Transcription factor paralogs may share a common role in staged or overlapping expression in specific tissues, as in the Hox family. In other cases, family members have distinct roles in a range of embryologic, differentiation or response pathways (as in the Tbx and Pax families). For the interferon regulatory factor (IRF) family of transcription factors, mice deficient in Irf1, Irf2, Irf3, Irf4, Irf5, Irf7, Irf8 or Irf9 have defects in the immune response but show no embryologic abnormalities. Mice deficient for Irf6 have not been reported, but in humans, mutations in IRF6 cause two mendelian orofacial clefting syndromes, and genetic variation in IRF6 confers risk for isolated cleft lip and palate. Here we report that mice deficient for Irf6 have abnormal skin, limb and craniofacial development. Histological and gene expression analyses indicate that the primary defect is in keratinocyte differentiation and proliferation. This study describes a new role for an IRF family member in epidermal development.

Tumor necrosis factor receptor-associated factor 3 (TRAF3) is an adaptor protein that directly binds to a number of receptors of the tumor necrosis factor receptor (TNF-R) superfamily. Despite in vitro evidence that TRAF3 plays diverse roles in different cell types, little is known about the in vivo functions of TRAF3. To address this gap in knowledge and to circumvent the early lethal effect of TRAF3 null mutations, we generated conditional TRAF3-deficient mice. B-cell-specific Traf3(-/-) mice displayed severe peripheral B cell hyperplasia, which culminated in hyperimmunoglobulinemia and increased T-independent antibody responses, splenomegaly and lymphadenopathy. Resting splenic B cells from these mice exhibited remarkably prolonged survival ex vivo independent of B cell activating factor and showed increased amounts of active nuclear factor-kappaB2 but decreased amounts of nuclear protein kinase Cdelta. Furthermore, these mice developed autoimmune manifestations as they aged. These findings indicate that TRAF3 is a critical regulator of peripheral B cell homeostasis and may be implicated in the regulation of peripheral self-tolerance induction.

Of the vertebrate senses, touch is the least understood at the molecular level The ion channels that form the core of the mechanosensory complex and confer touch sensitivity remain unknown. However, the similarity of the brain sodium channel 1 (BNC1) to nematode proteins involved in mechanotransduction indicated that it might be a part of such a mechanosensor. Here we show that disrupting the mouse BNC1 gene markedly reduces the sensitivity of a specific component of mechanosensation: low-threshold rapidly adapting mechanoreceptors. In rodent hairy skin these mechanoreceptors are excited by hair movement. Consistent with this function, we found BNC1 in the lanceolate nerve endings that lie adjacent to and surround the hair follicle. Although BNC1 has been proposed to have a role in pH sensing, the acid-evoked current in cultured sensory neurons and the response of acid-stimulated nociceptors were normal in BNC1 null mice. These data identify the BNC1 channel as essential for the normal detection of light touch and indicate that BNC1 may be a central component of a mechanosensory complex.

Bardet-Biedl syndrome (BBS) is a heterogeneous, pleiotropic human disorder characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, hypogenitalism, and an increased incidence of diabetes and hypertension. No information is available regarding the specific function of BBS2. We show that mice lacking Bbs2 gene expression have major components of the human phenotype, including obesity and retinopathy. In addition, these mice have phenotypes associated with cilia dysfunction, including retinopathy, renal cysts, male infertility, and a deficit in olfaction. With the exception of male infertility, these phenotypes are not caused by a complete absence of cilia. We demonstrate that BBS2 retinopathy involves normal retina development followed by apoptotic death of photoreceptors, the primary ciliated cells of the retina. Photoreceptor cell death is preceded by mislocalization of rhodopsin, indicating a defect in transport. We also demonstrate that Bbs2 ؊/؊ mice and a second BBS mouse model, Bbs4 ؊/؊ , have a defect in social function. The evaluation of Bbs2 ؊/؊ mice indicates additional phenotypes that should be evaluated in human patients, including deficits in social interaction and infertility.Bardet-Biedl syndrome ͉ mouse model ͉ obesity

Bardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder that results in retinal degeneration, obesity, cognitive impairment, polydactyly, renal abnormalities, and hypogenitalism. Of the 12 known BBS genes, BBS1 is the most commonly mutated, and a single missense mutation (M390R) accounts for Ϸ80% of BBS1 cases. To gain insight into the function of BBS1, we generated a Bbs1 M390R/M390R knockin mouse model. Mice homozygous for the M390R mutation recapitulated aspects of the human phenotype, including retinal degeneration, male infertility, and obesity. The obese mutant mice were hyperphagic and hyperleptinemic and exhibited reduced locomotor activity but no elevation in mean arterial blood pressure. Morphological evaluation of Bbs1 mutant brain neuroanatomy revealed ventriculomegaly of the lateral and third ventricles, thinning of the cerebral cortex, and reduced volume of the corpus striatum and hippocampus. Similar abnormalities were also observed in the brains of Bbs2 ؊/؊ , Bbs4 ؊/؊ , and Bbs6 ؊/؊ mice, establishing these neuroanatomical defects as a previously undescribed BBS mouse model phenotype. Ultrastructural examination of the ependymal cell cilia that line the enlarged third ventricle of the Bbs1 mutant brains showed that, whereas the 9 ؉ 2 arrangement of axonemal microtubules was intact, elongated cilia and cilia with abnormally swollen distal ends were present. Together with data from transmission electron microscopy analysis of photoreceptor cell connecting cilia, the Bbs1 M390R mutation does not affect axonemal structure, but it may play a role in the regulation of cilia assembly and/or function.B ardet-Biedl syndrome [BBS, Online Mendelian Inheritance in Man (OMIM) 209900] is a genetically heterogeneous autosomal recessive disorder characterized by obesity, retinal degeneration, polydactyly, cognitive impairment, hypogenitalism, and renal abnormalities, as well as susceptibility to hypertension, diabetes mellitus, olfaction deficits, and congenital cardiac defects. Twelve BBS genes (BBS1-12) have been identified to date (1-14). They encode a set of proteins thought to play a role in the structure or function of cilia, basal bodies, and intracellular transport (15, 16). Mutations in BBS1 are the most commonly observed in BBS. A single missense mutation that converts a methionine codon to an arginine codon (M390R) accounts for Ϸ80% of BBS1 mutations and is involved in 25% of all BBS cases (5). The M390R mutation occurs near predicted regions of coiled-coil protein domains and lies within a conserved predicted WD40-like protein motif. These protein motifs are involved in such basic biological processes as signal transduction, RNA synthesis/processing, chromatin assembly, vesicular trafficking, cytoskeletal assembly, cell cycle control, and apoptosis (17).Recently, BBS1, BBS2, BBS4, BBS5, BBS7, BBS8, and BBS9 were shown to form a stable Ϸ450-kDa protein complex called the BBSome in cultured retinal pigment epithelial (RPE) cells and mouse testes. Depletion of some components of the BBSome, ...

The leukocyte-specific adapter molecule SLP-76 (Src homology 2 domain-containing leukocyte protein of 76 kilodaltons) is rapidly phosphorylated on tyrosine residues after receptor ligation in several hematopoietically derived cell types. Mice made deficient for SLP-76 expression contained no peripheral T cells as a result of an early block in thymopoiesis. Macrophage and natural killer cell compartments were intact in SLP-76-deficient mice, despite SLP-76 expression in these lineages in wild-type mice. Thus, the SLP-76 adapter protein is required for normal thymocyte development and plays a crucial role in translating signals mediated by pre-T cell receptors into distal biochemical events.

The functions of the proteins encoded by the Bardet-Biedl syndrome (BBS) genes are unknown. Mutations in these genes lead to the pleiotropic human disorder BBS, which is characterized by obesity, retinopathy, polydactyly, renal and cardiac malformations, learning disabilities, and hypogenitalism. Secondary features include diabetes mellitus and hypertension. Recently, it has been suggested that the BBS phenotypes are the result of a lack of cilia formation or function. In this study, we show that mice lacking the Bbs4 protein have major components of the human phenotype, including obesity and retinal degeneration. We show that Bbs4-null mice develop both motile and primary cilia, demonstrating that Bbs4 is not required for global cilia formation. Interestingly, male Bbs4-null mice do not form spermatozoa flagella, and BBS4 retinopathy involves apoptotic death of photoreceptors, the primary ciliated cells of the retina. These mutation data demonstrate a connection between the function of a BBS protein and cilia. To further evaluate an association between cilia and BBS, we performed homology comparisons of BBS proteins in model organisms and find that BBS proteins are specifically conserved in ciliated organisms.B ardet-Biedl syndrome (BBS) is a genetically heterogeneous disorder with linkage to eight loci. Six BBS genes (BBS1, BBS2, BBS4, BBS6, BBS7, and BBS8) have been identified (1-7). With the exception of BBS6, which has similarity to type II chaperonins (8), BBS proteins do not show extensive homology with proteins of known function. BBS4 and BBS8 contain tetratricopeptide repeat domains, and a region of BBS8 shows similarity to the prokaryotic pilF domain. Based on the observations that BBS8 localizes to the basal body of ciliated cells and expression of the Caenorhabditis elegans orthologues of several BBS proteins are limited to ciliated cells, it has been hypothesized that BBS is the result of a defect in cilia assembly or function (7). We previously used positional cloning to identify the human BBS4 gene (3). To help elucidate the function of the BBS proteins, we have now targeted the Bbs4 gene in mice. Bbs4 Ϫ/Ϫ mice recapitulate aspects of the human phenotype: they become obese, fail to reproduce, and display retinal degeneration. We show the presence of both motile and primary cilia in these mice, demonstrating that Bbs4 deficiency does not prevent global ciliogenesis. Interestingly, male knockout mice have a complete lack of flagella, demonstrating that the Bbs4 protein is necessary for flagella formation during spermatogenesis. Furthermore, Bbs4-null mice undergo retinal degeneration due principally to photoreceptor cell loss associated with outer segment attenuation, suggesting a role for BBS proteins in maintenance of sensory cilia. These data demonstrate a role for BBS proteins in facets of cilia function. Materials and MethodsConstruction of the Bbs4 Gene Targeting Vector. BLAST analysis of the Celera mouse fragment database with the human BBS4 cDNA sequence identified sequences corresponding to...

The signaling lipid, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2), likely functions in multiple signaling pathways. Here, we report the characterization of a mouse mutant lacking Vac14, a regulator of PI(3,5)P 2 synthesis. The mutant mice exhibit massive neurodegeneration, particularly in the midbrain and in peripheral sensory neurons. Cell bodies of affected neurons are vacuolated, and apparently empty spaces are present in areas where neurons should be present. Similar vacuoles are found in cultured neurons and fibroblasts. Selective membrane trafficking pathways, especially endosome-to-TGN retrograde trafficking, are defective. This report, along with a recent report on a mouse with a null mutation in Fig4, presents the unexpected finding that the housekeeping lipid, PI(3,5)P 2, is critical for the survival of neural cells.T he low-abundance signaling lipids, phosphatidylinositol 3,5-bisphosphate (PI(3,5)P 2 ) and phosphatidylinositol 5-phosphate (PI(5)P), were discovered relatively recently (1-3). Because of their low abundance and the limited number of tools available for their study, relatively little is known about these lipids.An interesting property of PI(3,5)P 2 occurs in yeast, where a stimulus of hyperosmotic shock induces dramatic and transient changes in the levels of PI(3,5)P 2 . The levels of PI(3,5)P 2 transiently rise Ͼ20-fold (4). Within 1 minute, the levels rise 5-fold; by 5 minutes, they increase Ͼ20-fold; there is a short plateau of 10 min, and then PI(3,5)P 2 levels decrease at a rate similar to their increase. The rapid decrease in PI(3,5)P 2 levels occurs even though the cells remain in hyperosmotic media. Vacuole volume undergoes transient changes that parallel PI(3,5)P 2 levels. That these rapid and transient changes occur even in the presence of a sustained stimulus strongly suggests that PI(3,5)P 2 plays a major role in signaling pathways related to adaptation.Several proteins are required for the synthesis and turnover of PI(3,5)P 2 . PI(3,5)P 2 is synthesized from PI(3)P by the PI(3)P 5-kinase Fab1/PIKfyve/PIP5K3 (5, 6). Fab1 is stimulated by a regulatory complex that contains Vac14 (7, 8) and Fig4 (4, 9). Surprisingly, the Vac14/Fig4 complex plays two opposing roles in the regulation of steady-state levels of PI(3,5)P 2 . Vac14/Fig4 both activate Fab1 and also function in the breakdown of PI(3,5)P 2 through the lipid phosphatase activity of Fig4 (4, 9-11).In mammals, generation of PI(3,5)P 2 is predicted to impact PI(5)P production. In vitro studies have shown that PI(5)P can be generated from PI(3,5)P 2 through the PI(3,5)P 2 3-phosphatase activity of members of the myotubularin family and related proteins including MTM1, MTMR1, MTMR2, MTMR3, MTMR6, and hJUMPY/MTMR14 (12-15). In addition, PIKfyve/Fab1 can generate both PI(3,5)P 2 and PI(5)P in vitro (16). The source of PI(5)P in vivo has not been established. However, the generation of PI(5)P from either pathway requires PIKfyve/ Fab1 activity, either to produce the substrate for myotubularin [PI(3,5)P 2 ] or to produce PI(5...