Ribosome biogenesis underpins cell growth and division. Disruptions in ribosome biogenesis and translation initiation are deleterious to development and underlie a spectrum of diseases known collectively as ribosomopathies. Here, we describe a novel zebrafish mutant, titania (ttis450), which harbours a recessive lethal mutation in pwp2h, a gene encoding a protein component of the small subunit processome. The biochemical impacts of this lesion are decreased production of mature 18S rRNA molecules, activation of Tp53, and impaired ribosome biogenesis. In ttis450, the growth of the endodermal organs, eyes, brain, and craniofacial structures is severely arrested and autophagy is up-regulated, allowing intestinal epithelial cells to evade cell death. Inhibiting autophagy in ttis450 larvae markedly reduces their lifespan. Somewhat surprisingly, autophagy induction in ttis450 larvae is independent of the state of the Tor pathway and proceeds unabated in Tp53-mutant larvae. These data demonstrate that autophagy is a survival mechanism invoked in response to ribosomal stress. This response may be of relevance to therapeutic strategies aimed at killing cancer cells by targeting ribosome biogenesis. In certain contexts, these treatments may promote autophagy and contribute to cancer cells evading cell death.
Minor class or U12-type splicing is a highly conserved process required to remove a minute fraction of introns from human premRNAs. Defects in this splicing pathway have recently been linked to human disease, including a severe developmental disorder encompassing brain and skeletal abnormalities known as Taybi-Linder syndrome or microcephalic osteodysplastic primordial dwarfism 1, and a hereditary intestinal polyposis condition, Peutz-Jeghers syndrome. Although a key mechanism for regulating gene expression, the impact of impaired U12-type splicing on the transcriptome is unknown. Here, we describe a unique zebrafish mutant, caliban (clbn), with arrested development of the digestive organs caused by an ethylnitrosourea-induced recessive lethal point mutation in the rnpc3 [RNA-binding region (RNP1, RRM) containing 3] gene. rnpc3 encodes the zebrafish ortholog of human RNPC3, also known as the U11/U12 di-snRNP 65-kDa protein, a unique component of the U12-type spliceosome. The biochemical impact of the mutation in clbn is the formation of aberrant U11-and U12-containing small nuclear ribonucleoproteins that impair the efficiency of U12-type splicing. Using RNA sequencing and microarrays, we show that multiple genes involved in various steps of mRNA processing, including transcription, splicing, and nuclear export are disrupted in clbn, either through intron retention or differential gene expression. Thus, clbn provides a useful and specific model of aberrant U12-type splicing in vivo. Analysis of its transcriptome reveals efficient mRNA processing as a critical process for the growth and proliferation of cells during vertebrate development.S plicing, the excision of introns from pre-mRNA, is an essential step in gene expression and a major source of complexity in the transcriptome (1). The process is catalyzed by highly dynamic complexes of small nuclear ribonucleoproteins (snRNPs) called spliceosomes (2). Not widely appreciated is the coexistence of two types of introns in most eukaryotic genomes. The vast majority, the major class or U2-type introns, are marked by GT and AG at their 5′ and 3′ ends, respectively. Minor class or U12-type introns, of which there are ∼700 in the human genome, were initially recognized by the presence of AT and AC in these positions, prompting their original name of AT-AC introns (3). However, we now know that most of these introns contain the same GT-AG termini found in U2-type introns (4, 5) and are instead distinguished from them by two highly conserved motifs: one adjacent to the 5′ splice site (ss) and one corresponding to the branch point sequence (BPS), close to the 3′ ss (6, 7). These introns also lack the 3′ polypyrimidine tract characteristic of U2-type introns. Minor class introns are excised by U12-type spliceosomes, which are analogous in function and similar in composition to U2-type spliceosomes; each comprise five small nuclear RNAs (snRNAs) and hundreds of associated proteins (6). Although the U5 snRNA is shared between the two complexes, the U12-type spliceosome con...
This new method improves visualisation of the tricuspid valve and makes analysis easier and less prone to operator error than the current standard technique for MRI assessment of RV volumes.
Background & Aims Zebrafish mutants generated by ethylnitrosourea (ENU)-mutagenesis provide a powerful tool for dissecting the genetic regulation of developmental processes, including organogenesis. One zebrafish mutant, “flotte lotte” (flo), displays striking defects in intestinal, liver, pancreas and eye formation at 78hpf. In this study we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. Methods Positional cloning was employed to map the flo mutation. Sub-cellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, confocal and electron microscopy. Results The molecular lesion in flo is a nonsense mutation in the elys (embryonic large molecule derived from yolk sac) gene which encodes a severely truncated protein lacking the Elys C-terminal AT-hook DNA binding domain. Recently, ELYS has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Though elys mRNA is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally-expressed elys mRNA until 24hpf. From 72hpf, elys mRNA expression is restricted to proliferating tissues, including the intestinal epithelium, pancreas, liver and eye. Cells in these tissues display disrupted nuclear pore formation; ultimately intestinal epithelial cells undergo apoptosis. Conclusion Our results demonstrate that Elys regulates digestive organ formation.
A finite interval of initial swimbladder inflation in striped trumpeter Latris lineata larvae occurred over 4 days at 16 C. Water-surface films were removed on different days to form treatments: 4, 8, 9, 10, 11 and 12 days post hatching, dph (day 4, 8, 9, 10, 11 and 12 treatments, respectively). No swimbladder inflation was recorded prior to water-surface film removal. When the water-surface films were removed in day 4 and 8 treatments, initial swimbladder inflation was first recorded in larvae 9 dph at mean AE S.E. 35Á0 AE 5Á4% (n ¼ 4) and 45Á0 AE 7Á9%, respectively. Water-surface film removal at days 9, 10 and 11, resulted in initial swimbladder inflation the following day at 62Á5 AE 2Á5, 62Á5 AE 7Á2 and 11Á3 AE 5Á5% in larvae 10, 11 and 12 dph, respectively. No swimbladder inflation was recorded following water-surface film removal on day 12. There was no significant difference in initial inflation among larvae in day 4, 8, 9 and 10 treatments, ranging from 65Á0 AE 4Á1 to 73Á8 AE 6Á9% (P > 0Á05). Initial inflation was significantly lower in the day 11 treatment (11Á3 AE 5Á5%) (P < 0Á05). During the inflation interval (9-12 dph) swimbladders displayed one of three morphologies; liquid dilation, gas inflated and collapsed. Collapse of the swimbladder lumen was first apparent in larvae without swimbladder inflation from 11 dph and progressively developed thereafter in all larvae with non-inflated swimbladders. Larvae >6Á1 mm standard length lost the ability to undergo initial swimbladder inflation. This study demonstrates that the interval for initial swimbladder inflation in striped trumpeter is short, finite and related to larval size. The end of the inflation interval was marked by onset of abnormal swimbladder morphologies, but not to closure of the pneumatic duct. # 2005 The Fisheries Society of the British Isles
Summary This study examined swim bladder morphogenesis in three cohorts of striped trumpeter (Latris lineata), a euphysoclist species with physostomous larvae. The swim bladder was first discernible 1–2 days after hatching as an evagination on the dorsal surface of the incipient digestive tract. It comprised a cluster of mesenchymal cells surrounding an inner primordium of epithelial cells. At mouth opening in larvae of 5.3 mm standard length (SL), the swim bladder was noticeably enlarged. Histologically, the swim bladder lumen was dilated and liquid filled. The pneumatic duct was first seen during the dilation stage and the rete mirabile began forming among the connective tissue surrounding the swim bladder. Initial swim bladder inflation occurred on day 11 post‐hatching in Cohort 1, at 14°C, and day 9 post‐hatching, in Cohorts 2 and 3, at 16°C. Histologically, the lumens of inflated swim bladders were ellipsoid and the epithelium was squamous, except for cuboidal gas gland cells at the anterio‐ventral and anterio‐lateral regions of the swim bladder. During the initial inflation interval the pneumatic duct was dilated in larvae both with and without swim bladder inflation. The pneumatic duct began to regress in some larvae over 7.5 mm SL. The swim bladder of striped trumpeter was similar to larvae of other altricial perciform marine fish in respect to organ derivation, tissue differentiation, luminal dilation and initial gaseous inflation. However, variations, particularly the delay in initial swim bladder inflation until after the start of feeding, were observed that could be fundamental to problems encountered during larval rearing.
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