NOD/RIPK2 signalling pathway contributes to osteoarthritis susceptibility.

OBJECTIVES: How inflammatory signalling contributes to osteoarthritis (OA) susceptibility is undetermined. An allele encoding a hyperactive form of the Receptor Interacting Protein Kinase 2 (RIPK2) proinflammatory signalling intermediate has been associated with familial OA. To test whether altered nucleotide-binding oligomerisation domain (NOD)/RIPK2 pathway activity causes heightened OA susceptibility, we investigated whether variants affecting additional pathway components are associated with familial OA. To determine whether the Ripk2104Asp disease allele is sufficient to account for the familial phenotype, we determined the effect of the allele on mice. METHODS: Genomic analysis of 150 independent families with dominant inheritance of OA affecting diverse joints was used to identify coding variants that segregated strictly with occurrence of OA. Genome editing was used to introduce the OA-associated RIPK2 (p.Asn104Asp) allele into the genome of inbred mice. The consequences of the Ripk2104Asp disease allele on physiology and OA susceptibility in mice were measured by histology, immunohistochemistry, serum cytokine levels and gene expression. RESULTS: We identified six novel variants affecting components of the NOD/RIPK2 inflammatory signalling pathway that are associated with familial OA affecting the hand, shoulder or foot. The Ripk2104Asp allele acts dominantly to alter basal physiology and response to trauma in the mouse knee. Whereas the knees of uninjured Ripk2Asp104 mice appear normal histologically, the joints exhibit a set of marked gene expression changes reminiscent of overt OA. Although the Ripk2104Asp mice lack evidence of chronically elevated systemic inflammation, they do exhibit significantly increased susceptibility to post-traumatic OA (PTOA). CONCLUSIONS: Two types of data support the hypothesis that altered NOD/RIPK2 signalling confers susceptibility to OA.

Smoothened transduces Hedgehog signals via activity-dependent sequestration of PKA catalytic subunits.

The Hedgehog (Hh) pathway is essential for organ development, homeostasis, and regeneration. Dysfunction of this cascade drives several cancers. To control expression of pathway target genes, the G protein-coupled receptor (GPCR) Smoothened (SMO) activates glioma-associated (GLI) transcription factors via an unknown mechanism. Here, we show that, rather than conforming to traditional GPCR signaling paradigms, SMO activates GLI by binding and sequestering protein kinase A (PKA) catalytic subunits at the membrane. This sequestration, triggered by GPCR kinase (GRK)-mediated phosphorylation of SMO intracellular domains, prevents PKA from phosphorylating soluble substrates, releasing GLI from PKA-mediated inhibition. Our work provides a mechanism directly linking Hh signal transduction at the membrane to GLI transcription in the nucleus. This process is more fundamentally similar between species than prevailing hypotheses suggest. The mechanism described here may apply broadly to other GPCR- and PKA-containing cascades in diverse areas of biology.

A role for the MEGF6 gene in predisposition to osteoporosis.

Osteoporosis is a common skeletal disorder characterized by deterioration of bone tissue. The set of genetic factors contributing to osteoporosis is not completely specified. High-risk osteoporosis pedigrees were analyzed to identify genes that may confer susceptibility to disease. Candidate predisposition variants were identified initially by whole exome sequencing of affected-relative pairs, approximately cousins, from 10 pedigrees. Variants were filtered on the basis of population frequency, concordance between pairs of cousins, affecting a gene associated with osteoporosis, and likelihood to have functionally damaging, pathogenic consequences. Subsequently, variants were tested for segregation in 68 additional relatives of the index carriers. A rare variant in MEGF6 (rs755467862) showed strong evidence of segregation with the disease phenotype. Predicted protein folding indicated the variant (Cys200Tyr) may disrupt structure of an EGF-like calcium-binding domain of MEGF6. Functional analyses demonstrated that complete loss of the paralogous genes megf6a and megf6b in zebrafish resulted in significant delay of cartilage and bone formation. Segregation analyses, in silico protein structure modeling, and functional assays support a role for MEGF6 in predisposition to osteoporosis.

Crosstalk Between Retinoic Acid and Sex-Related Genes Controls Germ Cell Fate and Gametogenesis in Medaka.

Sex determination (SD) is a highly diverse and complex mechanism. In vertebrates, one of the first morphological differences between the sexes is the timing of initiation of the first meiosis, where its initiation occurs first in female and later in male. Thus, SD is intimately related to the responsiveness of the germ cells to undergo meiosis in a sex-specific manner. In some vertebrates, it has been reported that the timing for meiosis entry would be under control of retinoic acid (RA), through activation of Stra8. In this study, we used a fish model species for sex determination and lacking the stra8 gene, the Japanese medaka (Oryzias latipes), to investigate the connection between RA and the sex determination pathway. Exogenous RA treatments act as a stress factor inhibiting germ cell differentiation probably by activation of dmrt1a and amh. Disruption of the RA degrading enzyme gene cyp26a1 induced precocious meiosis and oogenesis in embryos/hatchlings of female and even some males. Transcriptome analyzes of cyp26a1-/-adult gonads revealed upregulation of genes related to germ cell differentiation and meiosis, in both ovaries and testes. Our findings show that germ cells respond to RA in a stra8 independent model species. The responsiveness to RA is conferred by sex-related genes, restricting its action to the sex differentiation period in both sexes.

Interactions among ryanodine receptor isotypes contribute to muscle fiber type development and function.

Mutations affecting ryanodine receptor (RyR) calcium release channels commonly underlie congenital myopathies. Although these channels are known principally for their essential roles in muscle contractility, mutations in the human RYR1 gene result in a broad spectrum of phenotypes, including muscle weakness, altered proportions of fiber types, anomalous muscle fibers with cores or centrally placed nuclei, and dysmorphic craniofacial features. Currently, it is unknown which phenotypes directly reflect requirements for RyRs and which result secondarily to aberrant muscle function. To identify biological processes requiring RyR function, skeletal muscle development was analyzed in zebrafish embryos harboring protein-null mutations. RyR channels contribute to both muscle fiber development and function. Loss of some RyRs had modest effects, altering muscle fiber-type specification in the embryo without compromising viability. In addition, each RyR-encoding gene contributed to normal swimming behavior and muscle function. The RyR channels do not function in a simple additive manner. For example, although isoform RyR1a is sufficient for muscle contraction in the absence of RyR1b, RyR1a normally attenuates the activity of the co-expressed RyR1b channel in slow muscle. RyR3 also acts to modify the functions of other RyR channels. Furthermore, diminished RyR-dependent contractility affects both muscle fiber maturation and craniofacial development. These findings help to explain some of the heterogeneity of phenotypes that accompany RyR1 mutations in humans.

tbx6l and tbx16 are redundantly required for posterior paraxial mesoderm formation during zebrafish embryogenesis.

BACKGROUND: T-box genes encode a large transcription factor family implicated in many aspects of development. We are focusing on two related zebrafish T-box genes, tbx6l and tbx16, that are expressed in highly overlapping patterns in embryonic paraxial mesoderm. tbx16 mutants are deficient in trunk, but not tail, somites; we explored whether presence of tail somites in tbx16 mutants was due to compensatory function provided by the tbx6l gene. RESULTS: We generated two zebrafish tbx6l mutant alleles. Loss of tbx6l has no apparent effect on embryonic development, nor does tbx6l loss enhance the phenotype of two other T-box gene mutants, ta and tbx6, or of the mesp family gene mutant msgn1. In contrast, loss of tbx6l function dramatically enhances the paraxial mesoderm deficiency of tbx16 mutants. CONCLUSIONS: These data demonstrate that tbx6l and tbx16 genes function redundantly to direct tail somite development. tbx6l single mutants develop normally because tbx16 fully compensates for loss of tbx6l function. However, tbx6l only partially compensates for loss of tbx16 function. These results resolve the question of why loss of function of tbx16 gene, which is expressed throughout the ventral and paraxial mesoderm, profoundly affects somite development in the trunk but not the tail. Developmental Dynamics 246:759-769, 2017. © 2017 Wiley Periodicals, Inc.

Comparing zinc finger nucleases and transcription activator-like effector nucleases for gene targeting in Drosophila.

Zinc-finger nucleases have proven to be successful as reagents for targeted genome manipulation in Drosophila melanogaster and many other organisms. Their utility has been limited, however, by the significant failure rate of new designs, reflecting the complexity of DNA recognition by zinc fingers. Transcription activator-like effector (TALE) DNA-binding domains depend on a simple, one-module-to-one-base-pair recognition code, and they have been very productively incorporated into nucleases (TALENs) for genome engineering. In this report we describe the design of TALENs for a number of different genes in Drosophila, and we explore several parameters of TALEN design. The rate of success with TALENs was substantially greater than for zinc-finger nucleases , and the frequency of mutagenesis was comparable. Knockout mutations were isolated in several genes in which such alleles were not previously available. TALENs are an effective tool for targeted genome manipulation in Drosophila.

Efficient identification of TALEN-mediated genome modifications using heteroduplex mobility assays.

The heteroduplex mobility assay (HMA) is widely used to characterize strain variants of human viruses. To determine whether it can detect small sequence differences in homologous templates, we constructed a series of deletion constructs (1-10 bp deletions) in the multiple cloning site (MCS) of pBluescript II. After PCR amplification of the MCS using a mixture of wild-type and one of the deletion constructs, the resulting PCR amplicons were electrophoresed using 15% polyacrylamide gels. Two types of heteroduplexes exhibited retarded electrophoretic migration compared with individual homoduplexes. Therefore, we applied this HMA to detect transcription activator-like effector nucleases (TALEN)-induced insertion and/or deletion (indel) mutations at an endogenous locus. We found that TALEN in vivo activity was easily estimated by the degree of multiple HMA profiles derived from TALEN-injected F0 embryos. Furthermore, TALEN-injected F0 founder fish produced several unique HMA profiles in F1 embryos. Sequence analysis confirmed that the different HMA profiles contained distinct indel mutations. Thus, HMA is a rapid and sensitive analytical method for the detection of the TALEN-mediated genome modifications.

Zebrafish foxP2 zinc finger nuclease mutant has normal axon pathfinding.

foxP2, a forkhead-domain transcription factor, is critical for speech and language development in humans, but its role in the establishment of CNS connectivity is unclear. While in vitro studies have identified axon guidance molecules as targets of foxP2 regulation, and cell culture assays suggest a role for foxP2 in neurite outgrowth, in vivo studies have been lacking regarding a role for foxP2 in axon pathfinding. We used a modified zinc finger nuclease methodology to generate mutations in the zebrafish foxP2 gene. Using PCR-based high resolution melt curve analysis (HRMA) of G0 founder animals, we screened and identified three mutants carrying nonsense mutations in the 2(nd) coding exon: a 17 base-pair (bp) deletion, an 8bp deletion, and a 4bp insertion. Sequence analysis of cDNA confirmed that these were frameshift mutations with predicted early protein truncations. Homozygous mutant fish were viable and fertile, with unchanged body morphology, and no apparent differences in CNS apoptosis, proliferation, or patterning at embryonic stages. There was a reduction in expression of the known foxP2 target gene cntnap2 that was rescued by injection of wild-type foxP2 transcript. When we examined axon pathfinding using a pan-axonal marker or transgenic lines, including a foxP2-neuron-specific enhancer, we did not observe any axon guidance errors. Our findings suggest that foxP2 is not necessary for axon pathfinding during development.

Wnt signaling regulates postembryonic hypothalamic progenitor differentiation.

Previous studies have raised the possibility that Wnt signaling may regulate both neural progenitor maintenance and neuronal differentiation within a single population. Here we investigate the role of Wnt/ß-catenin activity in the zebrafish hypothalamus and find that the pathway is first required for the proliferation of unspecified hypothalamic progenitors in the embryo. At later stages, including adulthood, sequential activation and inhibition of Wnt activity is required for the differentiation of neural progenitors and negatively regulates radial glia differentiation. The presence of Wnt activity is conserved in hypothalamic progenitors of the adult mouse, where it plays a conserved role in inhibiting the differentiation of radial glia. This study establishes the vertebrate hypothalamus as a model for Wnt-regulated postembryonic neural progenitor differentiation and defines specific roles for Wnt signaling in neurogenesis.

Hadp1, a newly identified pleckstrin homology domain protein, is required for cardiac contractility in zebrafish.

The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca(2+) handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca(2+) handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca(2+) handling in the heart through its interaction with phosphatidylinositols.

Trans-centromere effects on meiotic recombination in the zebrafish.

We report that lack of crossover along one chromosome arm is associated with high-frequency occurrence of recombination close to the opposing arm's centromere during zebrafish meiotic recombination. Our data indicate that recombination behavior on the two arms of a chromosome is linked. These results inform mapping strategies for telomeric mutants.

TIF1gamma controls erythroid cell fate by regulating transcription elongation.

Recent genome-wide studies have demonstrated that pausing of RNA polymerase II (Pol II) occurred on many vertebrate genes. By genetic studies in the zebrafish tif1gamma mutant moonshine we found that loss of function of Pol II-associated factors PAF or DSIF rescued erythroid gene transcription in tif1gamma-deficient animals. Biochemical analysis established physical interactions among TIF1gamma, the blood-specific SCL transcription complex, and the positive elongation factors p-TEFb and FACT. Chromatin immunoprecipitation assays in human CD34(+) cells supported a TIF1gamma-dependent recruitment of positive elongation factors to erythroid genes to promote transcription elongation by counteracting Pol II pausing. Our study establishes a mechanism for regulating tissue cell fate and differentiation through transcription elongation.

Optimization of volumetric computed tomography for skeletal analysis of model genetic organisms.

Forward and reverse genetics now allow researchers to understand embryonic and postnatal gene function in a broad range of species. Although some genetic mutations cause obvious morphological change, other mutations can be more subtle and, without adequate observation and quantification, might be overlooked. For the increasing number of genetic model organisms examined by the growing field of phenomics, standardized but sensitive methods for quantitative analysis need to be incorporated into routine practice to effectively acquire and analyze ever-increasing quantities of phenotypic data. In this study, we present platform-independent parameters for the use of microscopic x-ray computed tomography (microCT) for phenotyping species-specific skeletal morphology of a variety of different genetic model organisms. We show that microCT is suitable for phenotypic characterization for prenatal and postnatal specimens across multiple species.

SLC24A5, a putative cation exchanger, affects pigmentation in zebrafish and humans.

Lighter variations of pigmentation in humans are associated with diminished number, size, and density of melanosomes, the pigmented organelles of melanocytes. Here we show that zebrafish golden mutants share these melanosomal changes and that golden encodes a putative cation exchanger slc24a5 (nckx5) that localizes to an intracellular membrane, likely the melanosome or its precursor. The human ortholog is highly similar in sequence and functional in zebrafish. The evolutionarily conserved ancestral allele of a human coding polymorphism predominates in African and East Asian populations. In contrast, the variant allele is nearly fixed in European populations, is associated with a substantial reduction in regional heterozygosity, and correlates with lighter skin pigmentation in admixed populations, suggesting a key role for the SLC24A5 gene in human pigmentation.