MuSK is a BMP co-receptor that shapes BMP responses and calcium signaling in muscle cells.

Bone morphogenetic proteins (BMPs) function in most tissues but have cell type-specific effects. Given the relatively small number of BMP receptors, this exquisite signaling specificity requires additional molecules to regulate this pathway's output. The receptor tyrosine kinase MuSK (muscle-specific kinase) is critical for neuromuscular junction formation and maintenance. Here, we show that MuSK also promotes BMP signaling in muscle cells. MuSK bound to BMP4 and related BMPs with low nanomolar affinity in vitro and to the type I BMP receptors ALK3 and ALK6 in a ligand-independent manner both in vitro and in cultured myotubes. High-affinity binding to BMPs required the third, alternatively spliced MuSK immunoglobulin-like domain. In myoblasts, endogenous MuSK promoted BMP4-dependent phosphorylation of SMADs and transcription of Id1, which encodes a transcription factor involved in muscle differentiation. Gene expression profiling showed that MuSK was required for the BMP4-induced expression of a subset of genes in myoblasts, including regulator of G protein signaling 4 (Rgs4). In myotubes, MuSK enhanced the BMP4-induced expression of a distinct set of genes, including transcripts characteristic of slow muscle. MuSK-mediated stimulation of BMP signaling required type I BMP receptor activity but was independent of MuSK tyrosine kinase activity. MuSK-dependent expression of Rgs4 resulted in the inhibition of Ca(2+) signaling induced by the muscarinic acetylcholine receptor in myoblasts. These findings establish that MuSK has dual roles in muscle cells, acting both as a tyrosine kinase-dependent synaptic organizing molecule and as a BMP co-receptor that shapes BMP transcriptional output and cholinergic signaling.

The mouse hairy ears mutation exhibits an extended growth (anagen) phase in hair follicles and altered Hoxc gene expression in the ears.

The mouse In(15)2Rl (hairy ears, Eh) mutation is a paracentric inversion of the distal half of chromosome 15 (Chr 15). Heterozygous Eh/+ mice display misshaped and hairy ears that have more and longer hair than the ears of their wild-type littermates. We mapped, cloned and sequenced both inversion breakpoints. No protein-coding transcript was disrupted by either breakpoint. The proximal breakpoint is located between syntrophin basic 1 (Sntb1) and hyaluronan synthase 2 (Has2), and the distal breakpoint maps between homeobox C4 (Hoxc4) and single-strand selective monofunctional uracil DNA glycosylase (Smug1), near the middle and the telomere ends of Chr 15, respectively. The inversion spans ~47 megabases. Our genetic analysis suggests that the hairy-ear phenotype is caused by the proximal breakpoint of the inversion-bearing Chr 15. Quantitative RNA analysis by real-time polymerase chain reaction for the genes flanking the breakpoint indicated no changes in expression levels except for some homeobox C (Hoxc) genes whose expression was elevated in developing and mature skin of the ears but not of other body regions. The increased hair length on the ears of Eh/+ mice was due to an extension of the anagen stage in the hair cycle, as determined by histological analysis. Our data indicate that the Eh phenotype arises from mis-expression of Hoxc genes.

A position effect on TRPS1 is associated with Ambras syndrome in humans and the Koala phenotype in mice.

Ambras syndrome (AS) is a rare form of congenital hypertrichosis with excessive hair on the shoulders, face and ears. Cytogenetic studies have previously implicated an association with rearrangements of chromosome 8. Here we define an 11.5 Mb candidate interval for AS on chromosome 8q based on cytogenetic breakpoints in three patients. TRPS1, a gene within this interval, was deleted in a patient with an 8q23 chromosomal rearrangement, while its expression was significantly downregulated in another patient with an inversion breakpoint 7.3 Mb downstream of TRPS1. Here, we describe the first potential long-range position effect on the expression of TRPS1. To gain insight into the mechanisms by which Trps1 affects the hair follicle, we performed a detailed analysis of the hair abnormalities in Koa mice, a mouse model of hypertrichosis. We found that the proximal breakpoint of the Koa inversion is located 791 kb upstream of Trps1. Quantitative real-time polymerase chain reaction, in situ hybridization and immunofluorescence analysis revealed that Trps1 expression levels are reduced in Koa mutant mice at the sites of pathology for the phenotype. We determined that the Koa inversion creates a new Sp1 binding site and translocates additional Sp1 binding sites within a highly conserved stretch spanning the proximal breakpoint, providing a potential mechanism for the position effect. Collectively, these results describe a position effect that downregulates TRPS1 expression as the probable cause of hypertrichosis in AS in humans and the Koa phenotype in mice.

X-ray-induced deletion complexes in embryonic stem cells on mouse chromosome 15.

Chromosomal deletions have long been used as genetic tools in dissecting the functions of complex genomes, and new methodologies are still being developed to achieve the maximum coverage. In the mouse, where the chromosomal deletion coverage is far less extensive than that in Drosophila, substantial coverage of the genome with deletions is strongly desirable. This article reports the generation of three deletion complexes in the distal part of mouse Chromosome (Chr) 15. Chromosomal deletions were efficiently induced by X rays in embryonic stem (ES) cells around the Otoconin 90 (Oc 90), SRY-box-containing gene 10 (Sox 10), and carnitine palmitoyltransferase 1b (Cpt 1 b) loci. Deletions encompassing the Oc 90 and Sox 10 loci were transmitted to the offspring of the chimeric mice that were generated from deletion-bearing ES cells. Whereas deletion complexes encompassing the Sox 10 and the Cpt 1 b loci overlap each other, no overlap of the Oc 90 complex with the Sox 10 complex was found, possibly indicating the existence of a haploinsufficient gene located between Oc 90 and Sox 10. Deletion frequency and size induced by X rays depend on the selective locus, possibly reflecting the existence of haplolethal genes in the vicinity of these loci that yield fewer and smaller deletions. Deletions induced in ES cells by X rays vary in size and location of breakpoints, which makes them desirable for mapping and for functional genomics studies.

Modification of an existing chromosomal inversion to engineer a balancer for mouse chromosome 15.

Chromosomal inversions are valuable genetic tools for mutagenesis screens, where appropriately marked inversions can be used as balancer chromosomes to recover and maintain mutations in the corresponding chromosomal region. For any inversion to be effective as a balancer, it should exhibit both dominant and recessive visible traits; ideally the recessive trait should be a fully penetrant lethality in which inversion homozygotes die before birth. Unfortunately, most inversions recovered by classical radiation or chemical mutagenesis techniques do not have an overt phenotype in either the heterozygous or the homozygous state. However, they can be modified by relatively simple procedures to make them suitable as an appropriately marked balancer. We have used homologous recombination to modify, in embryonic stem cells, the recessive-lethal In(15)21Rk inversion to endow it with a dominant-visible phenotype. Several ES cell lines were derived from inversion heterozygotes, and a keratin-14 (K14) promoter-driven agouti minigene was introduced onto the inverted chromosome 15 in the ES cells by gene targeting. Mice derived from the targeted ES cells carry the inverted chromosome 15 and, at the same time, exhibit lighter coat color on their ears and tails, making this modified In(15)21Rk useful as a balancer for proximal mouse chromosome 15.

Overlapping deletions define novel embryonic lethal loci in the mouse t complex.

SUMMARY: The t complex region of mouse chromosome 17 contains genetic information critical for embryonic development. To identify and map loci required for normal embryogenesis, a set of overlapping deletions (D17Aus9(df10J), D17Aus9(df12J), and D17Aus9(df13J)) surrounding the D17Aus9 locus and one encompassing the T locus, Del(17)T(7J), were bred in various combinations and the consequences of nullizygosity in overlapping regions were examined. The results indicated that there are at least two functional units within 1 cM of D17Aus9. l17J1 is a peri-implantation lethal mutation within the region deleted in D17Aus9(df13J), whereas l17J2 is a later-acting lethal defined by the region of overlap between Del(17)T(7J) and D17Aus9(df12J). Del(17)T(7J)/D17Aus9(df12J) embryos die around 10.5 dpc. The development of the mutant embryos is characterized by lack of axial rotation, an abnormal notochord structure, and a ballooning pericardium. These studies demonstrate the value of overlapping deletion complexes, as opposed to individual deletion complexes, for the identification, mapping, and analysis of genes required for embryonic development.