Jean-Jacques Panthier

The mouse organellar biogenesis mutant buff results from a mutation in Vps33a, a homologue of yeast vps33 and Drosophila carnation.

In the mouse, more than 16 loci are associated with mutant phenotypes that include defective pigmentation, aberrant targeting of lysosomal enzymes, prolonged bleeding, and immunodeficiency, the result of defective biogenesis of cytoplasmic organelles: melanosomes, lysosomes, and various storage granules. Many of these mouse mutants are homologous to the human Hermansky–Pudlak syndrome (HPS), Chediak–Higashi syndrome, and Griscelli syndrome. We have mapped and positionally cloned one of these mouse loci, buff (bf), which has a mutant phenotype similar to that of human HPS. Mouse bf results from a mutation in Vps33a and thus is homologous to the yeast vacuolar protein-sorting mutant vps33 and Drosophila carnation (car). This is the first found defect of the class C vacuole/prevacuole-associated target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (t-SNARE) complex in mammals and the first mammalian mutant found that is directly homologous to a vps mutation of yeast. VPS33A thus is a good candidate gene for a previously uncharacterized form of human HPS.

Targeted disruption of Otog results in deafness and severe imbalance

Genes specifically expressed in the inner ear are candidates to underlie hereditary nonsyndromic deafness. The gene Otog has been isolated from a mouse subtractive cDNA cochlear library. It encodes otogelin, an N-glycosylated protein that is present in the acellular membranes covering the six sensory epithelial patches of the inner ear: in the cochlea (the auditory sensory organ), the tectorial membrane (TM) over the organ of Corti; and in the vestibule (the balance sensory organ), the otoconial membranes over the utricular and saccular maculae as well as the cupulae over the cristae ampullares of the three semi-circular canals. These membranes are involved in the mechanotransduction process. Their movement, which is induced by sound in the cochlea or acceleration in the vestibule, results in the deflection of the stereocilia bundle at the apex of the sensory hair cells, which in turn opens the mechanotransduction channels located at the tip of the stereo-cilia. We sought to elucidate the role of otogelin in the auditory and vestibular functions by generating mice with a targeted disruption of Otog. In Otog−/− mice, both the vestibular and the auditory functions were impaired. Histological analysis of these mutants demonstrated that in the vestibule, otogelin is required for the anchoring of the otoconial membranes and cupulae to the neuroepithelia. In the cochlea, ultrastructural analysis of the TM indicated that otogelin is involved in the organization of its fibrillar network. Otogelin is likely to have a role in the resistance of this membrane to sound stimulation. These results support OTOG as a possible candidate gene for a human nonsyndromic form of deafness.

Kit-negative fibroblast-like cells expressing SK3, a Ca2+-activated K+ channel, in the gut musculature in health and disease.

Abstract. The apamin-sensitive component of the inhibitory response of the gastrointestinal musculature involves the small conductance Ca2+-activated K+ channel SK3. Kit-immunoreactive (ir) interstitial cells of Cajal appear to be involved in nitrergic inhibition while the role of the recently described CD34-ir fibroblast-like cells adjacent to, but distinct from, the cells of Cajal remains elusive. The distribution of SK3 was studied by immunohistochemistry in the normal human gut, in motility disorders with a lack of cells of Cajal (infantile hypertrophic pyloric stenosis and Hirschsprungs disease) and in mice deficient in cells of Cajal. SK3 immunoreactivity was observed exclusively in Kit-negative interstitial cells adjacent to, but distinct from, the Kit-ir interstitial cells of Cajal in the normal gut. The distribution of SK3-ir cells was not altered in conditions where cells of Cajal were lacking. These cells were CD34-ir fibroblast-like cells in the human gut and in the mouse stomach, while SK3-ir cells in the mouse intestine were CD34 negative. As SK channels are reportedly involved in inhibitory neurotransmission, our morphological observations suggest that SK3-ir interstitial cells, distinct from the Kit-ir interstitial cells of Cajal, may represent a novel cellular component in the control of excitability of the digestive musculature. Further studies will be required to directly address the function of these cells.

Distribution and ultrastructure of interstitial cells of Cajal in the mouse colon, using antibodies to Kit and Kit(W-lacZ) mice.

Abstract. The roles of the interstitial cells of Cajal in the stomach and intestine are becoming increasingly clear. Interstitial cells of Cajal in the colon are less well known, however. We studied the development and distribution of the interstitial cells of Cajal in the mouse colon, using the tyrosine kinase receptor Kit as a marker. Sections and whole mounts were studied by confocal microscopy after double immunofluorescence with specific antibodies. The ultrastructure of Kit-expressing cells was examined by electron microcopy in KitW-lacz/+ transgenic mice, which carry the lacz gene inserted in place of the first exon of the Kit gene. In the subserosa, the interstitial cells of Cajal formed a two-dimensional plexus. In the myenteric area, the interstitial cells of Cajal formed a dense plexus that gradually merged with the interstitial cells of Cajal in the outer half of the circular muscle. The inner half of the circular layer was devoid of interstitial cells of Cajal whereas in the submuscular region the interstitial cells of Cajal formed a two-dimensional plexus. Tertiary nerves with various chemical codings closely followed interstitial cell of Cajal processes. By electron microscopy, Kit-expressing cells in the outer parts of the musculature had scattered caveolae, inconspicuous basal lamina and numerous mitochondria, whereas in the submuscular region they had more pronounced myoid features. Kit-expressing cells in the mouse colon are identifiable as interstitial cells of Cajal by their ultrastructure. The interstitial cells of Cajal in the mouse colon mature postnatally. They are organized into a characteristic plexus, close to the nerves with various chemical codings.

Melanoblasts' proper location and timed differentiation depend on Notch/RBP-J signaling in postnatal hair follicles.

The Notch/RBP-J pathway is involved in a variety of developmental processes and in tissue homeostasis. In the melanocyte lineage, it has been shown that Notch signaling acts through Hes1 to maintain the melanocyte stem cell population in the hair follicle. This study was designed to determine whether Notch signaling is implicated in other steps of melanocyte-lineage postnatal development. For this purpose, we developed mice in which the RBP-J gene was conditionally ablated in the melanocyte lineage and used the Dct-lacZ reporter transgene to track melanocytes and their precursors in individual hair follicles. We determine that Notch/RBP-J-deficient melanoblasts are in reduced number within the hair follicle and gather within its lower permanent part. Moreover, our results show that Notch signaling is necessary to prevent differentiation of melanocyte stem cells and of melanoblasts before they reach the hair bulb. Finally, our data show that Notch signaling is involved in proper location of melanoblasts in the outer root sheath and of melanocytes in the hair matrix. These findings reveal previously unrecognized roles for Notch signaling in the melanocyte lineage.

Gene expression changes in the host response between resistant and susceptible inbred mouse strains after influenza A infection

Inbred mouse strains exhibit differences in susceptibility to influenza A infections. However, the molecular mechanisms underlying these differences are unknown. Therefore, we infected a highly susceptible mouse strain (DBA/2J) and a resistant strain (C57BL/6J) with influenza A H1N1 (PR8) and performed genome-wide expression analysis. We found genes expressed in lung epithelium that were specifically down-regulated in DBA/2J mice, whereas a cluster of genes on chromosome 3 was only down-regulated in C57BL/6J. In both mouse strains, chemokines, cytokines and interferon-response genes were up-regulated, indicating that the main innate immune defense pathways were activated. However, many immune response genes were up-regulated in DBA/2J much stronger than in C57BL/6J, and several immune response genes were exclusively regulated in DBA/2J. Thus, susceptible DBA/2J mice showed a hyper-inflammatory response. This response is similar to infections with highly pathogenic influenza virus and may serve as a paradigm for a hyper-inflammatory host response to influenza A virus.

Analysis of major repetitive DNA sequences in the dog (Canis familiaris) genome

Abstract. A systematic screening and analysis of repeated DNA sequences from a dog genomic library composed of small DNA inserts enabled us to characterize abundant canine repetitive DNA families. Four main families were identified: i) a group of highly repeated tRNA-derived short interspersed repetitive DNA elements (tRNA-SINEs); ii) another type of SINE-like element that was mainly found inserted into long interspersed repetitive elements (LINEs); iii) LINEs of the L1 type; and iv) satellite or satellite-like DNA. Surprisingly, no SINEs derived from 7SL RNA were found in the dog genome. These data should help in the analysis of canine DNA sequences and in the design of canine genome mapping reagents.

A New Mouse Model Reveals a Critical Role for Host Innate Immunity in Resistance to Rift Valley Fever

Rift Valley fever (RVF) is an arthropod-borne viral disease repeatedly reported in many African countries and, more recently, in Saudi Arabia and Yemen. RVF virus (RVFV) primarily infects domesticated ruminants, resulting in miscarriage in pregnant females and death for newborns and young animals. It also has the ability to infect humans, causing a feverish syndrome, meningoencephalitis, or hemorrhagic fever. The various outcomes of RVFV infection in animals and humans argue for the existence of host genetic determinants controlling the disease. We investigated the susceptibility of inbred mouse strains to infection with the virulent RVFV ZH548 strain. Compared with classical BALB/cByJ mice, wild-derived Mus m. musculus MBT/Pas mice exhibited earlier and greater viremia and died sooner, a result in sharp contrast with their resistance to infection with West Nile virus and influenza A. Infection of mouse embryonic fibroblasts (MEFs) from MBT/Pas mice with RVFV also resulted in higher viral production. Microarray and quantitative RT-PCR experiments showed that BALB/cByJ MEFs displayed a significant activation of the type I IFN pathway. In contrast, MBT/Pas MEFs elicited a delayed and partial type I IFN response to RVFV infection. RNA interference-mediated inhibition of genes that were not induced by RVFV in MBT/Pas MEFs increased viral production in BALB/cByJ MEFs, thus demonstrating their functional importance in limiting viral replication. We conclude that the failure of MBT/Pas murine strain to induce, in due course, a complete innate immune response is instrumental in the selective susceptibility to RVF.

Interspecific Recombinant Congenic Strains Between C57BL/6 and Mice of the Mus spretus Species: A Powerful Tool to Dissect Genetic Control of Complex Traits

Complex traits are under the genetic control of multiple genes, often with weak effects and strong epistatic interactions. We developed two new collections of mouse strains to improve genetic dissection of complex traits. They are derived from several backcrosses of the Mus spretus SEG/Pas or STF/Pas strains on the C57BL/6J background. Each of the 55 interspecific recombinant congenic strains (IRCSs) carries up to eight SEG/Pas chromosomal segments with an average size of 11.7 Mb, totalizing 1.37% of the genome. The complete series covers 39.7% of the SEG/Pas genome. As a complementary resource, six partial or complete interspecific consomic strains were developed and increased genome coverage to 45.6%. To evaluate the usefulness of these strains for QTL mapping, 16 IRCSs were compared with C57BL/6J for seven hematological parameters. Strain 66H, which carries three SEG/Pas chromosomal segments, had lower red blood cell volume and higher platelet count than C57BL/6J. Each chromosomal segment was isolated in a congenic strain to evaluate individual effects. Congenic strains were combined to assess epistasis. Our data show that both traits were controlled by several genes with complex epistatic interactions. IRCSs are therefore useful to unravel QTL with small effects and gene-by-gene interactions.

Twister mutant mice are defective for otogelin, a component specific to inner ear acellular membranes.

Abstract. Deafness is a common sensory defect in human. Our understanding of the molecular bases of this pathology comes from the study of a few genes that have been identified in human and/or in mice. Indeed, deaf mouse mutants are good models for studying and identifying genes involved in human hereditary hearing loss. Among these mouse mutants, twister was initially reported to have abnormal behavior and thereafter to be deaf. The recessive twister (twt) mutation has been mapped on mouse Chromosome (Chr) 7, homologous to the long arm of human Chr 15 (15q11). Otog, the gene encoding otogelin, a glycoprotein specific to all the acellular membranes of the inner ear, is also localized to mouse Chr 7, but in a region more proximal to the twister mutation, homologous to the short arm of human Chr 11 (11p15) carrying the two deafness loci, DFNB18 and USH1C. Mutant mice resulting from the knock-out of Otog, the Otogtm1Prs mice, present deafness and severe imbalance. Although twt had been mapped distally to Otog, these data prompted us to test whether twt could be due to a mutation in the Otog locus. Here, we demonstrate by genetic analysis that twt is actually allelic to Otogtm1Prs. We further extend the phenotypical analysis of twister mice, documenting the association of a severe vestibular phenotype and moderate to severe form of deafness. Molecular analysis of the Otog gene revealed the absence of detectable expression of Otog in the twister mutant. The molecular and phenotypical description of the twt mouse mutation, Otogtwt, reported herein, highlights the importance of the acellular membranes in the inner ear mechanotransduction process.