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.
Anophthalmia (missing eye) describes a failure of early embryonic ocular development. Mutations in a relatively small set of genes account for 75% of bilateral anophthalmia cases, yet 25% of families ...currently are left without a molecular diagnosis. Here, we report our experimental work that aimed to uncover the developmental and genetic basis of the anophthalmia characterising the X-linked
(eye-ear reduction) X-ray-induced allele in mouse that was first identified in 1947. Histological analysis of the embryonic phenotype showed failure of normal eye development after the optic vesicle stage with particularly severe malformation of the ventral retina. Linkage analysis mapped this mutation to a ~6 Mb region on the X chromosome. Short- and long-read whole-genome sequencing (WGS) of affected and unaffected male littermates confirmed the
linkage but identified no plausible causative variants or structural rearrangements. These analyses did reduce the critical candidate interval and revealed evidence of multiple variants within the ancestral DNA, although none were found that altered coding sequences or that were unique to
. To investigate early embryonic events at a genetic level, we then generated mouse ES cells derived from male
embryos and wild type littermates. RNA-seq and accessible chromatin sequencing (ATAC-seq) data generated from cultured optic vesicle organoids did not reveal any large differences in gene expression or accessibility of putative cis-regulatory elements between
and wild type. However, an unbiased TF-footprinting analysis of accessible chromatin regions did provide evidence of a genome-wide reduction in binding of transcription factors associated with ventral eye development in
, and evidence of an increase in binding of the Zic-family of transcription factors, including Zic3, which is located within the
-refined critical interval. We conclude that the refined
critical region at chrX: 56,145,000-58,385,000 contains multiple genetic variants that may be linked to altered
regulation but does not contain a convincing causative mutation. Changes in the binding of key transcription factors to chromatin causing altered gene expression during development, possibly through a subtle mis-regulation of Zic3, presents a plausible cause for the anophthalmia phenotype observed in
, but further work is required to determine the precise causative allele and its genetic mechanism.
Translational control of many mRNAs in developing metazoan embryos is achieved by alterations in their poly(A) tail length. A family of cytoplasmic poly(A)-binding proteins (PABPs) bind the poly(A) ...tail and can regulate mRNA translation and stability. However, despite the extensive biochemical characterization of one family member (PABP1), surprisingly little is known about their in vivo roles or functional relatedness. Because no information is available in vertebrates, we address their biological roles, establishing that each of the cytoplasmic PABPs conserved in Xenopus laevis PABP1, embryonic PABP (ePABP), and PABP4 is essential for normal development. Morpholino-mediated knockdown of PABP1 or ePABP causes both anterior and posterior phenotypes and embryonic lethality. In contrast, depletion of PABP4 results mainly in anterior defects and lethality at later stages. Unexpectedly, cross-rescue experiments reveal that neither ePABP nor PABP4 can fully rescue PABP1 depletion, establishing that PABPs have distinct functions. Comparative analysis of the uncharacterized PABP4 with PABP1 and ePABP shows that it shares a mechanistically conserved core role in promoting global translation. Consistent with this analysis, each morphant displays protein synthesis defects, suggesting that their roles in mRNA-specific translational regulation and/or mRNA decay, rather than global translation, underlie the functional differences between PABPs. Domain-swap experiments reveal that the basis of the functional specificity is complex, involving multiple domains of PABPs, and is conferred, at least in part, by protein-protein interactions.
Abstract
The article clarifies the notion of the expert, observing that, in proceedings before the International Tribunal for the Law of the Sea (‘Tribunal’ or ITLOS), parties have made extensive use ...of expert evidence. The Tribunal’s Rules contain provisions to safeguard the impartiality and credibility of expert statements and to enable the Tribunal to play an active role in dealing with expert evidence. The function of the Tribunal in promoting impartial and credible expert statements is illustrated. These examples refer inter alia to the power of the Tribunal to assess the relevance—and thus admissibility—of expert statements, the need to avoid confusion between expert and party representative, the use of ‘voir dire’ in international proceedings, and the pro-active role the Tribunal may play in ensuring disclosure of relevant information. Thus, although the main responsibility of providing evidence lies with parties, the Tribunal may, under its Rules, play a useful role in ensuring that facts are based on reliable evidence.
We compared the toxicity of systemic local anesthetics bupivacaine and ropivacaine administered at equivalent and equipotent doses. In the first experiments, 18 male Wistar rats were anesthetized ...with thiopental and maintained under positive controlled ventilation. Electrocardiogram, electroencephalogram, and invasive arterial blood pressure were continuously recorded. The animals were randomly assigned to receive 3 mg x kg(-1) x min(-1) bupivacaine, 3 mg x kg(-1) x min(-1) ropivacaine IV (equivalent group), or 4.5 mg x kg(-1) x min(-1) ropivacaine (equipotent group). The timing of the occurrence of local anesthetic-induced toxic events (defined as the first QRS modification, dysrhythmia, seizures, moderate and severe bradycardia and hypotension, final systole) was recorded and the dose calculated. Eighteen additional rats, treated according to the same protocol were killed at the time of moderate, severe, and final hypotension for blood sampling and plasma bupivacaine and ropivacaine concentration measurement. In a third experiment, 15 awake rats (5 per group) received IV bupivacaine or ropivacaine (same infusion as in the first experiments) until seizure. At this moment, rats were allowed to recover from local anesthetic intoxication. In the first experiment, except for the first QRS modification, all the other toxic manifestations occurred at significantly larger doses (P<0.05) in the two ropivacaine groups in comparison to the bupivacaine group. In awake rats, all the animals intoxicated by ropivacaine easily recovered. In the bupivacaine group, two animals required cardiopulmonary resuscitation before any seizure activity could be detected, and only three rats survived. We conclude that, in the model used, ropivacaine, even at an equipotent dose, is less toxic than bupivacaine.
Somites give rise to a number of different embryonic cell types, including the precursors of skeletal muscle populations. The lateral aspect of amniote and fish somites have been shown to give rise ...specifically to hypaxial muscle, including the appendicular muscle that populates fins and limbs. We have investigated the morphogenetic basis for formation of specific hypaxial muscles within the zebrafish embryo and larvae. Transplantation experiments have revealed a developmentally precocious commitment of cells derived from pectoral fin level somites to forming hypaxial and specifically appendicular muscle. The fate of transplanted somites cannot be over-ridden by local inductive signals, suggesting that somitic tissue may be fixed at an early point in their developmental history to produce appendicular muscle. We further show that this restriction in competence is mirrored at the molecular level, with the exclusive expression of the receptor tyrosine kinase met within somitic regions fated to give rise to appendicular muscle. Loss-of-function experiments reveal that Met and its ligand, hepatocyte growth factor, are required for the correct morphogenesis of the hypaxial muscles in which met is expressed. Furthermore, we demonstrate a requirement for Met signaling in the process of proneuromast deposition from the posterior lateral line primordia.
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