-pair insert, plus putative murine orthologs of the chicken cell adhesion molecules Nr-CAM/Bravo and neurofascin. All four mRNAs are expressed in NSC-34 cells, but only neurofascin and the insert-minus form of L1 are expressed in its neuroblastoma parent, N18TG2. Analysis of RNA in neonatal tissues reveals expression largely restricted to the brain and spinal cord. In situ hybridization histochemistry of spinal cord shows that motoneurons express L1, Nr-CAM, and neurofascin as well as N-CAM. L1 and N-CAM RNAs are detected throughout the period studied (from embryonic day [E]11 to postnatal day [P]28), whereas Nr-CAM is expressed only at early ages (< E15) and neurofascin is predominantly expressed postnatally. Moreover, each CAM is expressed by distinct subsets of neighboring cells and at distinct times. For example, Nr-CAM mRNA is present in floor plate cells of embryonic spinal cord, whereas neurofascin is expressed by a subset of glia postnatally. Finally, we show that each CAM has a distinct spatiotemporal pattern of expression in dorsal root ganglia.

N-CAM, 43K-rapsyn, and S-laminin mRNAs are concentrated at synaptic sites in muscle fibers.

Several components of the postsynaptic apparatus are found highly concentrated at the motor endplate. Studies of the acetylcholine receptor have shown that selective transcription of its genes by synaptic nuclei contributes to its synaptic accumulation. We used the method of in situ hybridization to study the distribution of mRNAs encoding three other proteins localized to the motor endplate. We found preferential synaptic accumulation of mRNAs for a membrane-associated cell adhesion molecule (N-CAM) and for an acetylcholine receptor-associated cytoskeletal protein (43K-rapsyn). In contrast, RNAs encoding proteins present throughout the muscle were distributed all along the muscle fiber. RNA encoding a protein concentrated in synaptic basal lamina, s-laminin (laminin beta 2), was intermediate in distribution, detectable extrasynaptically but more abundant synaptically. Our data suggest that selective transcription by synaptic nuclei is a general mechanism that contributes to the concentration of specific proteins in the postsynaptic apparatus at the neuromuscular junction.