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A gain of function mutation causing skeletal overgrowth in the rapunzel mutant.

ABSTRACT: Mechanisms that regulate the growth and form of the vertebrate skeleton are largely unknown. The zebrafish mutant rapunzel has heterozygous defects in bone development, resulting in skeletal overgrowth, thus identification of the genetic lesion underlying rapunzel might provide insight into the molecular basis of skeletogenesis. In this report, we demonstrate that the rapunzel mutant results from a missense mutation in the previously uncharacterized rpz gene. This conclusion is supported by genetic mapping, identification of a missense mutation in rapunzel(c14) in a highly conserved region of the rpz gene, and suppression of the rapunzel homozygous embryonic phenotype with morpholino knockdown of rpz. In addition, rpz transcripts are identified in regions correlating with the homozygous embryonic phenotype (head, pectoral fin buds, somites and fin fold). This report provides the first gene identification for a mutation affecting segment number in the zebrafish fin and development of both the fin ray (dermal) and the axial skeleton.

SUBMITTER: Green J

PROVIDER: S-EPMC2756807 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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A gain of function mutation causing skeletal overgrowth in the rapunzel mutant.

Green Julie J Taylor Jennifer J JJ Hindes Anna A Johnson Stephen L SL Goldsmith Matthew I MI

Developmental biology 20090724 1

Mechanisms that regulate the growth and form of the vertebrate skeleton are largely unknown. The zebrafish mutant rapunzel has heterozygous defects in bone development, resulting in skeletal overgrowth, thus identification of the genetic lesion underlying rapunzel might provide insight into the molecular basis of skeletogenesis. In this report, we demonstrate that the rapunzel mutant results from a missense mutation in the previously uncharacterized rpz gene. This conclusion is supported by gene ...[more]

PMID: 19632218

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Project description:BACKGROUND: AMP-activated protein kinase (AMPK) is a heterotrimeric enzyme that is evolutionarily conserved from yeast to mammals and functions to maintain cellular and whole body energy homeostasis. Studies in experimental animals demonstrate that activation of AMPK in skeletal muscle protects against insulin resistance, type 2 diabetes and obesity. The regulatory gamma(3) subunit of AMPK is expressed exclusively in skeletal muscle; however, its importance in controlling overall AMPK activity is unknown. While evidence is emerging that gamma subunit mutations interfere specifically with AMP activation, there remains some controversy regarding the impact of gamma subunit mutations. Here we report the first gain-of-function mutation in the muscle-specific regulatory gamma(3) subunit in humans. METHODS AND FINDINGS: We sequenced the exons and splice junctions of the AMPK gamma(3) gene (PRKAG3) in 761 obese and 759 lean individuals, identifying 87 sequence variants including a novel R225W mutation in subjects from two unrelated families. The gamma(3) R225W mutation is homologous in location to the gamma(2)R302Q mutation in patients with Wolf-Parkinson-White syndrome and to the gamma(3)R225Q mutation originally linked to an increase in muscle glycogen content in purebred Hampshire Rendement Napole (RN-) pigs. We demonstrate in differentiated muscle satellite cells obtained from the vastus lateralis of R225W carriers that the mutation is associated with an approximate doubling of both basal and AMP-activated AMPK activities. Moreover, subjects bearing the R225W mutation exhibit a approximately 90% increase of skeletal muscle glycogen content and a approximately 30% decrease in intramuscular triglyceride (IMTG). CONCLUSIONS: We have identified for the first time a mutation in the skeletal muscle-specific regulatory gamma(3) subunit of AMPK in humans. The gamma(3)R225W mutation has significant functional effects as demonstrated by increases in basal and AMP-activated AMPK activities, increased muscle glycogen and decreased IMTG. Overall, these findings are consistent with an important regulatory role for AMPK gamma(3) in human muscle energy metabolism.

Project description:Four patients with overhydrated cation leak stomatocytosis (OHSt) exhibited the heterozygous RhAG missense mutation F65S. OHSt erythrocytes were osmotically fragile, with elevated Na and decreased K contents and increased cation channel-like activity. Xenopus oocytes expressing wild-type RhAG and RhAG F65S exhibited increased ouabain and bumetanide-resistant uptake of Li(+) and (86)Rb(+), with secondarily increased (86)Rb(+) influx sensitive to ouabain and to bumetanide. Increased RhAG-associated (14)C-methylammonium (MA) influx was severely reduced in RhAG F65S-expressing oocytes. RhAG-associated influxes of Li(+), (86)Rb(+), and (14)C-MA were pharmacologically distinct, and Li(+) uptakes associated with RhAG and RhAG F65S were differentially inhibited by NH(4)(+) and Gd(3+). RhAG-expressing oocytes were acidified and depolarized by 5 mM bath NH(3)/NH(4)(+), but alkalinized and depolarized by subsequent bath exposure to 5 mM methylammonium chloride (MA/MA(+)). RhAG F65S-expressing oocytes exhibited near-wild-type responses to NH(4)Cl, but MA/MA(+) elicited attenuated alkalinization and strong hyperpolarization. Expression of RhAG or RhAG F65S increased steady-state cation currents unaltered by bath Li(+) substitution or bath addition of 5 mM NH(4)Cl or MA/MA(+). These oocyte studies suggest that 1) RhAG expression increases oocyte transport of NH(3)/NH(4)(+) and MA/MA(+); 2) RhAG F65S exhibits gain-of-function phenotypes of increased cation conductance/permeability, and loss-of-function phenotypes of decreased and modified MA/MA(+) transport, and decreased NH(3)/NH(4)(+)-associated depolarization; and 3) RhAG transports NH(3)/NH(4)(+) and MA/MA(+) by distinct mechanisms, and/or the substrates elicit distinct cellular responses. Thus, RhAG F65S is a loss-of-function mutation for amine transport. The altered oocyte intracellular pH, membrane potential, and currents associated with RhAG or RhAG F65S expression may reflect distinct transport mechanisms.

Dataset Information

A gain of function mutation causing skeletal overgrowth in the rapunzel mutant.

Publications

A gain of function mutation causing skeletal overgrowth in the rapunzel mutant.

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