Yves De Repentigny

Transgenic Expression of the Activating Natural Killer Receptor Ly49H Confers Resistance to Cytomegalovirus in Genetically Susceptible Mice

Natural resistance to infection with mouse cytomegalovirus (MCMV) is controlled by a dominant locus, Cmv1. Cmv1 is linked to the Ly49 family of natural killer receptors on distal chromosome 6. While some studies localized Cmv1 as distal to the Ly49 gene cluster, genetic and functional analysis identified Ly49h as a pivotal factor in resistance to MCMV. The role of these two independent genomic domains in MCMV resistance was evaluated by functional complementation using transgenesis of bacterial artificial chromosomes (BAC) in genetically susceptible mice. Phenotypic and genetic characterization of the transgenic animals traced the resistance gene to a single region spanning the Ly49h gene. The appearance of the Ly49H protein in NK cells of transgenic mice coincided with the emergence of MCMV resistance, and there was a threshold Ly49H protein level associated with full recovery. Finally, transgenic expression of Ly49H in the context of either of the two independent susceptibility alleles, Cmv1 sBALB or Cmv1 sFVB, conferred resistance to MCMV infection. These results demonstrate that Ly49h is necessary and sufficient to confer MCMV resistance, and formally demonstrate allelism between Cmv1 and Ly49h. This panel of transgenic animals provides a unique resource to study possible pleiotropic effect of Cmv1.

Dystonin Is Essential for Maintaining Neuronal Cytoskeleton Organization.

The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons-cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology. Copyright 1998 Academic Press.

Cdx2 regulation of posterior development through non-Hox targets

The homeodomain transcription factors Cdx1, Cdx2 and Cdx4 play essential roles in anteroposterior vertebral patterning through regulation of Hox gene expression. Cdx2 is also expressed in the trophectoderm commencing at E3.5 and plays an essential role in implantation, thus precluding assessment of the cognate-null phenotype at later stages. Cdx2 homozygous null embryos generated by tetraploid aggregation exhibit an axial truncation indicative of a role for Cdx2 in elaborating the posterior embryo through unknown mechanisms. To better understand such roles, we developed a conditional Cdx2 floxed allele in mice and effected temporal inactivation at post-implantation stages using a tamoxifen-inducible Cre. This approach yielded embryos that were devoid of detectable Cdx2 protein and exhibited the axial truncation phenotype predicted from previous studies. This phenotype was associated with attenuated expression of genes encoding several key players in axial elongation, including Fgf8, T, Wnt3a and Cyp26a1, and we present data suggesting that T, Wnt3a and Cyp26a1 are direct Cdx2 targets. We propose a model wherein Cdx2 functions as an integrator of caudalizing information by coordinating axial elongation and somite patterning through Hox-independent and -dependent pathways, respectively.

Wnt11 Promotes Cardiomyocyte Development by Caspase-Mediated Suppression of Canonical Wnt Signals

ABSTRACT Specification and early patterning of the vertebrate heart are dependent on both canonical and noncanonical wingless (Wnt) signal pathways. However, the impact of each Wnt pathway on the later stages of myocardial development and differentiation remains controversial. Here, we report that the components of each Wnt signal conduit are expressed in the developing and postnatal heart, yet canonical/β-catenin activity is restricted to nonmyocardial regions. Subsequently, we observed that noncanonical Wnt (Wnt11) enhanced myocyte differentiation while preventing stabilization of the β-catenin protein, suggesting active repression of canonical Wnt signals. Wnt11 stimulation was synonymous with activation of a caspase 3 signal cascade, while inhibition of caspase activity led to accumulation of β-catenin and a dramatic reduction in myocyte differentiation. Taken together, these results suggest that noncanonical Wnt signals promote myocyte maturation through caspase-mediated inhibition of β-catenin activity.

Dystonin expression in the developing nervous system predominates in the neurons that degenerate in dystonia musculorum mutant mice.

Dystonia musculorum (dt) is an inherited neurodegenerative disorder in mice. The dt gene product, dystonin, contains the bullous pemphigoid antigen 1 coding region at its C-terminus and an actin binding domain at its N-terminus. We demonstrate that dystonin expression throughout mouse development predominates in neurons of the cranial and spinal sensory ganglia. These structures are the most severely affected in dystonic mice which could explain their severe sensory ataxia. Since we show expression in sensory neurons with small and large axoplasmic volumes, but degeneration is restricted primarily to the latter type, we suggest that caliber and size of the axon is an important factor in the disease process. Dystonin is also expressed in the extrapyramidal motor system and in the cerebellum. Functional defects in these cell types could account for the dystonic symptoms of dt mice not explained by simple sensory denervation. We also detect dystonin expression in motor neurons most of which are unaffected by the degenerative process in dt mice.

Disruption of MEF2 activity in cardiomyoblasts inhibits cardiomyogenesis

Myocyte enhancer factors (MEF2s) bind to muscle-specific promoters and activate transcription. Drosophila Mef2 is essential for Drosophila heart development, however, neither MEF2C nor MEF2B are essential for the early stages of murine cardiomyogenesis. Although Mef2c-null mice were defective in the later stages of heart morphogenesis, differentiation of cardiomyocytes still occurred. Since there are four isoforms of MEF2 factors (MEF2A, MEF2B, MEF2C and MEF2D), the ability of cells to differentiate may have been confounded by genetic redundancy. To eliminate this variable, the effect of a dominant-negative MEF2 mutant (MEF2C/EnR) during cardiomyogenesis was examined in transgenic mice and P19 cells. Targeting the expression of MEF2C/EnR to cardiomyoblasts using an Nkx2-5 enhancer in the P19 system resulted in the loss of both cardiomyocyte development and the expression of GATA4, BMP4, Nkx2-5 and MEF2C. In transiently transgenic mice, MEF2C/EnR expression resulted in embryos that lacked heart structures and exhibited defective differentiation. Our results show that MEF2C, or genes containing MEF2 DNA-binding sites, is required for the efficient differentiation of cardiomyoblasts into cardiomyocytes, suggesting conservation in the role of MEF2 from Drosophila to mammals.

Acf7 (MACF) is an actin and microtubule linker protein whose expression predominates in neural, muscle, and lung development.

Several proteins belonging to the plakin family of cytoskeletal linker proteins have recently been identified, including dystonin/Bpag1 and plectin. These proteins are unique in their abilities to form bridges between different cytoskeletal elements through specialized modular domains. We have previously reported the cloning and partial characterization of Acf7, a novel member of the plakin family. More recently, the full‐length cDNA for mouse Acf7 has been reported. Acf7 has a hybrid composition, with extended homology to dystonin/Bpag1 and plectin in the N‐terminal half, and to dystrophin in the central and C‐terminal half. Recent studies have demonstrated that Acf7 has functional actin and microtubule binding domains. Here, we describe the developmental expression profile for mouse Acf7. RNA in situ hybridization experiments revealed Acf7 transcripts in the dermomyotome and neural fold of day 8.5 mouse embryos. Later in development, Acf7 expression was predominant in neural and muscle tissues and was strongly up‐regulated just before birth in type II alveolar cells of the lung. Altogether, our results suggest that Acf7 functions as a versatile cytoskeletal linker protein and plays an important role in neural, muscle, and lung development.

Microtubule stability, Golgi organization, and transport flux require dystonin-a2–MAP1B interaction

Loss of interaction between the dystonin-a2 isoform and the microtubule-associated protein MAP1B induces microtubule instability and trafficking defects that may underlie certain neuropathies.

Myogenic program dysregulation is contributory to disease pathogenesis in spinal muscular atrophy

Mutations in the survival motor neuron (SMN1) gene lead to the neuromuscular disease spinal muscular atrophy (SMA). Although SMA is primarily considered as a motor neuron disease, the importance of muscle defects in its pathogenesis has not been fully examined. We use both primary cell culture and two different SMA model mice to demonstrate that reduced levels of Smn lead to a profound disruption in the expression of myogenic genes. This disruption was associated with a decrease in myofiber size and an increase in immature myofibers, suggesting that Smn is crucial for myogenic gene regulation and early muscle development. Histone deacetylase inhibitor trichostatin A treatment of SMA model mice increased myofiber size, myofiber maturity and attenuated the disruption of the myogenic program in these mice. Taken together, our work highlights the important contribution of myogenic program dysregulation to the muscle weakness observed in SMA.

Snf2h-mediated chromatin organization and histone H1 dynamics govern cerebellar morphogenesis and neural maturation

Chromatin compaction mediates progenitor to post-mitotic cell transitions and modulates gene expression programs, yet the mechanisms are poorly defined. Snf2h and Snf2l are ATP-dependent chromatin remodelling proteins that assemble, reposition and space nucleosomes, and are robustly expressed in the brain. Here we show that mice conditionally inactivated for Snf2h in neural progenitors have reduced levels of histone H1 and H2A variants that compromise chromatin fluidity and transcriptional programs within the developing cerebellum. Disorganized chromatin limits Purkinje and granule neuron progenitor expansion, resulting in abnormal post-natal foliation, while deregulated transcriptional programs contribute to altered neural maturation, motor dysfunction and death. However, mice survive to young adulthood, in part from Snf2l compensation that restores Engrailed-1 expression. Similarly, Purkinje-specific Snf2h ablation affects chromatin ultrastructure and dendritic arborization, but alters cognitive skills rather than motor control. Our studies reveal that Snf2h controls chromatin organization and histone H1 dynamics for the establishment of gene expression programs underlying cerebellar morphogenesis and neural maturation.