Belinda S. Harris

Protein accumulation and neurodegeneration in the woozy mutant mouse is caused by disruption of SIL1, a cochaperone of BiP.

Endoplasmic reticulum (ER) chaperones and ER stress have been implicated in the pathogenesis of neurodegenerative disorders, such as Alzheimer and Parkinson diseases, but their contribution to neuron death remains uncertain. In this study, we establish a direct in vivo link between ER dysfunction and neurodegeneration. Mice homozygous with respect to the woozy (wz) mutation develop adult-onset ataxia with cerebellar Purkinje cell loss. Affected cells have intracellular protein accumulations reminiscent of protein inclusions in both the ER and the nucleus. In addition, upregulation of the unfolded protein response, suggestive of ER stress, occurs in mutant Purkinje cells. We report that the wz mutation disrupts the gene Sil1 that encodes an adenine nucleotide exchange factor of BiP, a crucial ER chaperone. These findings provide evidence that perturbation of ER chaperone function in terminally differentiated neurons leads to protein accumulation, ER stress and subsequent neurodegeneration.

The Netrin 1 Receptors Unc5h3 and Dcc Are Necessary at Multiple Choice Points for the Guidance of Corticospinal Tract Axons

Migrating axons require the correct presentation of guidance molecules, often at multiple choice points, to find their target. Netrin 1, a bifunctional cue involved in both attracting and repelling axons, is involved in many cell migration and axon pathfinding processes in the CNS. The netrin 1 receptor DCC and itsCaenorhabditis elegans homolog UNC-40 have been implicated in directing the guidance of axons toward netrin sources, whereas the C. elegans UNC-6 receptor, UNC-5 is necessary for migrations away from UNC-6. However, a role of vertebrate UNC-5 homologs in axonal migration has not been demonstrated. We demonstrate that the Unc5h3 gene product, shown previously to regulate cerebellar granule cell migrations, also controls the guidance of the corticospinal tract, the major tract responsible for coordination of limb movements. Furthermore, we show that corticospinal tract fibers respond differently to loss of UNC5H3. In addition, we observe corticospinal tract defects in mice homozygous for a spontaneous mutation that truncates the Dcctranscript. Postnatal day 0 netrin 1 mutant mice also demonstrate corticospinal tract abnormalities. Last, interactions between the Dcc and Unc5h3 mutations were observed in gene dosage experiments. This is the first evidence of an involvement in axon guidance for any member of the vertebrateunc-5 family and confirms that both the cellular and axonal guidance functions of C. elegans unc-5 have been conserved in vertebrates.

Mouse H6 Homeobox 1 (Hmx1) mutations cause cranial abnormalities and reduced body mass.

BackgroundThe H6 homeobox genes Hmx1, Hmx2, and Hmx3 (also known as Nkx5-3; Nkx5-2 and Nkx5-1, respectively), compose a family within the NKL subclass of the ANTP class of homeobox genes. Hmx gene family expression is mostly limited to sensory organs, branchial (pharyngeal) arches, and the rostral part of the central nervous system. Targeted mutation of either Hmx2 or Hmx3 in mice disrupts the vestibular system. These tandemly duplicated genes have functional overlap as indicated by the loss of the entire vestibular system in double mutants. Mutants have not been described for Hmx1, the most divergent of the family.ResultsDumbo (dmbo) is a semi-lethal mouse mutation that was recovered in a forward genetic mutagenesis screen. Mutants exhibit enlarged ear pinnae with a distinctive ventrolateral shift. Here, we report on the basis of this phenotype and other abnormalities in the mutant, and identify the causative mutation as being an allele of Hmx1. Examination of dumbo skulls revealed only subtle changes in cranial bone morphology, namely hyperplasia of the gonial bone and irregularities along the caudal border of the squamous temporal bone. Other nearby otic structures were unaffected. The semilethality of dmbo/dmbo mice was found to be ~40%, occured perinatally, and was associated with exencephaly. Surviving mutants of both sexes exhibited reduced body mass from ~3 days postpartum onwards. Most dumbo adults were microphthalmic. Recombinant animals and specific deletion-bearing mice were used to map the dumbo mutation to a 1.8 Mb region on Chromosome 5. DNA sequencing of genes in this region revealed a nonsense mutation in the first exon of H6 Homeobox 1 (Hmx1; also Nkx5-3). An independent spontaneous allele called misplaced ears (mpe) was also identified, confirming Hmx1 as the responsible mutant gene.ConclusionThe divergence of Hmx1 from its paralogs is reflected by different and diverse developmental roles exclusive of vestibular involvement. Additionally, these mutant Hmx1 alleles represent the first mouse models of a recently-discovered Oculo-Auricular syndrome caused by mutation of the orthologous human gene.

A novel null allele of mouse Dscam survives to adulthood on an inbred C3H background with reduced phenotypic variability

DSCAMs are cell adhesion molecules that play several important roles in neurodevelopment. Mouse alleles of Dscam identified to date do not survive on an inbred C57BL/6 background, complicating analysis of DSCAM‐dependent developmental processes because of phenotypic variability related to the segregating backgrounds needed for postnatal survival. A novel spontaneous allele of Dscam, hereafter referred to as Dscam2J, has been identified. This allele contains a four base pair duplication in exon 19, leading to a frameshift and truncation of the open reading frame. Mice homozygous for the Dscam2J mutant allele survive into adulthood on the C3H/HeJ background on which the mutation was identified. Using the Dscam2J allele, retinal phenotypes that have variable severity on a segregating background were examined. A neurite lamination defect similar to that described in chick was discovered in mice. These results indicate that, in the retina, additional DSCAM‐dependent processes can be found by analysis of mutations on different genetic backgrounds.

Forebrain overgrowth (fog): A new mutation in the mouse affecting neural tube development

Forebrain overgrowth, fog, is a spontaneous autosomal recessive mutation in the mouse producing forebrain, lumbo-sacral, and facial defects. The defects appear to result from excessive growth or cellular proliferation leading to abnormalities in neural tube closure. Three unique features of the mutant are: (1) the growth of telencephalon cells into the surrounding mesenchyme, (2) presence of an encephalocele through the midline cleft in some mutants, and (3) dissociation of the tail defect from the caudal neural tube defect. We used an intersubspecific intercross between mice carrying the fog mutation and mice from an inbred Mus musculus castaneus strain (CAST/Ei) to map the fog mutation to mouse Chromosome 10 near D10Mit262 and D10Mit230 in a region with several potential candidate genes.

Mapping the Bst mutation on mouse Chromosome 16: a model for human optic atrophy

Antosomal dominant optic atrophy (OPAl) is the most common form of hereditary optic atrophy in humans, with an incidence of 1:50,000 (165500; GDB 1995). A recent study has localized OPAl to Chromosome (Chr) 3 between q28-qter (Eiberg et al. 1994). OPAl is characterized by a loss of visual acuity, deficits in color vision, and scotomas of varying size (Eliott et al. 1993). Retinas of patients with OPAl have a reduction in the number of retinal ganglion cells and a decrease in myelin content in the optic nerves, chiasrn, and tracts (Johnston et al. 1979; Kjer et al. 1983). Neurons that are in the main target of the retinal ganglion cell projection, the dorsal lateral geniculate nucleus, are also atrophic (Kjer et al. 1983). Other cell populations in the retina of humans with OPAl appear to be normal (Johnston et al. 1979; Kjer et al. 1983). OPAl is a dominant mutation, but the expression of the phenotype is highly variable both within and among families (Kline and Glaser 1979). The loss of visual acuity and the atrophy of the optic nerves often varies between right and left sides (Kline and Glaser 1979; Kjer et al. 1983). Recently, we have identified a striking abnormality in optic nerves of mice that are heterozygous for the spontaneous mutation belly spot and tail (Bst). Bst is a semi-dominant, homozygous lethal mutation that arose in the inbred strain C57BLKS (BKS; previously denoted C57BL/Ks). Heterozygous mice have a kinky tail, white feet, and a white spot at the ventral midline. In approximately 50% of the Bst/+ mice, there is a reduction or a complete absence of the pupillary light reflex in one or both eyes (Rice et al. 1993). This neurological phenotype is associated with a unilateral or bilateral atrophy of the optic nerves. As in humans with OPAl, the severity of the atrophy of the optic nerves is highIy variable-ranging from a slight reduction in the number of ganglion cell axons in one optic nerve to a complete elimination of both optic nerves. The surface area of the retina and the appearance of the inner and outer nuclear layers are qualitatively normal (Rice et al. 1993). The Bst locus has been mapped previously as the distal-most locus of a three-point cross in relation to IgIt (immunoglobin lambda-1) and md (mohaganoid) on Chr 16 (Epstein et al. 1986). Harris et al. (1989) subsequently mapped Bst in a two-point cross with Sodl (superoxide dismutase-1). Collectively, these studies place the Bst locus 25 to 42 cM distal to the centromere and proximal to Sodl. This region of mouse Chr 16 is conserved in human Chr 3 (Reeves and Citron 1994). Given the marked phenotypic similarity of retinal phenotypes between OPAl and Bst and the chromosomal homology, we have generated a higher resolution map of Bst on Chr 16 using an intraspecific backcross. F1

The podosomal-adaptor protein SH3PXD2B is essential for normal postnatal development.

Podosome-type adhesions are actin-based membrane protrusions involved in cell-matrix adhesion and extracellular matrix degradation. Despite growing knowledge of many proteins associated with podosome-type adhesions, much remains unknown concerning the function of podosomal proteins at the level of the whole animal. In this study, the spontaneous mouse mutant nee was used to identify a component of podosome-type adhesions that is essential for normal postnatal growth and development. Mice homozygous for the nee allele exhibited runted growth, craniofacial and skeletal abnormalities, ocular anterior segment dysgenesis, and hearing impairment. Adults also exhibited infertility and a form of lipodystrophy. Using genetic mapping and DNA sequencing, the cause of nee phenotypes was identified as a 1-bp deletion within the Sh3pxd2b gene on mouse Chromosome 11. Whereas the wild-type Sh3pxd2b gene is predicted to encode a protein with one PX domain and four SH3 domains, the nee mutation is predicted to cause a frameshift and a protein truncation altering a portion of the third SH3 domain and deleting all of the fourth SH3 domain. The SH3PXD2B protein is believed to be an important component of podosomes likely to mediate protein-protein interactions with membrane-spanning metalloproteinases. Testing this directly, SH3PXD2B localized to podosomes in constitutively active Src-transfected fibroblasts and through its last SH3 domain associated with a transmembrane member of a disintegrin and metalloproteinase family of proteins, ADAM15. These results identify SH3PXD2B as a podosomal-adaptor protein required for postnatal growth and development, particularly within physiologic contexts involving extracellular matrix regulation.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Curly bare (cub), a new mouse mutation on chromosome 11 causing skin and hair abnormalities, and a modifier gene (mcub) on chromosome 5

In the outcrossing of a new recessive mouse mutation causing hair loss, a new wavy-coated phenotype appeared. The two distinct phenotypes were shown to be alternative manifestations of the same gene mutation and attributable to a single modifier locus. The new mutation, curly bare (cub), was mapped to distal Chr 11 and the modifier (mcub) was mapped to Chr 5. When homozygous for the recessive mcub allele, cub/cub mice appear hairless. A single copy of the dominant Mcub allele confers a full, curly coat in cub/cub mice. Reciprocal transfer of full-thickness skin grafts between mutant and control animals showed that the skin phenotype was tissue autonomous. The hairless cub/cub mcub/mcub mice show normal contact sensitivity responses to oxazolone. The similarity of the wavy coat phenotype to those of Tgfa and Egfr mutations and the map positions of cub and mcub suggest candidate genes that interact in the EGF receptor signal transduction pathway.

A Novel Mouse Dscam Mutation Inhibits Localization and Shedding of DSCAM.

The differential adhesion hypothesis of development states that patterning of organisms, organs and tissues is mediated in large part by expression of cell adhesion molecules. The cues provided by cell adhesion molecules are also hypothesized to facilitate specific connectivity within the nervous system. In this study we characterize a novel mouse mutation in the gene Dscam (Down Syndrome Cell Adhesion Molecule). Vertebrate DSCAM is required for normal development of the central nervous system and has been best characterized in the visual system. In the visual system DSCAM is required for regulation of cell number, mosaic formation, laminar specificity, and refinement of retinal-tectal projections. We have identified a novel mutation in Dscam that results in a single amino acid substitution, R1018P, in the extracellular domain of the DSCAM protein. Mice homozygous for the R1018P mutation develop a subset of defects observed in Dscam null mice. In vitro analysis identified defects in DSCAMR1018P localization to filopodia. We also find that wild type DSCAM protein is constitutively cleaved and shed from transfected cells. This secretion is inhibited by the R1018P mutation. We also characterized a novel splice isoform of Dscam and identified defects in lamination of type 2 and type 6 cone bipolar cells in Dscam mutant mice. The identification and characterization of partial loss of function mutations in genes such as Dscam will be helpful in predicting signs and symptoms that may be observed in human patients with partial loss of DSCAM function.