Mutation of FOP/FGFR1OP in mice recapitulates human short rib-polydactyly ciliopathy.

Skeletal dysplasias are a clinically and genetically heterogeneous group of bone and cartilage disorders. A total of 436 skeletal dysplasias are listed in the 2015 revised version of the nosology and classification of genetic skeletal disorders, of which nearly 20% are still genetically and molecularly uncharacterized. We report the clinical and molecular characterization of a lethal skeletal dysplasia of the short-rib group caused by mutation of the mouse Fop gene. Fop encodes a centrosomal and centriolar satellite (CS) protein. We show that Fop mutation perturbs ciliogenesis in vivo and that this leads to the alteration of the Hedgehog signaling pathway. Fop mutation reduces CSs movements and affects pericentriolar material composition, which probably participates to the ciliogenesis defect. This study highlights the role of a centrosome and CSs protein producing phenotypes in mice that recapitulate a short rib-polydactyly syndrome when mutated.

Direct and heterologous approaches to identify the LET-756/FGF interactome.

BACKGROUND: Fibroblast growth factors (FGFs) are multifunctional proteins that play important roles in cell communication, proliferation and differentiation. However, many aspects of their activities are not well defined. LET-756, one of the two C. elegans FGFs, is expressed throughout development and is essential for worm development. It is both expressed in the nucleus and secreted. RESULTS: To identify nuclear factors associated with LET-756, we used three approaches. First, we screened a two-hybrid cDNA library derived from mixed stages worms and from a normalized library, using LET-756 as bait. This direct approach allowed the identification of several binding partners that play various roles in the nucleus/nucleolus, such as PAL-1, a transcription regulator, or RPS-16, a component of the small ribosomal subunit. The interactions were validated by co-immunoprecipitation and determination of their site of occurrence in mammalian cells. Second, because patterns of protein interactions may be conserved throughout species, we searched for orthologs of known mammalian interactors and measured binary interaction with these predicted candidates. We found KIN-3 and KIN-10, the orthologs of CK2alpha and CK2beta, as new partners of LET-756. Third, following the assumption that recognition motifs mediating protein interaction may be conserved between species, we screened a two-hybrid cDNA human library using LET-756 as bait. Among the few FGF partners detected was 14-3-3beta. In support of this interaction we showed that the two 14-3-3beta orthologous proteins, FTT-1 and FTT-2/PAR-5, interacted with LET-756. CONCLUSION: We have conducted the first extensive search for LET-756 interactors using a multi-directional approach and established the first interaction map of LET-756/FGF with other FGF binding proteins from other species. The interactors identified play various roles in developmental process or basic biochemical events such as ribosome biogenesis.

Caenorhabditis elegans receptors related to mammalian vascular endothelial growth factor receptors are expressed in neural cells.

Through a genomic survey of the Caenorhabditis elegans genome for genes encoding tyrosine kinase receptors (RTK) we identified a family of four RTKs which are structurally related to vascular endothelial growth factor receptors (VEGFRs). We named this family the ver gene family (for Vascular Endothelial growth factor receptor Related). It was intriguing to find this type of RTK in an animal devoid of a vascular system. The common sites of expression of the ver genes are specialized cells of neural origin: ver-1 (T17A3.1) is expressed in the support (glial) cells of amphid and phasmid neurons, ver-2 (T17A3.8) in ADL, a pair of chemosensorial neurons, and ver-3 (F59F3.1) in the ALA neuron. In mammals, the VEGFRs are associated with angiogenesis and neurogenesis. We provide here the first observation that these molecules may be primarily and solely involved in neurogenesis in a living organism.

Myomegalin is necessary for the formation of centrosomal and Golgi-derived microtubules.

The generation of cellular microtubules is initiated at specific sites such as the centrosome and the Golgi apparatus that contain nucleation complexes rich in γ-tubulin. The microtubule growing plus-ends are stabilized by plus-end tracking proteins (+TIPs), mainly EB1 and associated proteins. Myomegalin was identified as a centrosome/Golgi protein associated with cyclic nucleotide phosphodiesterase. We show here that Myomegalin exists as several isoforms. We characterize two of them. One isoform, CM-MMG, harbors a conserved domain (CM1), recently described as a nucleation activator, and is related to a family of γ-tubulin binding proteins, which includes Drosophila centrosomin. It localizes at the centrosome and at the cis-Golgi in an AKAP450-dependent manner. It recruits γ-tubulin nucleating complexes and promotes microtubule nucleation. The second isoform, EB-MMG, is devoid of CM1 domain and has a unique N-terminus with potential EB1-binding sites. It localizes at the cis-Golgi and can localize to microtubule plus-ends. EB-MMG binds EB1 and affects its loading on microtubules and microtubule growth. Depletion of Myomegalin by small interfering RNA delays microtubule growth from the centrosome and Golgi apparatus, and decreases directional migration of RPE1 cells. In conclusion, the Myomegalin gene encodes different isoforms that regulate microtubules. At least two of these have different roles, demonstrating a previously unknown mechanism to control microtubules in vertebrate cells.

Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals.

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.

A pair as a minimum: the two fibroblast growth factors of the nematode Caenorhabditis elegans.

Fibroblast growth factors (FGFs) regulate many important developmental and homeostatic physiological events. The FGF superfamily contains several families. In this review, we present recent findings on the two FGFs of the nematode Caenorhabditis elegans from both functional and phylogenic points of view. C. elegans has a single FGFR (EGL-15) with two functionally exclusive isoforms, and two FGFs (LET-756 and EGL-17), which play distinct roles: an essential function for the former, and guidance of the migrating sex myoblasts for the latter. Regulation of homeostasis by control of the fluid balance could be the basis for the essential function of LET-756. Phylogenetic and functional studies suggest that LET-756, like vertebrate FGF9, -16, and -20, belongs to the FGF9 family, whereas EGL-17, like vertebrate FGF8, -17, and -18, could be included in the FGF8 family.

An evolutionary history of the FGF superfamily.

Fibroblast growth factors (FGF) are associated with multiple developmental and metabolic processes in triploblasts, and perhaps also in diploblasts. The evolution of the FGF superfamily has accompanied the major morphological and functional innovations of metazoan species. The study of FGFs throughout species shows that the FGF superfamily can be subdivided in eight families in present-day organisms and has evolved through phases of gene duplications and gene losses. At least two major expansions of the superfamily can be recognized: a first expansion increased the number of FGFs from one or few archeo-FGFs to eight proto-FGFs, prototypic of the eight families. A second expansion, which took place during euchordate evolution, is associated with genome duplications. It increased the number of members in the families. Subsequent losses reduced that number to the present-day figures.

Functional phylogeny relates LET-756 to fibroblast growth factor 9.

Fibroblast growth factors (FGFs) are secreted regulatory proteins involved in various developmental processes. In vertebrates, the FGF superfamily comprises 22 members. In non-vertebrates, six FGF genes have been identified in Ciona intestinalis, three in Drosophila melanogaster, and two (let-756 and egl-17) in Caenorhabditis elegans. The core of LET-756 shares a 30-50% sequence identity with the various members of the superfamily. The relationships between vertebrate and non-vertebrate FGFs are not clear. We made chimeric FGFs by replacing the core region of LET-756 by the cores of various mammalian, fly, and worm FGFs. LET-756 deleted in its core region was no longer able to rescue the lethal phenotype of a let-756 null mutant, and only chimeras containing the cores of FGFs 9, 16, and 20 showed rescue capacity. This core contains an internal motif of six amino acid residues (EFISIA) whose deletion or mutation abolished both the rescue activity and FGF secretion in the supernatant of transfected COS-1 cells. Chimera containing the core of C. intestinalis FGF9/16/20, a potential ortholog of FGF9 lacking the complete EFISIA motif, was not able to rescue the lethal phenotype or be secreted. However, the introduction of the EFISIA motif restored both activities. The data show that the EFISIA motif in the core of LET-756 is essential for its biological activity and that FGFs 9, 16, and 20, which contain that motif, are functionally close to LET-756 and may be evolutionary related. This non-classical mode of secretion using an internal motif is conserved throughout evolution.