William Poueymirou

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.

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.

Conditional Activation of Akt in Adult Skeletal Muscle Induces Rapid Hypertrophy

ABSTRACT Skeletal muscle atrophy is a severe morbidity caused by a variety of conditions, including cachexia, cancer, AIDS, prolonged bedrest, and diabetes. One strategy in the treatment of atrophy is to induce the pathways normally leading to skeletal muscle hypertrophy. The pathways that are sufficient to induce hypertrophy in skeletal muscle have been the subject of some controversy. We describe here the use of a novel method to produce a transgenic mouse in which a constitutively active form of Akt can be inducibly expressed in adult skeletal muscle and thereby demonstrate that acute activation of Akt is sufficient to induce rapid and significant skeletal muscle hypertrophy in vivo, accompanied by activation of the downstream Akt/p70S6 kinase protein synthesis pathway. Upon induction of Akt in skeletal muscle, there was also a significant decrease in adipose tissue. These findings suggest that pharmacologic approaches directed toward activating Akt will be useful in inducing skeletal muscle hypertrophy and that an increase in lean muscle mass is sufficient to decrease fat storage.

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.

Efficient differentiation and function of human macrophages in humanized CSF-1 mice

Humanized mouse models are useful tools to understand pathophysiology and to develop therapies for human diseases. While significant progress has been made in generating immunocompromised mice with a human hematopoietic system, there are still several shortcomings, one of which is poor human myelopoiesis. Here, we report that human CSF-1 knockin mice show augmented frequencies and functions of human myeloid cells. Insertion of human CSF1 into the corresponding mouse locus of Balb/c Rag2(-/-) γc(-/-) mice through VELOCIGENE technology resulted in faithful expression of human CSF-1 in these mice both qualitatively and quantitatively. Intra-hepatic transfer of human fetal liver derived hematopoietic stem and progenitor cells (CD34(+)) in humanized CSF-1 (CSF1(h/h)) newborn mice resulted in more efficient differentiation and enhanced frequencies of human monocytes/macrophages in the bone marrow, spleens, peripheral blood, lungs, liver and peritoneal cavity. Human monocytes/macrophages obtained from the humanized CSF-1 mice show augmented functional properties including migration, phagocytosis, activation and responses to LPS. Thus, humanized mice engineered to express human cytokines will significantly help to overcome the current technical challenges in the field. In addition, humanized CSF-1 mice will be a valuable experimental model to study human myeloid cell biology.

Mice with megabase humanization of their immunoglobulin genes generate antibodies as efficiently as normal mice

Significance The accompanying paper describes the precise, in situ replacement of six megabases of mouse immune genes with the corresponding human immune genes. This manuscript shows that this genomic engineering feat resulted in a unique kind of “HumAb” mouse. Dubbed VelocImmune, these mice efficiently generate antibodies that can be rapidly reformatted into therapeutics. VelocImmune mice have proven to be extraordinarily efficient and productive, generating over a dozen therapeutic candidates that have already progressed into human clinical trials for a variety of important diseases. Mice genetically engineered to be humanized for their Ig genes allow for human antibody responses within a mouse background (HumAb mice), providing a valuable platform for the generation of fully human therapeutic antibodies. Unfortunately, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which their genetic humanization was carried out. Heretofore, HumAb mice have been generated by disrupting the endogenous mouse Ig genes and simultaneously introducing human Ig transgenes at a different and random location; KO-plus-transgenic humanization. As we describe in the companion paper, we attempted to make mice that more efficiently use human variable region segments in their humoral responses by precisely replacing 6 Mb of mouse Ig heavy and kappa light variable region germ-line gene segments with their human counterparts while leaving the mouse constant regions intact, using a unique in situ humanization approach. We reasoned the introduced human variable region gene segments would function indistinguishably in their new genetic location, whereas the retained mouse constant regions would allow for optimal interactions and selection of the resulting antibodies within the mouse environment. We show that these mice, termed VelocImmune mice because they were generated using VelociGene technology, efficiently produce human:mouse hybrid antibodies (that are rapidly convertible to fully human antibodies) and have fully functional humoral immune systems indistinguishable from those of WT mice. The efficiency of the VelocImmune approach is confirmed by the rapid progression of 10 different fully human antibodies into human clinical trials.

C9orf72 ablation causes immune dysregulation characterized by leukocyte expansion, autoantibody production, and glomerulonephropathy in mice

The expansion of a hexanucleotide (GGGGCC) repeat in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Both the function of C9ORF72 and the mechanism by which the repeat expansion drives neuropathology are unknown. To examine whether C9ORF72 haploinsufficiency induces neurological disease, we created a C9orf72-deficient mouse line. Null mice developed a robust immune phenotype characterized by myeloid expansion, T cell activation, and increased plasma cells. Mice also presented with elevated autoantibodies and evidence of immune-mediated glomerulonephropathy. Collectively, our data suggest that C9orf72 regulates immune homeostasis and an autoimmune response reminiscent of systemic lupus erythematosus (SLE) occurs in its absence. We further imply that haploinsufficiency is unlikely to be the causative factor in C9ALS/FTD pathology.

Precise and in situ genetic humanization of 6 Mb of mouse immunoglobulin genes

Significance This paper describes a major advance in genomic engineering, describing by far the largest genetic humanization of the mouse ever attempted. Six megabases of mouse immune genes were replaced in a precise manner and “in situ” (in the orthologous position) with the corresponding human immune genes using largechimeric bacterial artificial chromosome targeting vectors. The accompanying manuscript demonstrates that the engineered mice function with high efficiency and have already generated therapeutic candidates that have progressed into human trials. Genetic humanization, which involves replacing mouse genes with their human counterparts, can create powerful animal models for the study of human genes and diseases. One important example of genetic humanization involves mice humanized for their Ig genes, allowing for human antibody responses within a mouse background (HumAb mice) and also providing a valuable platform for the generation of fully human antibodies as therapeutics. However, existing HumAb mice do not have fully functional immune systems, perhaps because of the manner in which they were genetically humanized. Heretofore, most genetic humanizations have involved disruption of the endogenous mouse gene with simultaneous introduction of a human transgene at a new and random location (so-called KO-plus-transgenic humanization). More recent efforts have attempted to replace mouse genes with their human counterparts at the same genetic location (in situ humanization), but such efforts involved laborious procedures and were limited in size and precision. We describe a general and efficient method for very large, in situ, and precise genetic humanization using large compound bacterial artificial chromosome–based targeting vectors introduced into mouse ES cells. We applied this method to genetically humanize 3-Mb segments of both the mouse heavy and κ light chain Ig loci, by far the largest genetic humanizations ever described. This paper provides a detailed description of our genetic humanization approach, and the companion paper reports that the humoral immune systems of mice bearing these genetically humanized loci function as efficiently as those of WT mice.