Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning

During limb outgrowth, signaling by bone morphogenetic proteins (BMPs) must be moderated to maintain the signaling loop between the zone of polarizing activity (ZPA) and the apical ectodermal ridge (AER). Gremlin, an extracellular Bmp antagonist, has been proposed to fulfill this function and therefore be important in limb patterning. We tested this model directly by mutating the mouse gene encoding gremlin (Cktsf1b1, herein called gremlin). In the mutant limb, the feedback loop between the ZPA and the AER is interrupted, resulting in abnormal skeletal pattern. We also show that the gremlin mutation is allelic to the limb deformity mutation (ld). Although Bmps and their antagonists have multiple roles in limb development, these experiments show that gremlin is the principal BMP antagonist required for early limb outgrowth and patterning.     

Fgf8 signalling from the AER is essential for normal limb development

Vertebrate limb development depends on signals from the apical ectodermal ridge (AER), which rims the distal tip of the limb bud. Removal of the AER in chick results in limbs lacking distal skeletal elements. Fibroblast growth factor (FGF) proteins can substitute for the AER (refs 4-7), suggesting that FGF signalling mediates AER activity. Of the four mouse Fgf genes (Fgf4 , Fgf8, Fgf9, Fgf17) known to display AER-specific expression domains within the limb bud (AER-Fgfs), only Fgf8 is expressed throughout the AER. Moreover, Fgf8 expression precedes that of other AER-Fgfs (refs 8-13), suggesting that Fgf8 may perform unique functions early in limb development. In mice, loss of function of Fgf4 (refs 13,14), Fgf9 (D. Ornitz, pers. comm.) or Fgf17 (ref. 15) has no effect on limb formation. We report here that inactivating Fgf8 in early limb ectoderm causes a substantial reduction in limb-bud size, a delay in Shh expression, misregulation of Fgf4 expression, and hypoplasia or aplasia of specific skeletal elements. Our data identify Fgf8 as the only known AER-Fgf individually necessary for normal limb development, and provide insight into the function of Fgf signalling from the AER in the normal outgrowth and patterning of the limb.     

Fgf10 regulates hepatopancreatic ductal system patterning and differentiation

During organogenesis, the foregut endoderm gives rise to the many different cell types that comprise the hepatopancreatic system, including hepatic, pancreatic and gallbladder cells, as well as the epithelial cells of the hepatopancreatic ductal system that connects these organs together and with the intestine. However, the mechanisms responsible for demarcating ducts versus organs are poorly understood. Here, we show that Fgf10 signaling from the adjacent mesenchyme is responsible for refining the boundaries between the hepatopancreatic duct and organs. In zebrafish fgf10 mutants, the hepatopancreatic ductal epithelium is severely dysmorphic, and cells of the hepatopancreatic ductal system and adjacent intestine misdifferentiate toward hepatic and pancreatic fates. Furthermore, Fgf10 also functions to prevent the differentiation of the proximal pancreas and liver into hepatic and pancreatic cells, respectively. These data shed light onto how the multipotent cells of the foregut endoderm, and subsequently those of the hepatopancreatic duct, are directed toward different organ fates.     

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.     

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.     

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.     

Rom-1 is required for rod photoreceptor viability and the regulation of disk morphogenesis

The homologous membrane proteins Rom-1 and peripherin-2 are localized to the disk rims of photoreceptor outer segments (OSs), where they associate as tetramers and larger oligomers. Disk rims are thought to be critical for disk morphogenesis, OS renewal and the maintenance of OS structure, but the molecules which regulate these processes are unknown. Although peripherin-2 is known to be required for OS formation (because Prph2-/- mice do not form OSs; ref. 6), and mutations in RDS (the human homologue of Prph2) cause retinal degeneration, the relationship of Rom-1 to these processes is uncertain. Here we show that Rom1-/- mice form OSs in which peripherin-2 homotetramers are localized to the disk rims, indicating that peripherin-2 alone is sufficient for both disk and OS morphogenesis. The disks produced in Rom1-/- mice were large, rod OSs were highly disorganized (a phenotype which largely normalized with age) and rod photoreceptors died slowly by apoptosis. Furthermore, the maximal photoresponse of Rom1-/- rod photoreceptors was lower than that of controls. We conclude that Rom-1 is required for the regulation of disk morphogenesis and the viability of mammalian rod photoreceptors, and that mutations in human ROM1 may cause recessive photoreceptor degeneration.     

nagie oko, encoding a MAGUK-family protein, is essential for cellular patterning of the retina

A layered organization of cells is a common architectural feature of many neuronal formations. Mutations of the zebrafish gene nagie oko (nok) produce a severe disruption of retinal architecture, indicating a key role for this locus in neuronal patterning. We show that nok encodes a membrane-associated guanylate kinase-family scaffolding protein. Nok localizes to the vicinity of junctional complexes in retinal neuroepithelium and in the photoreceptor cell layer. Mosaic analysis indicates that the nok retinal patterning phenotype is not cell-autonomous. We propose that nok function in patterning of postmitotic neurons is mediated through neuroepithelial cells and is necessary for guiding neurons to their proper destinations in retinal laminae.     

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.     

A structural-maintenance-of-chromosomes hinge domain-containing protein is required for RNA-directed DNA methylation

RNA-directed DNA methylation (RdDM) is a process in which dicer-generated small RNAs guide de novo cytosine methylation at the homologous DNA region. To identify components of the RdDM machinery important for Arabidopsis thaliana development, we targeted an enhancer active in meristems for methylation, which resulted in silencing of a downstream GFP reporter gene. This silencing system also features secondary siRNAs, which trigger methylation that spreads beyond the targeted enhancer region. A screen for mutants defective in meristem silencing and enhancer methylation retrieved six dms complementation groups, which included the known factors DRD1 (ref. 3; a SNF2-like chromatin-remodeling protein) and Pol IVb subunits. Additionally, we identified a previously unknown gene DMS3 (At3g49250), encoding a protein similar to the hinge-domain region of structural maintenance of chromosomes (SMC) proteins. This finding implicates a putative chromosome architectural protein that can potentially link nucleic acids in facilitating an RNAi-mediated epigenetic modification involving secondary siRNAs and spreading of DNA methylation.