MYBPC1 mutations impair skeletal muscle function in zebrafish models of arthrogryposis.

Myosin-binding protein C1 (MYBPC1) is an abundant skeletal muscle protein that is expressed predominantly in slow-twitch muscle fibers. Human MYBPC1 mutations are associated with distal arthrogryposis type 1 and lethal congenital contracture syndrome type 4. As MYBPC1 function is incompletely understood, the mechanism by which human mutations result in contractures is unknown. Here, we demonstrate using antisense morpholino knockdown, that mybpc1 is required for embryonic motor activity and survival in a zebrafish model of arthrogryposis. Mybpc1 morphant embryos have severe body curvature, cardiac edema, impaired motor excitation and are delayed in hatching. Myofibril organization is selectively impaired in slow skeletal muscle and sarcomere numbers are greatly reduced in mybpc1 knockdown embryos, although electron microscopy reveals normal sarcomere structure. To evaluate the effects of human distal arthrogryposis mutations, mybpc1 mRNAs containing the corresponding human W236R and Y856H MYBPC1 mutations were injected into embryos. Dominant-negative effects of these mutations were suggested by the resultant mild bent body curvature, decreased motor activity, as well as impaired overall survival compared with overexpression of wild-type RNA. These results demonstrate a critical role for mybpc1 in slow skeletal muscle development and establish zebrafish as a tractable model of human distal arthrogryposis.

Kinesin family member 6 (kif6) is necessary for spine development in zebrafish.

BACKGROUND: Idiopathic scoliosis is a form of spinal deformity that affects 2-3% of children and results in curvature of the spine without structural defects of the vertebral units. The pathogenesis of idiopathic scoliosis remains poorly understood, in part due to the lack of a relevant animal model. RESULTS: We performed a forward mutagenesis screen in zebrafish to identify new models for idiopathic scoliosis. We isolated a recessive zebrafish mutant, called skolios, which develops isolated spinal curvature that arises independent of vertebral malformations. Using meiotic mapping and whole genome sequencing, we identified a nonsense mutation in kinesin family member 6 (kif6(gw326) ) unique to skolios mutants. Three additional kif6 frameshift alleles (gw327, gw328, gw329) were generated with transcription activator-like effector nucleases (TALENs). Zebrafish homozygous or compound heterozygous for kif6 frameshift mutations developed a scoliosis phenotype indistinguishable from skolios mutants, confirming that skolios is caused by the loss of kif6. Although kif6 may play a role in cilia, no evidence for cilia dysfunction was seen in kif6(gw326) mutants. CONCLUSIONS: Overall, these findings demonstrate a novel role for kif6 in spinal development and identify a new candidate gene for human idiopathic scoliosis.

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

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.

Severe ceftriaxone-induced hemolysis complicated by diffuse cerebral ischemia in a child with sickle cell disease.

Ceftriaxone-induced hemolytic anemia is a rare and often fatal phenomenon. We report here the case of a 6-year-old female with sickle cell disease who survived a brisk and profound hemolytic reaction, resulting in hemoglobin of 0.4 g/dL, after ceftriaxone infusion. Ongoing hemolysis was abrogated with aggressive supportive care, but the patient suffered extensive neurologic sequelae as a result of the event. Serologic testing confirmed the presence of ceftriaxone antibodies.

A chemical screen to identify novel inhibitors of fin regeneration in zebrafish.

We performed a chemical screen to look for novel inhibitors of zebrafish caudal fin regeneration. In a pilot screen, 520 compounds were tested. Two compounds, budesonide and AGN192403, abrogated fin regeneration. One compound in particular, AGN192403, targets the imidazoline receptor, a pathway not previously linked to fin regeneration. In addition to inhibiting regeneration of the adult fin, AGN192403 also blocked regeneration of the larval fin fold. Finally, the inhibitory effect of AGN192403 on fin regeneration persisted after removal of the drug. These studies demonstrate that chemical screening is feasible in adult zebrafish and that it is a reasonable strategy to use for exploring the biology of regeneration.

A developmental transition in growth control during zebrafish caudal fin development.

A long-standing question in developmental biology is how do growing and developing animals achieve form and then maintain it. We have revealed a critical transition in growth control during zebrafish caudal fin development, wherein a switch from allometric to isometric growth occurs. This morphological transition led us to hypothesize additional physiological changes in growth control pathways. To test this, we fasted juvenile and adult zebrafish. Juvenile fins continued allometric growth until development of the mature bi-lobed shape was completed. In contrast, the isometric growth of mature adult fins arrested within days of initiating a fast. We explored the biochemical basis of this difference in physiology between the two phases by assessing the sensitivity to rapamycin, a drug that blocks a nutrient-sensing pathway. We show that the nutrition-independent, allometric growth phase is resistant to rapamycin at 10-fold higher concentrations than are effective at arresting growth in the nutrition-dependent, isometric growth phase. We thus link a morphological transition in growth control between allometric and isometric growth mechanisms to different physiological responses to nutritional state of the animal and finally to different pharmacological responses to a drug (rapamycin) that affects the nutrition-sensing mechanism described from yeast to human.

Saltatory control of isometric growth in the zebrafish caudal fin is disrupted in long fin and rapunzel mutants.

Zebrafish fins grow by sequentially adding new segments of bone to the distal end of each fin ray. In wild type zebrafish, segment addition is regulated such that an isometric relationship is maintained between fin length and body length over the lifespan of the growing fish. Using a novel, surrogate marker for fin growth in conjunction with cell proliferation assays, we demonstrate here that segment addition is not continuous, but rather proceeds by saltation. Saltation is a fundamental growth mechanism shared by disparate vertebrates, including humans. We further demonstrate that segment addition proceeds in conjunction with cyclic bursts of cell proliferation in the distal fin ray mesenchyme. In contrast, cells in the distal fin epidermis proliferate at a constant rate throughout the fin ray growth cycle. Finally, we show that two separate fin overgrowth mutants, long fin and rapunzel, bypass the stasis phase of the fin ray growth cycle to develop asymmetrical and symmetrical fin overgrowth, respectively.