Kay E. Davies

A systematic, genome-wide, phenotype-driven mutagenesis programme for gene function studies in the mouse.

As the human genome project approaches completion, the challenge for mammalian geneticists is to develop approaches for the systematic determination of mammalian gene function. Mouse mutagenesis will be a key element of studies of gene function. Phenotype-driven approaches using the chemical mutagen ethylnitrosourea (ENU) represent a potentially efficient route for the generation of large numbers of mutant mice that can be screened for novel phenotypes. The advantage of this approach is that, in assessing gene function, no a priori assumptions are made about the genes involved in any pathway. Phenotype-driven mutagenesis is thus an effective method for the identification of novel genes and pathways. We have undertaken a genome-wide, phenotype-driven screen for dominant mutations in the mouse. We generated and screened over 26,000 mice, and recovered some 500 new mouse mutants. Our work, along with the programme reported in the accompanying paper, has led to a substantial increase in the mouse mutant resource and represents a first step towards systematic studies of gene function in mammalian genetics.

Utrophin-Dystrophin-Deficient Mice as a Model for Duchenne Muscular Dystrophy

The absence of dystrophin at the muscle membrane leads to Duchenne muscular dystrophy (DMD), a severe muscle-wasting disease that is inevitably fatal in early adulthood. In contrast, dystrophin-deficient mdx mice appear physically normal despite their underlying muscle pathology. We describe mice deficient for both dystrophin and the dystrophin-related protein utrophin. These mice show many signs typical of DMD in humans: they show severe progressive muscular dystrophy that results in premature death, they have ultrastructural neuromuscular and myotendinous junction abnormalities, and they aberrantly coexpress myosin heavy chain isoforms within a fiber. The data suggest that utrophin and dystrophin have complementing roles in normal functional or developmental pathways in muscle. Detailed study of these mice should provide novel insights into the pathogenesis of DMD and provide an improved model for rapid evaluation of gene therapy strategies.

Genomic Variation and Gene Conversion in Spinal Muscular Atrophy: Implications for Disease Process and Clinical Phenotype

Autosomal recessive spinal muscular atrophy (SMA) is classified, on the basis of age at onset and severity, into three types: type I, severe; type II, intermediate; and type III, mild. The critical region in 5q13 contains an inverted repeat harboring several genes, including the survival motor neuron (SMN) gene, the neuronal apoptosis inhibitory protein (NAIP) gene, and the p44 gene, which encodes a transcription-factor subunit. Deletion of NAIP and p44 is observed more often in severe SMA, but there is no evidence that these genes play a role in the pathology of the disease. In > 90% of all SMA patients, exons 7 and 8 of the telomeric SMN gene (SMNtel) are not detectable, and this is also observed in some normal siblings and parents. Point mutations and gene conversions in SMNtel suggest that it plays a major role in the disease. To define a correlation between genotype and phenotype, we mapped deletions, using pulsed-field gel electrophoresis. Surprisingly, our data show that mutations in SMA types II and III, previously classed as deletions, are in fact due to gene-conversion events in which SMNtel is replaced by its centromeric counterpart, SMNcen. This results in a greater number of SMNcen copies in type II and type III patients compared with type I patients and enables a genotype/phenotype correlation to be made. We also demonstrate individual DNA-content variations of several hundred kilobases, even in a relatively isolated population from Finland. This explains why no consensus map of this region has been produced. This DNA variation may be due to a midisatellite repeat array, which would promote the observed high deletion and gene-conversion rate.

Molecular mechanisms of muscular dystrophies: old and new players

The study of the muscle cell in the muscular dystrophies (MDs) has shown that mutant proteins result in perturbations of many cellular components. MDs have been associated with mutations in structural proteins, signalling molecules and enzymes as well as mutations that result in aberrant processing of mRNA or alterations in post-translational modifications of proteins. These findings have not only revealed important insights for cell biologists, but have also provided unexpected and exciting new approaches for therapy.

PHARMACOLOGICAL STRATEGIES FOR MUSCULAR DYSTROPHY

Duchenne muscular dystrophy (DMD) is a fatal, genetic disorder whose relentless progression underscores the urgency for developing a cure. Although Duchenne initiated clinical trials roughly 150 years ago, therapies for DMD remain supportive rather than curative. A paradigm shift towards developing rational therapeutic strategies occurred with identification of the DMD gene. Gene- and cell-based therapies designed to replace the missing gene and/or dystrophin protein have achieved varying degrees of success. However, pharmacological strategies not designed to replace dystrophin per se appear promising, and can circumvent many hurdles hampering gene- and cell-based therapy. Here, we will review present pharmacological strategies, in particular those dealing with functional substitution of dystrophin by utrophin and enhancing muscle progenitor commitment by myostatin blockade, with a view toward facilitating drug discovery for DMD.

The European dimension for the mouse genome mutagenesis program

The European Mouse Mutagenesis Consortium is the European initiative contributing to the international effort on functional annotation of the mouse genome. Its objectives are to establish and integrate mutagenesis platforms, gene expression resources, phenotyping units, storage and distribution centers and bioinformatics resources. The combined efforts will accelerate our understanding of gene function and of human health and disease.

Duchenne muscular dystrophy and dystrophin: pathogenesis and opportunities for treatment.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene that encodes the 427‐kDa cytoskeletal protein dystrophin. Increased knowledge of the function of dystrophin and its role in muscle has led to a greater understanding of the pathogenesis of DMD. This, together with advances in the genetic toolkit of the molecular biologist, are leading to many different approaches to treatment. Gene therapy can be achieved using plasmids or viruses, mutations can be corrected using chimaeraplasts and short DNA fragments, exon skipping of mutations can be induced using oligonucleotides and readthrough of nonsense mutations can be achieved using aminoglycoside antibiotics. Blocking the proteasome degradation pathway can stabilize any truncated dystrophin protein, and upregulation of other proteins can also prevent the dystrophic process. Muscle can be repopulated with myoblasts or stem cells. All, or a combination, of these approaches hold great promise for the treatment of this devastating disease.

Activation of the lectin DC-SIGN induces an immature dendritic cell phenotype triggering Rho-GTPase activity required for HIV-1 replication.

DC-SIGN, a C-type lectin expressed on dendritic cells (DCs), can sequester human immunodeficiency virus (HIV) virions in multivesicular bodies. Here, using large-scale gene expression profiling and tyrosine-phosphorylated proteome analyses, we characterized signaling mediated by DC-SIGN after activation by either HIV or a DC-SIGN-specific antibody. Activation of DC-SIGN resulted in downregulation of genes encoding major histocompatibility complex class II, Jagged 1 and interferon-response molecules and upregulation of the gene encoding transcription factor ATF3. Phosphorylated proteome analysis showed that HIV- or antibody-stimulated DC-SIGN signaling was mediated by the Rho guanine nucleotide–exchange factor LARG and led to increased Rho-GTPase activity. Activation of LARG in DCs exposed to HIV was required for the formation of virus–T cell synapses. Thus, HIV sequestration by and stimulation of DC-SIGN helps HIV evade immune responses and spread to cells.

High-Throughput Analysis of Promoter Occupancy Reveals Direct Neural Targets of FOXP2, a Gene Mutated in Speech and Language Disorders

We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.

Alternative splicing events are a late feature of pathology in a mouse model of spinal muscular atrophy.

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT–PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival.