The SRC homology 2 domain protein Shep1 plays an important role in the penetration of olfactory sensory axons into the forebrain.

Shep1 is a multidomain signaling protein that forms a complex with Cas, a key scaffolding component of integrin signaling pathways, to promote the migration of non-neuronal cells. However, the physiological function of Shep1 in the nervous system remains unknown. Interestingly, we found that Shep1 and Cas are both concentrated in the axons of developing olfactory sensory neurons (OSNs). These neurons extend their axons from the olfactory epithelium to the olfactory bulb located at the anterior tip of the forebrain. However, in developing Shep1 knock-out mice, we did not detect penetration of OSN axons across the pial basement membrane surrounding the olfactory bulb, suggesting that Shep1 function is important for the establishment of OSN connections with the olfactory bulb. Interestingly, we observed reduced levels of Cas tyrosine phosphorylation in OSN axons of Shep1 knock-out mice, suggesting compromised Cas signaling function. Indeed, when embedded in a three-dimensional gel of basement membrane proteins, explants from Shep1 knock-out olfactory epithelium extend neuronal processes less efficiently than explants from control epithelium. Furthermore, ectopic expression of Shep1 in non-neuronal cells promotes cell migration through a collagen gel. Later in development, loss of Shep1 function also causes a marked reduction in olfactory bulb size and disruption of bulb lamination, which may be primarily attributable to the defective innervation. The greatly reduced OSN connections and hypoplasia of the olfactory bulb, likely resulting in anosmia, are reminiscent of the symptoms of Kallmann syndrome, a human developmental disease that can be caused by mutations in a growing number of genes.

Genomic plasticity of the immune-related Mhc class I B region in macaque species.

BACKGROUND: In sharp contrast to humans and great apes, the expanded Mhc-B region of rhesus and cynomolgus macaques is characterized by the presence of differential numbers and unique combinations of polymorphic class I B genes per haplotype. The MIB microsatellite is closely linked to the single class I B gene in human and in some great apes studied. The physical map of the Mhc of a heterozygous rhesus monkey provides unique material to analyze MIB and Mamu-B copy number variation and then allows one to decipher the compound evolutionary history of this region in primate species. RESULTS: In silico research pinpointed 12 MIB copies (duplicons), most of which are associated with expressed B-genes that cluster in a separate clade in the phylogenetic tree. Generic primers tested on homozygous rhesus and pedigreed cynomolgus macaques allowed the identification of eight to eleven MIB copies per individual. The number of MIB copies present per haplotype varies from a minimum of three to six in cynomolgus macaques and from five to eight copies in rhesus macaques. Phylogenetic analyses highlight a strong transpecific sharing of MIB duplicons. Using the physical map, we observed that, similar to MIB duplicons, highly divergent Mamu-B genes can be present on the same haplotype. Haplotype variation as reflected by the copy number variation of class I B loci is best explained by recombination events, which are found to occur between MIBs and Mamu-B. CONCLUSION: The data suggest the existence of highly divergent MIB and Mamu-B lineages on a given haplotype, as well as variable MIB and B copy numbers and configurations, at least in rhesus macaque. Recombination seems to occur between MIB and Mamu-B loci, and the resulting haplotypic plasticity at the individual level may be a strategy to better cope with pathogens. Therefore, evolutionary inferences based on the multiplicated MIB loci but also other markers close to B-genes appear to be promising for the study of B-region organization and evolution in primates.

Splice variants and expression patterns of SHEP1, BCAR3 and NSP1, a gene family involved in integrin and receptor tyrosine kinase signaling.

SHEP1, BCAR3 and NSP1 are the three members of a family of cytoplasmic proteins involved in cell adhesion/migration and antiestrogen resistance. All three proteins contain an SH2 domain and an exchange factor-like domain that binds both Ras GTPases and the scaffolding protein Cas. SHEP1, BCAR3 and NSP1 mRNAs are widely expressed in tissues, and SHEP1 and BCAR3 have multiple splice variants that differ in their 5' untranslated regions and in some cases the beginning of their coding regions. Interestingly, our data suggest that SHEP1 is highly expressed in blood vessels in mouse breast cancer models. In contrast, BCAR3 and NSP1 are more highly expressed than SHEP1 in breast cancer cells. These expression patterns suggest differential roles for the three genes during breast cancer progression in either the vasculature or the tumor cells.

Branchio-oto-renal syndrome: the mutation spectrum in EYA1 and its phenotypic consequences.

EYA1 mutations cause branchio-oto-renal (BOR) syndrome. These mutations include single nucleotide transitions and transversions, small duplications and deletions, and complex genomic rearrangements. The last cannot be detected by coding sequence analysis of EYA1. We sought to refine the clinical diagnosis of BOR syndrome by analyzing phenotypic data from families segregating EYA1 disease-causing mutations. Based on genotype-phenotype analyses, we propose new criteria for the clinical diagnosis of BOR syndrome. We found that in approximately 40% of persons meeting our criteria, EYA1 mutations were identified. Of these mutations, 80% were coding sequence variants identified by SSCP, and 20% were complex genomic rearrangements identified by a semiquantitative PCR-based screen. We conclude that genetic testing of EYA1 should include analysis of the coding sequence and a screen for complex rearrangements.

Genomic rearrangements of EYA1 account for a large fraction of families with BOR syndrome.

Branchio-Oto-Renal (BOR) syndrome is transmitted as an autosomal dominant disorder, affects an estimated 2% of profoundly deaf children, and is caused by mutations in the human EYA1 gene. However, in up to half of the reported cases, EYA1 mutation screening is negative. This finding has been taken as evidence of genetic heterogeneity. Mutation screening of the coding region of EYA1 in a panel of families linked to chromosome 8 was conducted using SSCP and direct sequencing. Only one point mutation in five probands was detected. However, complex rearrangements, such as inversions or large deletions, were discovered in the other four patients using Southern blot analysis. These data suggest that more complex rearrangements may remain undetected in EYA1 since SSCP and sequencing were commonly used to detect mutations in this gene.

Characterization of a human import component of the mitochondrial outer membrane, TOMM70A.

Functional mitochondria require up to 1000 proteins to function properly, with 99% synthesized as precursors in the cytoplasm and transported into the mitochondria with the aid of cytosolic chaperones and mitochondrial translocators (import components). Proteins to be imported are chaperoned to the mitochondria by the cytosolic heat shock protein (cHSP70) and are immediately pursued by Translocators of the Outer Membrane (TOMs), followed by transient interactions of the unfolded proteins with Translocators of the Inner Membrane (TIMs). In the present study, we describe a human gene, TOMM70A, orthologous to the yeast Tom70 import component. TOMM70A is ubiquitously expressed in human tissues, maps on chromosome 3q13.1-q13.2 and consists of 12 coding exons spanning over 37 kb. TOMM70A localizes in the mitochondria of COS-7 cells, and in organello import assays confirmed its presence in the Outer Mitochondrial membrane (OM) of rat liver mitochondria. TOMM70A could play a significant role in the import of nuclear-encoded mitochondrial proteins with internal targeting sites such as ADP/ATP carriers and the uncoupling proteins.

The SH2 domain protein Shep1 regulates the in vivo signaling function of the scaffolding protein Cas.

The members of the p130Cas (Cas) family are important scaffolding proteins that orchestrate cell adhesion, migration and invasiveness downstream of integrin adhesion receptors and receptor tyrosine kinases by recruiting enzymes and structural molecules. Shep1, BCAR3/AND-34 and NSP1 define a recently identified family of SH2 domain-containing proteins that constitutively bind Cas proteins through a Cdc25-type nucleotide exchange factor-like domain. To gain insight into the functional interplay between Shep1 and Cas in vivo, we have inactivated the Shep1 gene in the mouse through Cre-mediated deletion of the exon encoding the SH2 domain. Analysis of Cas tyrosine phosphorylation in the brains of newborn mice, where Shep1 is highly expressed, revealed a strong decrease in Cas substrate domain phosphorylation in knockout compared to wild-type brains. Src family kinases bind to Cas via their SH3 and SH2 domains, which contributes to their activation, and phosphorylate multiple tyrosines in the Cas substrate domain. These tyrosine-phosphorylated motifs represent docking sites for the Crk adaptor, linking Cas to the downstream Rac1 and Rap1 GTPases to regulate cell adhesion and actin cytoskeleton organization. Accordingly, we detected lower Cas-Crk association and lower phosphorylation of the Src activation loop in Shep1 knockout brains compared to wild-type. Conversely, Shep1 transfection in COS cells increases Cas tyrosine phosphorylation. The SH2 domain is likely critical for the effects of Shep1 on Cas and Src signaling because the knockout mice express Shep1 fragments that lack the amino-terminal region including the SH2 domain, presumably due to aberrant translation from internal ATG codons. These fragments retain the ability to increase Cas levels in transfected cells, similar to full-length Shep1. However, they do not affect Cas phosphorylation on their own or in the presence of co-transfected full-length Shep1. They also do not show dominant negative effects on the activity of full-length Shep1 in vivo because the heterozygous mice, which express the fragments, have a normal life span. This is in contrast to the homozygous knockout mice, most of which die soon after birth. These data demonstrate that Shep1 plays a critical role in the in vivo regulation of Src activity and Cas downstream signaling through Crk, and suggest that the SH2 domain of Shep1 is critical for these effects.

A novel Flk1-TVA transgenic mouse model for gene delivery to angiogenic vasculature.

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.

POMGnT1 gene alterations in a family with neurological abnormalities.

Muscle-eye-brain disease (MEB), is caused by mutations in the POMGnT1 gene. We describe a white family with two siblings affected with congenital hypotonia early-onset glaucoma, and psychomotor delays. Brain magnetic resonance images (MRIs) showed hydrocephalus, bilateral frontal polymicrogyria, abnormal cerebellum, and characteristic flattened dystrophic pons. We identified novel POMGnT1 gene alterations in this family. Both affected siblings were found to be compound hetrozygotes and carried two missense changes inherited from their mother and one missense change (p.R442C) inherited from their father. Our findings further define the phenotypic spectrum of MEB and its occurrence in the US population.

AGTR2 mutations in X-linked mental retardation.

Two angiotensin II (Ang II)-specific receptors, AGTR1 and AGTR2, are expressed in the mammalian brain. Ang II actions on blood pressure regulation, water electrolyte balance, and hormone secretion are primarily mediated by AGTR1. The function of AGTR2 remains unclear. Here, we show that expression of the AGTR2 gene was absent in a female patient with mental retardation (MR) who had a balanced X;7 chromosomal translocation. Additionally, 8 of 590 unrelated male patients with MR were found to have sequence changes in the AGTR2 gene, including one frameshift and three missense mutations. These findings indicate a role for AGTR2 in brain development and cognitive function.