Thomas M. DeChiara

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.

Agrin Acts via a MuSK Receptor Complex

Formation of th neuromuscular junction depends upon reciprocal inductive interactions between the developing nerve and muscle, resulting in the precise juxtaposition of a differentiated nerve terminal with a highly specialized patch on the muscle membrane, termed the motor endplate. Agrin is a nerve-derived factor that can induced molecular reorganizations at the motor endplate, but the mechanism of action of agrin remains poorly understood. MuSK is a receptor tyrosine kinase localized to the motor endplate, seemingly well positioned to receive a key nerve-derived signal. Mice lacking either agrin or MuSK have recently been generated and exhibit similarly profound defects in their neuromuscular junctions. Here we demonstrate that agrin acts via a receptor complex that includes MuSK as well as a myotube-specific accessory component.

High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis

One of the most effective approaches for determining gene function involves engineering mice with mutations or deletions in endogenous genes of interest. Historically, this approach has been limited by the difficulty and time required to generate such mice. We describe the development of a high-throughput and largely automated process, termed VelociGene, that uses targeting vectors based on bacterial artificial chromosomes (BACs). VelociGene permits genetic alteration with nucleotide precision, is not limited by the size of desired deletions, does not depend on isogenicity or on positive–negative selection, and can precisely replace the gene of interest with a reporter that allows for high-resolution localization of target-gene expression. We describe custom genetic alterations for hundreds of genes, corresponding to about 0.5–1.0% of the entire genome. We also provide dozens of informative expression patterns involving cells in the nervous system, immune system, vasculature, skeleton, fat and other tissues.*Note: In the author list of the AOP version of this article, the name of author Rostislav Chernomorsky was misspelled Rostislav Chernomorski. This has been corrected in the online and print versions of the article.

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.

Dominant mutations in ROR2 , encoding an orphan receptor tyrosine kinase, cause brachydactyly type B

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus was previously mapped to chromosome 9q22 within an interval of 7.5 cM (refs 9,10). Here we describe mutations in ROR2, which encodes the orphan receptor tyrosine kinase ROR2 (ref. 11), in three unrelated families with BDB1. We identified distinct heterozygous mutations (2 nonsense, 1 frameshift) within a 7–amino-acid segment of the 943–amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. We obtained further evidence for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse orthologue, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning.

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.

F0 generation mice fully derived from gene-targeted embryonic stem cells allowing immediate phenotypic analyses

A useful approach for exploring gene function involves generating mutant mice from genetically modified embryonic stem (ES) cells. Recent advances in genetic engineering of ES cells have shifted the bottleneck in this process to the generation of mice. Conventional injections of ES cells into blastocyst hosts produce F0 generation chimeras that are only partially derived from ES cells, requiring additional breeding to obtain mutant mice that can be phenotyped. The tetraploid complementation approach directly yields mice that are almost entirely derived from ES cells, but it is inefficient, works only with certain hybrid ES cell lines and suffers from nonspecific lethality and abnormalities, complicating phenotypic analyses. Here we show that laser-assisted injection of either inbred or hybrid ES cells into eight cell–stage embryos efficiently yields F0 generation mice that are fully ES cell–derived and healthy, exhibit 100% germline transmission and allow immediate phenotypic analysis, greatly accelerating gene function assignment.

Loss-of-Function Analysis of EphA Receptors in Retinotectal Mapping

EphA tyrosine kinases are thought to act as topographically specific receptors in the well-characterized projection map from the retina to the tectum. Here, we describe a loss-of-function analysis of EphA receptors in retinotectal mapping. Expressing patches of a cytoplasmically truncated EphA3 receptor in chick retina caused temporal axons to have reduced responsiveness to posterior tectal repellent activity in vitro and to shift more posteriorly within the map in vivo. A gene disruption of mouse EphA5, replacing the intracellular domain with β-galactosidase, reduced in vitro responsiveness of temporal axons to posterior target membranes. It also caused map abnormalities in vivo, with temporal axons shifted posteriorly and nasal axons anteriorly, but with the entire target still filled by retinal axons. The anterior shift of nasal axons was not accompanied by increased responsiveness to tectal repellent activity, in contrast to the comparable anterior shift in ephrin-A knock-outs, helping to resolve a previous ambiguity in interpreting the ephrin gene knock-outs. The results show the functional requirement for endogenous EphA receptors in retinotectal mapping, show that the receptor intracellular domain is required for a forward signaling response to topographic cues, and provide new evidence for a role of axon competition in topographic mapping.

Distinct phenotypes of mutant mice lacking agrin, MuSK, or rapsyn.

Differentiation of the postsynaptic membrane at the neuromuscular junction requires agrin, a nerve-derived signal; MuSK, a critical component of the agrin receptor in muscle; and rapsyn, a protein that interacts with acetylcholine receptors (AChRs). We showed previously that nerve-induced AChR aggregation is dramatically impaired in knockout mice lacking agrin, MuSK, or rapsyn. However, the phenotypes of these mutants differed in several respects, suggesting that the pathway from agrin to MuSK to rapsyn is complex. Here, we compared the effects of these mutations on two aspects of synaptic differentiation: AChR clustering and transcriptional specialization of synapse-associated myonuclei. First, we show that a plant lectin, VVA-B4, previously shown to act downstream of agrin, can induce AChR clusters on MuSK-deficient but not rapsyn-deficient myotubes in culture. Thus, although both MuSK and rapsyn are required for AChR clustering in vivo, only rapsyn is essential for cluster formation per se. Second, we show that neuregulin, a nerve-derived inducer of AChR gene expression, activates AChR gene expression in cultured agrin- and MuSK-deficient myotubes, even though synapse-specific transcriptional specialization is disrupted in agrin and MuSK mutants in vivo. We propose that agrin works through MuSK to determine a synaptogenic region within which synaptic differentiation occurs.

Conditionals by inversion provide a universal method for the generation of conditional alleles

Significance We describe conditional by inversion (COIN), a new design for conditional alleles that uses an optimized conditional gene trap module (COIN module) inserted into the target gene in an orientation opposite to the gene’s direction of transcription. Activation by Cre recombinase inverts the COIN module, resulting in expression of a reporter and termination of transcription, thereby inactivating the target gene while marking the cells where the conditional event has occurred. Creation of COIN alleles for more than 20 genes showed that it is a robust and universal method—applicable to any gene regardless of exon–intron structure—that overcomes the limitations of previous conditional approaches. Conditional mutagenesis is becoming a method of choice for studying gene function, but constructing conditional alleles is often laborious, limited by target gene structure, and at times, prone to incomplete conditional ablation. To address these issues, we developed a technology termed conditionals by inversion (COIN). Before activation, COINs contain an inverted module (COIN module) that lies inertly within the antisense strand of a resident gene. When inverted into the sense strand by a site-specific recombinase, the COIN module causes termination of the target gene’s transcription and simultaneously provides a reporter for tracking this event. COIN modules can be inserted into natural introns (intronic COINs) or directly into coding exons as part of an artificial intron (exonic COINs), greatly simplifying allele design and increasing flexibility over previous conditional KO approaches. Detailed analysis of over 20 COIN alleles establishes the reliability of the method and its broad applicability to any gene, regardless of exon–intron structure. Our extensive testing provides rules that help ensure success of this approach and also explains why other currently available conditional approaches often fail to function optimally. Finally, the ability to split exons using the COIN’s artificial intron opens up engineering modalities for the generation of multifunctional alleles.