The smad5 mutation somitabun blocks Bmp2b signaling during early dorsoventral patterning of the zebrafish embryo

Signaling by members of the TGFbeta superfamily is thought to be transduced by Smad proteins. Here, we describe a zebrafish mutant in smad5, designated somitabun (sbn). The dominant maternal and zygotic effect of the sbntc24 mutation is caused by a change in a single amino acid in the L3 loop of Smad5 protein which transforms Smad5 into an antimorphic version, inhibiting wild-type Smad5 and related Smad proteins. sbn mutant embryos are strongly dorsalized, similarly to mutants in Bmp2b, its putative upstream signal. Double mutant analyses and RNA injection experiments show that sbn and bmp2b interact and that sbn acts downstream of Bmp2b signaling to mediate Bmp2b autoregulation during early dorsoventral (D-V) pattern formation. Comparison of early marker gene expression patterns, chimera analyses and rescue experiments involving temporally controlled misexpression of bmp or smad in mutant embryos reveal three phases of D-V patterning: an early sbn- and bmp2b-independent phase when a coarse initial D-V pattern is set up, an intermediate sbn- and bmp2b-dependent phase during which the putative morphogenetic Bmp2/4 gradient is established, and a later sbn-independent phase during gastrulation when the Bmp2/4 gradient is interpreted and cell fates are specified.     

Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo

A class of recessive lethal zebrafish mutations has been identified in which normal skeletal muscle differentiation is followed by a tissue-specific degeneration that is reminiscent of the human muscular dystrophies. Here, we show that one of these mutations, sapje, disrupts the zebrafish orthologue of the X-linked human Duchenne muscular dystrophy (DMD) gene. Mutations in this locus cause Duchenne or Becker muscular dystrophies in human patients and are thought to result in a dystrophic pathology through disconnecting the cytoskeleton from the extracellular matrix in skeletal muscle by reducing the level of dystrophin protein at the sarcolemma. This is thought to allow tearing of this membrane, which in turn leads to cell death. Surprisingly, we have found that the progressive muscle degeneration phenotype of sapje mutant zebrafish embryos is caused by the failure of embryonic muscle end attachments. Although a role for dystrophin in maintaining vertebrate myotendinous junctions (MTJs) has been postulated previously and MTJ structural abnormalities have been identified in the Dystrophin-deficient mdx mouse model, in vivo evidence of pathology based on muscle attachment failure has thus far been lacking. This zebrafish mutation may therefore provide a model for a novel pathological mechanism of Duchenne muscular dystrophy and other muscle diseases.     

Lrrc10 is required for early heart development and function in zebrafish

Leucine-rich Repeat Containing protein 10 (LRRC10) has recently been identified as a cardiac-specific factor in mice. However, the function of this factor remains to be elucidated. In this study, we investigated the developmental roles of Lrrc10 using zebrafish as an animal model. Knockdown of Lrrc10 in zebrafish embryos (morphants) using morpholinos caused severe cardiac morphogenic defects including a cardiac looping failure accompanied by a large pericardial edema, and embryonic lethality between day 6 and 7 post fertilization. The Lrrc10 morphants exhibited cardiac functional defects as evidenced by a decrease in ejection fraction and cardiac output. Further investigations into the underlying mechanisms of the cardiac defects revealed that the number of cardiomyocyte was reduced in the morphants. Expression of two cardiac genes was deregulated in the morphants including an increase in atrial natriuretic factor, a hallmark for cardiac hypertrophy and failure, and a decrease in cardiac myosin light chain 2, an essential protein for cardiac contractility in zebrafish. Moreover, a reduced fluorescence intensity from NADH in the morphant heart was observed in live zebrafish embryos as compared to control. Taken together, the present study demonstrates that Lrrc10 is necessary for normal cardiac development and cardiac function in zebrafish embryos, which will enhance our understanding of congenital heart defects and heart disease.     

NXT2 is required for embryonic heart development in zebrafish

Background  

NXT2 is a member of NXT family proteins that are generally involved in exporting nuclear RNA in eukaryotic cells. It is not known if NXT2 has any function in specific biological processes.     

Otopetrin 1 is required for otolith formation in the zebrafish Danio rerio

Orientation with respect to gravity is essential for the survival of complex organisms. The gravity receptor is one of the phylogenetically oldest sensory systems, and special adaptations that enhance sensitivity to gravity are highly conserved. The fish inner ear contains three large extracellular biomineral particles, otoliths, which have evolved to transduce the force of gravity into neuronal signals. Mammalian ears contain thousands of small particles called otoconia that serve a similar function. Loss or displacement of these structures can be lethal for fish and is responsible for benign paroxysmal positional vertigo (BPPV) in humans. The distinct morphologies of otoconial particles and otoliths suggest divergent developmental mechanisms. Mutations in a novel gene Otopetrin 1 (Otop1), encoding multi-transmembrane domain protein, result in nonsyndromic otoconial agenesis and a severe balance disorder in mice. Here we show that the zebrafish, Danio rerio, contains a highly conserved gene, otop1, that is essential for otolith formation. Morpholino-mediated knockdown of zebrafish Otop1 leads to otolith agenesis without affecting the sensory epithelium or other structures within the inner ear. Despite lack of otoliths in early development, otolith formation partially recovers in some fish after 2 days. However, the otoliths are malformed, misplaced, lack an organic matrix, and often consist of inorganic calcite crystals. These studies demonstrate that Otop1 has an essential and conserved role in the timing of formation and the size and shape of the developing otolith.     

Connexin 48.5 is required for normal cardiovascular function and lens development in zebrafish embryos

Gap junctions are composed of connexin (Cx) proteins and mediate intercellular communication required for many developmental and physiological processes. Here we describe the isolation and characterization of Cx48.5, a zebrafish connexin with the highest sequence identity to mammalian Cx46. Expression analysis showed that Cx48.5 is expressed in the adult and embryonic lens and heart, adult testis, and transiently in the embryonic otic vesicles. Injection of Cx48.5 cRNA into Xenopus oocytes elicited intercellular electrical coupling with voltage sensitivity similar to mammalian Cx46. In single oocytes, Cx48.5 also induced large outward currents on depolarization, consistent with gap-junctional hemichannels. Disruption of Cx48.5 expression in embryos with antisense morpholino oligos (morpholinos) revealed that Cx48.5 has an essential role in the maintenance of lens homeostasis. The morpholino-treated embryos also developed small lenses and eyes as well as severe cardiovascular abnormalities.