The SH2 tyrosine phosphatase shp2 is required for mammalian limb development

The tyrosine phosphatase Shp2 is recruited into tyrosine-kinase signalling pathways through binding of its two amino-terminal SH2 domains to specific phosphotyrosine motifs, concurrent with its re-localization and stimulation of phosphatase activity. Shp2 can potentiate signalling through the MAP-kinase pathway and is required during early mouse development for gastrulation. Chimaeric analysis can identify, by study of phenotypically normal embryos, tissues that tolerate mutant cells (and therefore do not require the mutated gene) or lack mutant cells (and presumably require the mutated gene during their developmental history). We therefore generated chimaeric mouse embryos to explore the cellular requirements for Shp2. This analysis revealed an obligatory role for Shp2 during outgrowth of the limb. Shp2 is specifically required in mesenchyme cells of the progress zone (PZ), directly beneath the distal ectoderm of the limb bud. Comparison of Ptpn11 (encoding Shp2)-mutant and Fgfr1 (encoding fibroblast growth factor receptor-1)-mutant chimaeric limbs indicated that in both cases mutant cells fail to contribute to the PZ of phenotypically normal chimaeras, leading to the hypothesis that a signal transduction pathway, initiated by Fgfr1 and acting through Shp2, is essential within PZ cells. Rather than integrating proliferative signals, Shp2 probably exerts its effects on limb development by influencing cell shape, movement or adhesion. Furthermore, the branchial arches, which also use Fgfs during bud outgrowth, similarly require Shp2. Thus, Shp2 regulates phosphotyrosine-signalling events during the complex ectodermal-mesenchymal interactions that regulate mammalian budding morphogenesis.     

Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.     

AID is required for germinal center-derived lymphomagenesis

Most human B cell non-Hodgkin's lymphomas (B-NHLs) derive from germinal centers (GCs), the structure in which B cells undergo somatic hypermutation (SHM) and class switch recombination (CSR) before being selected for high-affinity antibody production. The pathogenesis of B-NHL is associated with distinct genetic lesions, including chromosomal translocations and aberrant SHM, which arise from mistakes occurring during CSR and SHM. A direct link between these DNA remodeling events and GC lymphoma development, however, has not been demonstrated. Here we have crossed three mouse models of B cell lymphoma driven by oncogenes (Myc, Bcl6 and Myc/Bcl6; refs. 5,6) with mice lacking activation-induced cytidine deaminase (AID), the enzyme required for both CSR and SHM. We show that AID deficiency prevents Bcl6-dependent, GC-derived B-NHL, but has no impact on Myc-driven, pre-GC lymphomas. Accordingly, abrogation of AID is associated with the disappearance of CSR- and SHM-mediated structural alterations. These results show that AID is required for GC-derived lymphomagenesis, supporting the notion that errors in AID-mediated antigen-receptor gene modification processes are principal contributors to the pathogenesis of human B-NHL.     

Dax1 is required for testis determination

The orphan nuclear receptor, Dax1, was originally proposed to act as an 'anti-testis' factor. We find, however, that Nr0b1 (also called Dax1 and Ahch, which encodes Dax1) is in fact required for testis differentiation.     

Epidermal growth factor receptor function is necessary for normal craniofacial development and palate closure.

Craniofacial malformations are among the most frequent congenital birth defects in humans; cleft palate, that is inadequate fusion of the palatal shelves, occurs with an annual incidence of 1 in 700 to 1 in 1,000 live births among individuals of European descent. The secondary palate arises as bilateral outgrowths from the maxillary processes, and its formation depends on the coordinated development of craniofacial structures including the Meckel's cartilage and the mandible. Cleft lip and palate syndromes in humans are associated with polymorphisms in the gene (TGFA) encoding transforming growth factor-alpha (TGF-alpha), an epidermal growth factor receptor (EGFR) ligand made by most epithelia. Here we have characterized craniofacial development in Egfr-deficient (Egfr-/-) mice. Newborn Egfr-/- mice have facial mediolateral defects including narrow, elongated snouts, underdeveloped lower jaw and a high incidence of cleft palate. Palatal shelf explants from Egfr-/- mice fused, but frequently had residual epithelium in the midline. In addition, morphogenesis of Meckel's cartilage was deficient in cultured mandibular processes from Egfr-/- embryos. The secretion of matrix metalloproteinases (MMPs) was diminished in Egfr-/- explants, consistent with the ability of EGF to increase MMP secretion and with the decreased MMP expression caused by inhibition of Egfr signalling in wild-type explants. Accordingly, inactivation of MMPs in wild-type explants phenocopied the defective morphology of Meckel's cartilage seen in Egfr-/- explants. Our results indicate that EGFR signalling is necessary for normal craniofacial development and that its role is mediated in part by its downstream targets, the MMPs, and may explain the genetic correlation of human cleft palate with polymorphisms in TGFA.     

Chfr is required for tumor suppression and Aurora A regulation

Tumorigenesis is a consequence of loss of tumor suppressors and activation of oncogenes. Expression of the mitotic checkpoint protein Chfr is lost in 20-50% of primary tumors and tumor cell lines. To explore whether downregulation of Chfr contributes directly to tumorigenesis, we generated Chfr knockout mice. Chfr-deficient mice are cancer-prone, develop spontaneous tumors and have increased skin tumor incidence after treatment with dimethylbenz(a)anthracene. Chfr deficiency leads to chromosomal instability in embryonic fibroblasts and regulates the mitotic kinase Aurora A, which is frequently upregulated in a variety of tumors. Chfr physically interacts with Aurora A and ubiquitinates Aurora A both in vitro and in vivo. Collectively, our data suggest that Chfr is a tumor suppressor and ensures chromosomal stability by controlling the expression levels of key mitotic proteins such as Aurora A.     

Fgf8 is required for outgrowth and patterning of the limbs

The expression pattern and activity of fibroblast growth factor-8 (FGF8) in experimental assays indicate that it has important roles in limb development, but early embryonic lethality resulting from mutation of Fgf8 in the germ line of mice has prevented direct assessment of these roles. Here we report that conditional disruption of Fgf8 in the forelimb of developing mice bypasses embryonic lethality and reveals a requirement for Fgf8 in the formation of the stylopod, anterior zeugopod and autopod. Lack of Fgf8 in the apical ectodermal ridge (AER) alters expression of other Fgf genes, Shh and Bmp2.     

BMP2 activity, although dispensable for bone formation, is required for the initiation of fracture healing

Adult bones have a notable regenerative capacity. Over 40 years ago, an intrinsic activity capable of initiating this reparative response was found to reside within bone itself, and the term bone morphogenetic protein (BMP) was coined to describe the molecules responsible for it. A family of BMP proteins was subsequently identified, but no individual BMP has been shown to be the initiator of the endogenous bone repair response. Here we demonstrate that BMP2 is a necessary component of the signaling cascade that governs fracture repair. Mice lacking the ability to produce BMP2 in their limb bones have spontaneous fractures that do not resolve with time. In fact, in bones lacking BMP2, the earliest steps of fracture healing seem to be blocked. Although other osteogenic stimuli are still present in the limb skeleton of BMP2-deficient mice, they cannot compensate for the absence of BMP2. Collectively, our results identify BMP2 as an endogenous mediator necessary for fracture repair.     

The mismatch repair system is required for S-phase checkpoint activation

Defective S-phase checkpoint activation results in an inability to downregulate DNA replication following genotoxic insult such as exposure to ionizing radiation. This 'radioresistant DNA synthesis' (RDS) is a phenotypic hallmark of ataxia-telangiectasia, a cancer-prone disorder caused by mutations in ATM. The mismatch repair system principally corrects nucleotide mismatches that arise during replication. Here we show that the mismatch repair system is required for activation of the S-phase checkpoint in response to ionizing radiation. Cells deficient in mismatch repair proteins showed RDS, and restoration of mismatch repair function restored normal S-phase checkpoint function. Catalytic activation of ATM and ATM-mediated phosphorylation of the protein NBS1 (also called nibrin) occurred independently of mismatch repair. However, ATM-dependent phosphorylation and activation of the checkpoint kinase CHK2 and subsequent degradation of its downstream target, CDC25A, was abrogated in cells lacking mismatch repair. In vitro and in vivo approaches both show that MSH2 binds to CHK2 and that MLH1 associates with ATM. These findings indicate that the mismatch repair complex formed at the sites of DNA damage facilitates the phosphorylation of CHK2 by ATM, and that defects in this mechanism form the molecular basis for the RDS observed in cells deficient in mismatch repair.     

CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium

Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.