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Abstract

Zebrafish mutants generated by ethylnitrosourea-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 78 hours postfertilization (hpf). In this study, we sought to identify the underlying mutated gene in flo and link the genetic lesion to its phenotype. Positional cloning was employed to map the flo mutation. Subcellular characterization of flo embryos was achieved using histology, immunocytochemistry, bromodeoxyuridine incorporation analysis, and confocal and electron microscopy. 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, the human ELYS protein has been shown to play a critical, and hitherto unsuspected, role in nuclear pore assembly. Although elys messenger RNA (mRNA) is expressed broadly during early zebrafish development, widespread early defects in flo are circumvented by the persistence of maternally expressed elys mRNA until 24 hpf. From 72 hpf, 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. Our results demonstrate that Elys regulates digestive organ formation.

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... Mouse embryos homozygous for Elys die during early development due to apoptosis of the inner cells (14), while its specific depletion in the developing intestinal epithelium causes extensive apoptosis of crypt progenitor cells in juvenile mice (15). Zebrafish elys mutants also show widespread apoptosis of larval retinal and intestinal epithelial cells with altered Nups distribution (16,17). Interestingly, fly seh1 homozygous mutants are viable but mutant females have reduced fecundity (18). ...
... Alcian Blue Staining, Histology, and Immunoblotting-Alcian Blue staining, histology, and immunoblotting were performed as previously described with modifications (17,26,27). For immunoblotting, about 80 live embryos at indicated stages were anesthetized for each group and their heads were dissected. ...
... The requirement of the Nup107-160 complex components for development of specific tissues has been demonstrated in a few cases. For example, zebrafish flo/elys mutant embryos show defects in the intestine, liver, pancreas, and eyes (16,17); Fly Seh1 mutant females have some of oocytes developed as pseudo-nurse cells but their somatic tissues are unaffected (18). We have demonstrated in this study that zygotic deficiency of Nup107 in zebrafish embryos results in loss of pharyngeal skeletons in addition to degeneration of intestinal and retinal epithelia. ...
Article
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The Nup107-160 multiprotein subcomplex is essential for the assembly of nuclear pore complexes. The developmental functions of individual constituents of this subcomplex in vertebrates remain elusive. In particular, it is unknown whether Nup107 plays an important role in development of vertebrate embryos. Zebrafish nup107 is maternally expressed and its zygotic expression becomes prominent in the head region and the intestine from 24 hours postfertilization (hpf) onward. In this study, we generate a zebrafish mutant line, nup107tsu068Gt, in which the nup107 locus is disrupted by an insertion of Tol2 transposon element in the first intron and as a result it fails to produce normal transcripts. Homozygous nup107tsu068Gt mutant embryos exhibit tissue-specific defects after 3 days postfertilization (dpf), including loss of the pharyngeal skeletons, degeneration of the intestine, absence of the swim bladder, and smaller eyes. These mutants die at 5-6 days. Extensive apoptosis occurs in the affected tissues, which is partially dependent on p53 apoptotic pathways. In cells of the defective tissues, FG-repeat nucleoporins are disturbed and nuclear pore number is reduced, leading to impaired translocation of mRNAs from the nucleus to the cytoplasm. Our findings shed new light on developmental function of Nup107 in vertebrates.
... The main role of the nuclear pore is to provide a passage to facilitate the nuclear and cytoplasmic transport of mRNA and proteins. Different NPC components have been found to have critical roles in diverse developmental processes, including oogenesis (18), gastrula-* This work was supported by "973 Program" Grant 2012CB944550 and tion (19,20), neurogenesis (21), and the formation of digestive organs, pharyngeal cartilage, and eyes (22)(23)(24). Specifically, the study of a zebrafish flotte lotte (flo) mutant that carries a mutation in the elys gene showed that, in addition to displaying hypoplastic digestive organs, the flo mutant also exhibited a malformed retina due to the failure of proliferating precursors to differentiate neurons from their stem cell niche (22)(23)(24). By studying the zebrafish nup107 mutant, Zheng et al. (25) showed that Nup107 is essential for the development of most organs, including pharyngeal cartilage, intestines, and eyes. ...
... Different NPC components have been found to have critical roles in diverse developmental processes, including oogenesis (18), gastrula-* This work was supported by "973 Program" Grant 2012CB944550 and tion (19,20), neurogenesis (21), and the formation of digestive organs, pharyngeal cartilage, and eyes (22)(23)(24). Specifically, the study of a zebrafish flotte lotte (flo) mutant that carries a mutation in the elys gene showed that, in addition to displaying hypoplastic digestive organs, the flo mutant also exhibited a malformed retina due to the failure of proliferating precursors to differentiate neurons from their stem cell niche (22)(23)(24). By studying the zebrafish nup107 mutant, Zheng et al. (25) showed that Nup107 is essential for the development of most organs, including pharyngeal cartilage, intestines, and eyes. ...
... Previous reports have demonstrated that NPC activity is essential for development of retinal architecture. For example, mutation of the elys gene, which encodes a component of NPCs, not only results in defective digestive organs but also leads to a disrupted retinal structure due to the failure to differentiate the proliferating precursors to neurons from their stem cell niche (22)(23)(24). We found that the sec13 sq198 mutant, but not the sec31a/sec31b double morphant or the embryo treated with BFA, lacks a nuclear pore structure when examined by TEM and immunostaining of Nup153, Nup214, and Nup358. ...
Article
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Sec13 is a dual function protein, being a core component of both the COPII coat, which mediates protein trafficking from the endoplasmic reticulum to the Golgi apparatus, and the nuclear pore complex (NPC), which facilitates nucleo-cytoplasmic traffic. Here, we present a genetic model to differentiate the roles of these two functions of Sec13 in vivo. We report that sec13sq198 mutant embryos develop small eyes that exhibit disrupted retinal lamination and that the mutant retina contains an excessive number of apoptotic cells. Surprisingly, we found that loss of COPII function by oligonucleotide-mediated gene knockdown of sec31a and sec31b or brefeldin A treatment did not disrupt retinal lamination, although it did result in digestive organ defects similar to those seen in sec13sq198, suggesting that the digestive organ defects observed in sec13sq198 are due to loss of COPII function, whereas the retinal lamination defects are due to loss of the NPC function. We showed that the retinal cells of sec13sq198 failed to form proper nuclear pores, leading to a nuclear accumulation of total mRNA and abnormal activation of the p53-dependent apoptosis pathway, causing the retinal defect in sec13sq198. Furthermore, we found that a mutant lacking Nup107, a key NPC-specific component, phenocopied the retinal lamination phenotype as observed in sec13sq198. Our results demonstrate a requirement for the nuclear pore function of Sec13 in development of the retina and provide the first genetic evidence to differentiate the contributions of the NPC and the COPII functions of Sec13 during organogenesis.
... In this study, we use the zebrafish flotte lotte (flo) mutant to explore the mechanisms that regulate terminal cell cycle exit of RPCs in the CMZ. The flo locus encodes Elys (also known as Ahctf1) (Davuluri et al., 2008;de Jong-Curtain et al., 2009), a component of the Nup107-160 complex that localises to kinetochores during mitosis and participates in the reformation of functional nuclear pores immediately after mitosis (Rasala et al., 2006). Elys is required for cell cycle progression, playing key roles in kinetochore function (Mishra et al., 2010) and in the regulation of mitotic entrance and exit (Davuluri et al., 2008;Fernandez and Piano, 2006;Franz et al., 2007;Galy et al., 2006;Gillespie et al., 2007). ...
... AB and tupl wild-type and flotte lotte (flo ti262c ) zebrafish (Danio rerio) strains were bred and maintained according to standard procedures (Westerfield, 2000). Embryos were genotyped as described (de Jong-Curtain et al., 2009), except that the digestion products were resolved on a 2.5% Metasieve agarose (Flowgen) gel in Tris-borate-EDTA buffer (Sigma). ...
... Apoptosis at the interface between the CMZ and central retina is responsible for decreased eye growth in the flo mutant flo ti262c/ti262c embryos are initially indistinguishable from wild-type siblings, but their eyes exhibit growth defects that are evident by 2-3 days post-fertilisation (dpf) (Fig. 1A,B) (Wallace et al., 2005). Other than small eyes, mutants show no overt phenotypes until later stages, when defects in intestinal development lead to larval death (Davuluri et al., 2008;de Jong-Curtain et al., 2009;Wallace et al., 2005). In histological sections, the phenotype of flo mutants could first be identified at 2 dpf by the presence of occasional acellular holes in the retina (Fig. 1C,D). ...
Article
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It is currently unclear how intrinsic and extrinsic mechanisms cooperate to control the progression from self-renewing to neurogenic divisions in retinal precursor cells. Here, we use the zebrafish flotte lotte (flo) mutant, which carries a mutation in the elys (ahctf1) gene, to study the relationship between cell cycle progression and neuronal differentiation by investigating how proliferating progenitor cells transition towards differentiation in a retinal stem cell niche termed the ciliary marginal zone (CMZ). In zebrafish embryos without Elys, CMZ cells retain the capacity to proliferate but lose the ability to enter their final neurogenic divisions to differentiate as neurons. However, mosaic retinae composed of wild-type and flo cells show that despite inherent cell cycle defects, flo mutant cells progress from proliferation to differentiation when in the vicinity of wild-type retinal neurons. We propose that the differentiated retinal environment limits the proliferation of precursors emerging from the CMZ in a manner that explains the spatial organisation of cells in the CMZ and ensures that proliferative retinal progenitors are driven towards differentiation.
... Galy and Peter Askjaer, former Mattaj lab post-docs, identified a locus in C. elegans whose deletion resulted in pronuclear appearance defects. The gene, later termed mel-28, encodes a 200 kDa AT-hook-containing protein and has orthologues of similar size in most higher eukaryotes (de Jong-Curtain et al., 2008). Filamentous fungi and S.pombe possess significantly smaller MEL-28 orthologoues around 35 kDa (Liu et al., 2009). ...
... My work has extended her results and addressed novel aspects of MEL-28 function, as explained in subsequent parts of this thesis. Since I began working on MEL-28, five studies from other research groups dealing with MEL-28/ELYS have been published (Rasala et al., 2006;Gillespie et al., 2007;Rasala et al., 2008;Davuluri et al., 2008;de Jong-Curtain et al., 2008). ...
... MEL-28/ELYS is essential for nuclear integrity in invertebrates (Galy et al., 2006;Fernandez and Piano, 2006) and vertebrates (Davuluri et al., 2008;de Jong-Curtain et al., 2008). A point mutation in the zebrafish ELYS gene, leading to a stop codon within the coding region, gives rise to the flotte lotte (flo) mutant phenotype. ...
Article
The nuclear envelope (NE) is a highly specialized membrane system that surrounds the interphase nucleus of eukaryotic cells. Nuclear pore complexes (NPCs) form gated channels through the NE and mediate nucleocytoplasmic transport. In metazoan cells, the NE breaks down and reforms during each cell cycle. These events are tightly coordinated in space and time with the formation of the mitotic spindle and the segregation of chromosomes to the two daughter cells. At the end of mitosis, new NPCs begin to assemble on chromatin and an intact NE reforms around the decondensing chromosomes. MEL-28/ELYS is a recently identified NE protein essential for nuclear integrity and function in many organisms. Genetic mutation or RNAi depletion of MEL-28 severely impair nuclear morphology and lead to loss of NPCs from the NE in a variety of cells and organisms. Our work and that of others shows that MEL-28 is critically involved in postmitotic NPC formation, but at the same time links between MEL-28 and other cellular processes are emerging. This thesis aims at thoroughly characterizing the role of MEL-28 in nuclear assembly. It addresses the function of MEL-28 in living cells and examines the contribution of MEL-28 to nuclear assembly in vitro. MEL-28 is an NPC/INM protein in interphase and partly localizes to kinetochores in mitosis. RNAi knockdown of MEL-28 in human cells results in loss of nucleoporins (nups) from the NE, but leaves the NE membranes intact, suggesting that it is specifically involved in NPC assembly. This phenotype is mirrored by employing MEL-28-immunodepleted Xenopus laevis egg extract for nuclear assembly in vitro, which gives rise to nuclei devoid of pores. MEL-28 acts in NPC formation by targeting nups to chromatin. It interacts with a subset of nups, the Nup107-160 complex, which is a central building block of the NPC. MEL-28 binds directly to chromatin through its AT hook and additional chromatin binding motifs in its C-terminus. My data show that MEL-28 anchors the forming NPC to chromatin. Addition of high concentrations of AT hook to a nuclear assembly reaction leads to inhibition of NPC assembly and recapitulates the MEL-28 depletion phenotype. Recombinant MEL-28 rescues the recruitment of the Nup107-160 complex to chromatin, indicating that the depletion phenotype in nuclear assembly can be specifically attributed to MEL-28. The function of MEL-28 is under control of the Ran GTPase. RanGTP enhances MEL-28 and nup binding to chromatin and thus triggers NPC formation. Moreover, MEL-28 chromatin binding is regulated during the cell cycle, possibly by phosphorylation. In conclusion, this study extends our current model of postmitotic NPC formation by demonstrating that targeting of nups to chromatin is mediated by and requires MEL-28. MEL-28 function is regulated spatially by the Ran GTPase and coordinated temporally with the cell cycle. The involvement of MEL-28 in NPC formation is its best characterized function to date, but it is likely that MEL-28 has additional roles in other cellular processes. In addition, this thesis contains an initial characterization of NET5, a conserved transmembrane protein of the INM. NET5 has a well defined domain topology and localizes to foci in the NE which are not identical to nuclear pores. RNAi knockdown of NET5 in human cells perturbs nuclear integrity and leads to distortion of the NE, suggesting that it has an essential role in nuclear organization. Die Kernhülle stellt ein hochgradig spezialisiertes Membransystem dar, welches den Zellkern eukaryontischer Zellen in der Interphase umgibt. Kernporenkomplexe bilden Kanäle durch die Kernhülle und vermitteln den Transport von Molekülen zwischen Zellkerninnerem und Zytoplasma. In Vielzellern wird die Kernhülle bei jeder Zellteilung abgebaut, und bildet sich danach neu. Diese Vorgänge sind räumlich und zeitlich eng mit der Bildung der mitotischen Spindel und der Verteilung der Chromosomen auf die beiden Tochterzellen abgestimmt. Am Ende der Mitose bilden sich neue Kernporenkomplexe auf dem Chromatin, und eine intakte Kernhülle baut sich um die dekondensierenden Chromosomen auf. MEL-28/ELYS ist ein kürzlich identifiziertes Kernhüllenprotein, das in zahlreichen Organismen für die Integrität und Funktion des Zellkerns essentiell ist. Die genetische Mutation oder RNAi Depletion von MEL-28 beeinträchtigen die Gestalt des Zellkerns und führen in einer Vielzahl von Zellen und Organismen zu einem Verlust von Kernporenkomplexen von der Kernhülle. Unsere Arbeit und diejenige anderer zeigen, dass MEL-28 an der Bildung von Kernporenkomplexen am Ende der Mitose beteiligt ist. Gleichzeitig wird jedoch, deutlich, dass MEL-28 auch in andere zelluläre Vorgängen eingebunden ist. Die vorliegende Arbeit hatte das Ziel, die Rolle von MEL-28 bei der Bildung des Zellkerns umfassend zu charakterisieren. Sie beinhaltet Experimente zur Funktion von MEL-28 in lebenden Zellen und zum Beitrag von MEL-28 zur Zellkernbildung in vitro. MEL-28 ist Teil der Kernpore und der inneren Kernmembran in der Interphase, und ein Teil des zellulären MEL-28 bindet an Kinetochore in der Mitose. Die RNAi Depletion von MEL-28 in menschlichen Zellen hat den Verlust von Nukleoporinen von der Kernhülle zur Folge, nimmt jedoch keinen Einfluss auf die Kernmembran. Dieses Ergebnis legt nahe, dass MEL-28 spezifisch an der Bildung von Kernporenkomplexen beteiligt ist. Ein vergleichbares Ergebnis erhält man mit dem in vitro Kernbildungssystem, bei dem die Verwendung von MEL-28-immundepletiertem Xenopus laevis Eiextrakt zur Bildung von kernporenfreien Zellkernen führt. Die Funktion von MEL-28 besteht darin, Nukleoporine bei der Bildung des Kernporenkomplexes zum Chromatin zu führen. MEL-28 interagiert mit einer Gruppe von Nukleoporinen, dem Nup107-160 Komplex, einem zentralen Baustein des Kernporenkomplexes. Es bindet mittels seines AT hook Motifs und weiterer Chromatinbindungsmotive in seinem C-Terminus direkt an Chromatin. Meine Daten zeigen, dass MEL-28 den sich bildenden Kernporenkomplex am Chromatin verankert. Die Zugabe hoher Konzentrationen von AT hook zu einer Kernbildungsreaktion hemmt die Bildung von Kernporenkomplexen und führt zum gleichen Phänotyp wie die Depletion von MEL-28. Rekombinantes MEL-28 rettet die Bindung des Nup107-160 Komplexes an Chromatin, was verdeutlicht, dass der Depletionsphänotyp bei der Zellkernbildung spezifisch auf MEL-28 zurückzuführen ist. Die Funktion von MEL-28 wird durch die GTPase Ran reguliert. RanGTP verstärkt die Bindung von MEL-28 und Nukleoporinen an Chromatin und fördert so die Bildung von Kernporenkomplexen. Zusätzlich ist die Bindung von MEL-28 an Chromatin im Laufe des Zellzyklus reguliert, möglicherweise durch Phosphorylierung. Die vorliegende Arbeit erweitert unser Modell der Bildung von Kernporenkomplexen um die Erkenntnis, dass MEL-28 die Bindung von Nukleoporinen an Chromatin vermittelt. Die Funktion von MEL-28 ist räumlich durch die GTPase Ran reguliert und zeitlich mit dem Zellzyklus abgestimmt. Die Beteiligung an der Kernporenbildung ist die bisher bestcharakterisierte Funktion von MEL-28, aber es ist wahrscheinlich, dass es an weiteren zellulären Vorgängen beteiligt ist. Diese Arbeit umfasst zusätzlich eine initiale Charakterisierung von NET5, einem konservierten Protein der inneren Kernmembran. NET5 verfügt über eine klar definierte Topologie und ist in Foci in der Kernhülle zu finden, bei denen es sich nicht um Kernporen handelt. Die RNAi Depletion von NET5 in menschlichen Zellen zerstört die Integrität des Zellkerns und führt zu einer Auffaltung der Kernhülle. Diese Ergebnisse legen nahe, dass NET5 eine essentielle Funktion für die Organisation des Zellkerns besitzt.
... The developmental regulation of the EIT remains poorly understood, despite its detailed description decades ago (Coulombre and Coulombre, 1958;Trier and Moxey, 1979;Chalmers and Slack, 1998;Wallace et al., 2005). A promising approach to identifying genes required for the EIT has been classical genetic analysis using the zebrafish (Allende et al., 1996;Mayer and Fishman, 2003;Amsterdam et al., 2004;Yee et al., 2007;Davuluri et al., 2008;de Jong-Curtain et al., 2009). The zebrafish has distinct advantages in that morphogenesis can be quickly scored in the live embryo, and the cellular architecture of the intestine is simpler compared with mammals. ...
... The zebrafish has distinct advantages in that morphogenesis can be quickly scored in the live embryo, and the cellular architecture of the intestine is simpler compared with mammals. However, one pitfall of this approach may be that the genes implicated are required for downstream cellular processes such as ribosome biogenesis, RNA processing and transport, and protein biosynthesis, rather than serving in regulatory roles (Allende et al., 1996;Pack et al., 1996;Mayer and Fishman, 2003;Amsterdam et al., 2004;Yee et al., 2007;Davuluri et al., 2008;de Jong-Curtain et al., 2009). Nonetheless, these cited studies offer a hint that the EIT might be regulated by a common upstream pathway that controls anabolic growth, such as TOR kinase (Hwang et al., 2008). ...
Article
The intestinal epithelium arises from undifferentiated endoderm via a developmental program known as the endoderm-intestine transition (EIT). Previously we found that the target of rapamycin complex 1 (TORC1) regulates intestinal growth and differentiation during the EIT in zebrafish. Here we address a possible role for the tumor-suppressor kinase Lkb1 in regulating TORC1 in this context. We find that TORC1 activity is transiently upregulated during the EIT in both zebrafish and mouse. Concomitantly, Lkb1 becomes transiently localized to the nucleus, suggesting that these two phenomena may be linked. Morpholino-mediated knockdown of lkb1 stimulated intestinal growth via upregulation of TORC1, and also induced precocious intestine-specific gene expression in the zebrafish gut epithelium. Knockdown of tsc2, which acts downstream of lkb1, likewise induced early expression of intestine-specific genes. These data suggest that programmed localization of Lkb1 could represent a novel mechanism for regulating the EIT during intestinal development in vertebrates.
... The NPC defect within these cells causes development arrest and apoptotic cell death. A non-complementing zebrafish elys allele was also reported to disrupt NPC assembly and was associated with epithelial cell apoptosis in the developing intestine 9 . We have shown that apoptosis in flo intestinal progenitors occurs independently of p53 and is potentiated by DNA replication inhibitors, such as hydroxyurea and UV-irradiation 2 . ...
... The distribution of FG-Nups in Elys-deficient intestinal epithelial cells at all stages examined was indistinguishable from control intestinal cells ( Figure 3A, B). Cytoplasmic membrane bound nucleoporins (annulate lamellae) indicative of defective NPC assembly were not detected in the Elys-deficient intestines, whereas they are abundant in zebrafish flo mutants and Elys-deficient mammalian cells 2,4,7,9 . Western blot analyses of nuclear and cytoplasmic fractions showed only minimal levels of cytoplasmic FG-nup nucleoporins in mutant cells ( Figure 3D). ...
Article
Elys is a conserved protein that directs nuclear pore complex (NPC) assembly in mammalian cell lines and developing worms and zebrafish. Related studies in these systems indicate a role for Elys in DNA replication and repair. Intestinal epithelial progenitors of zebrafish elys mutants undergo apoptosis early in development. However, it is not known whether loss of Elys has a similar effect in the mammalian intestine or whether the NPC and DNA repair defects each contribute to the overall phenotype. We developed mice in which a conditional Elys allele was inactivated in the developing intestinal epithelium and during preimplantation development. Phenotypes of conditional mutant mice were determined using immunohistochemical analysis for nuclear pore proteins, electron microscopy, and immunoblot analysis of DNA replication and repair proteins. Conditional inactivation of the Elys locus in the developing mouse intestinal epithelium led to a reversible delay in growth in juvenile mice that was associated with epithelial architecture distortion and crypt cell apoptosis. The phenotype was reduced in adult mutant mice, which were otherwise indistinguishable from wild-type mice. All mice had activated DNA damage responses but no evidence of NPC assembly defects. In mice, Elys maintains genome stability in intestinal epithelial progenitor cells, independent of its role in NPC assembly in zebrafish.
... Alternatively, Nol9 may have tissue-specific interacting partners and extra-ribosomal functions in the digestive organs and in HSPCs [56,57]. Furthermore, it has been proposed that the spectrum of translated mRNAs might change with the overall reduction of fully functional cytoplasmic ribosomes or specific ribosomal biogenesis proteins, suggesting that ribosome composition may vary between distinct cell types [56][57][58]. Another explanation for the tissue specific effect of nol9 depletion stems from the high proliferative rate of the affected tissues. ...
... The reason for this is twofold. Firstly, it is reasonable to speculate, that highly proliferative cell populations, such as pancreatic cells and HSPCs, deplete maternally-derived Nol9 protein earlier than other cell populations [58]. And secondly, defects in ribosomal biogenesis affect primarily highly proliferative cells due to their high demand for ribosome production. ...
Article
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Ribosome biogenesis is a ubiquitous and essential process in cells. Defects in ribosome biogenesis and function result in a group of human disorders, collectively known as ribosomopathies. In this study, we describe a zebrafish mutant with a loss-of-function mutation in nol9, a gene that encodes a non-ribosomal protein involved in rRNA processing. nol9sa1022/sa1022 mutants have a defect in 28S rRNA processing. The nol9sa1022/sa1022 larvae display hypoplastic pancreas, liver and intestine and have decreased numbers of hematopoietic stem and progenitor cells (HSPCs), as well as definitive erythrocytes and lymphocytes. In addition, ultrastructural analysis revealed signs of pathological processes occurring in endothelial cells of the caudal vein, emphasizing the complexity of the phenotype observed in nol9sa1022/sa1022 larvae. We further show that both the pancreatic and hematopoietic deficiencies in nol9sa1022/sa1022 embryos were due to impaired cell proliferation of respective progenitor cells. Interestingly, genetic loss of Tp53 rescued the HSPCs but not the pancreatic defects. In contrast, activation of mRNA translation via the mTOR pathway by L-Leucine treatment did not revert the erythroid or pancreatic defects. Together, we present the nol9sa1022/sa1022 mutant, a novel zebrafish ribosomopathy model, which recapitulates key human disease characteristics. The use of this genetically tractable model will enhance our understanding of the tissue-specific mechanisms following impaired ribosome biogenesis in the context of an intact vertebrate.
... The nodal factors Cyclop and Squint, and the Sox-type factors, Casanova and Sox17, are essential for the specification of the whole endoderm. In contrast, Gata factors (Gata4/5/6), FoxA factors (FoxA1/2/3), and other factors, such as Def, Npo and Elys, are essential for the normal organogenesis of the entire digestive system (Chen et al., 2005;de Jong-Curtain et al., 2009;Mayer and Fishman, 2003). The signaling molecules Bmp2a/b, Fgf, and Wnt2bb are apparently crucial factors for the development of the liver and pancreas (Huang et al., 2008;Ober et al., 2006;Shin et al., 2007). ...
... Therefore, whether this function of Sec13 is related to the observed organ-specific subcellular defects in the mutant fish will be of great interest in our future study. Meanwhile, it would be interesting to use sec13 sq198 to study the role of the nuclear pore complex structure in organogenesis in the future (de Jong-Curtain et al., 2009). Although the currently known function of Sec31 is solely attributed to form the COPII complex we cannot exclude the possibility that Sec31 might function independently in the organogenesis of the digestive organs. ...
Article
Full-text available
The Sec13-Sec31 heterotetramer serves as the outer coat in the COPII complex, which mediates protein trafficking from the endoplasmic reticulum (ER) to the Golgi apparatus. Although it has been studied in depth in yeast and cultured cells, the role of COPII in organogenesis in a multicellular organism has not. We report here that a zebrafish sec13(sq198) mutant, which exhibits a phenotype of hypoplastic digestive organs, has a mutation in the sec13 gene. The mutant gene encodes a carboxyl-terminus-truncated Sec13 that loses its affinity to Sec31a, which leads to disintegration of the ER structure in various differentiated cells in sec13(sq198), including chondrocytes, intestinal epithelial cells and hepatocytes. Disruption of the ER structure activates an unfolded protein response that eventually causes the cells to undergo cell-cycle arrest and cell apoptosis, which arrest the growth of developing digestive organs in the mutant. Our data provide the first direct genetic evidence that COPII function is essential for the organogenesis of the digestive system.
... ELYS orthologue in zebrafish, flotte lotte (flo), is critically required for early embryonic and pharyngeal skeleton development (15). Additionally, flo deletion disrupted NPC formation and defective nuclear import induced replication stress in intestinal progenitor cells (16). ...
... The ubiquitous knockdown of dElys, induced developmental defects, and lethality in Drosophila (Fig. 2) mirroring the defects observed in other organisms following the conserved function of ELYS molecules (6,11,53,54). The dElys phenocopies nucleoporin functions of ELYS molecule characterized in other organisms and is involved in nucleo-cytoplasmic transport regulation, NPC assembly and interacts with nuclear lamina proteins (Fig. 3) as reported in mammalian cell lines, C. elegans and Xenopus egg extract studies (6,7,9,16,28,53), suggesting a conserved functional role in Drosophila. Nucleocytoplasmic transport defect observed in dElys depletion highlights its conserved role as a nucleoporin (7,10,55-57). ...
Article
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Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes (NPCs) into functional nuclear pores toward the end of mitosis. Using cellular, molecular and genetic tools, including fluorescence and electron microscopy, quantitative PCR and RNAi mediated depletion, here; we report that ELYS ortholog ( d Elys) plays critical roles during Drosophila development. d Elys localized to the nuclear rim in interphase cells, but during mitosis, it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid . The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that d Elys has conserved structural features, but contains a non-canonical AT-hook like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by d Elys during development.
... A few studies have proposed the implications of nucleoporins in PA formation [32,59], but functions and mechanisms of nucleoporins during the morphogenesis of craniofacial cartilages in vertebrates remain to be well explored. In this study, we demonstrated the pivotal roles of zebrafish Nup62l in the development of PA cartilages. ...
Article
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We have previously observed the predominant expression of nucleoporin 62-like (Nup62l) mRNA in the pharyngeal region of zebrafish, which raises the question whether Nup62l has important implications in governing the morphogenesis of pharyngeal arches (PA) in zebrafish. Herein, we explored the functions of Nup62l in PA development. The disruption of Nup62l with a CRISPR/Cas9-dependent gene knockout approach led to defective PA, which was characterized by a thinned and shortened pharyngeal region and a significant loss of pharyngeal cartilages. During pharyngeal cartilage formation, prechondrogenic condensation and chondrogenic differentiation were disrupted in homozygous nup62l-mutants, while the specification and migration of cranial neural crest cells (CNCCs) were unaffected. Mechanistically, the impaired PA region of nup62l-mutants underwent extensive apoptosis, which was mainly dependent on activation of p53-dependent apoptotic pathway. Moreover, aberrant activation of a series of apoptotic pathways in nup62l-mutants is closely associated with the inactivation of Wnt/β-catenin signaling. Thus, these findings suggest that the regulation of Wnt/β-catenin activity by Nup62l is crucial for PA formation in zebrafish.
... Another example is mouse Nup133, which was shown to play a role in embryonic development of the neural lineage (Lupu et al., 2008). Furthermore, genetic mutation of the zebrafish ELYS inhibits normal development and proliferation of the retina and the intestine (Davuluri et al., 2008;de Jong-Curtain et al., 2008). It remains to be seen if these effects reflect a role for these Nups in gene regulation. ...
Article
Over the last decade, the nuclear envelope (NE) has emerged as a key component in the organization and function of the nuclear genome. As many as 100 different proteins are thought to specifically localize to this double membrane that separates the cytoplasm and the nucleoplasm of eukaryotic cells. Selective portals through the NE are formed at sites where the inner and outer nuclear membranes are fused, and the coincident assembly of approximately 30 proteins into nuclear pore complexes occurs. These nuclear pore complexes are essential for the control of nucleocytoplasmic exchange. Many of the NE and nuclear pore proteins are thought to play crucial roles in gene regulation and thus are increasingly linked to human diseases.
... Based on larval survival and the appearance of skin damage, we carried out subsequent analyzes at 3 days of exposure to 50 mg/ml TNBS, unless otherwise indicated. Development of the eye, containing rapidly proliferative tissue, was used as an indicator of extra-intestinal effects of TNBS exposure on growth rate (de Jong-Curtain et al., 2009;Parichy et al., 2009). Measurement of larval eye diameter revealed no significant difference between TNBS-treated and control samples (97.4% 6 1.0% and 100% 6 1.2%, respectively, P ¼ 0.10). ...
Article
Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens.
... The Drosophila homolog of Nup155, Nup154, has also been shown to have a tissue-specific role, but here instead affecting gametogenesis in both sexes (Gigliotti et al. 1998;Grimaldi et al. 2007). Furthermore, a mutation in the zebrafish ELYS was described to affect normal development and proliferation of the retina and the intestine (Davuluri et al. 2008;de Jong-Curtain et al. 2008). Mutations in several plant NUPs have been reported to cause diverse developmental defects, which often appear comparatively mild and relatively specific (Meier and Brkljacic 2009). ...
Article
Eukaryotic cell function depends on the physical separation of nucleoplasmic and cytoplasmic components by the nuclear envelope (NE). Molecular communication between the two compartments involves active, signal-mediated trafficking, a function that is exclusively performed by nuclear pore complexes (NPCs). The individual NPC components and the mechanisms that are involved in nuclear trafficking are well documented and have become textbook knowledge. However, in addition to their roles as nuclear gatekeepers, NPC components-nucleoporins-have been shown to have critical roles in chromatin organization and gene regulation. These findings have sparked new enthusiasm to study the roles of this multiprotein complex in nuclear organization and explore novel functions that in some cases appear to go beyond a role in transport. Here, we discuss our present view of NPC biogenesis, which is tightly linked to proper cell cycle progression and cell differentiation. In addition, we summarize new data suggesting that NPCs represent dynamic hubs for the integration of gene regulation and nuclear transport processes.
... colourless, one of the first ENS mutants to be isolated, results from a lesion in the sox10 gene (Dutton et al., 2001;Kelsh and Eisen, 2000) which was already known to be important in ENS development in mammals (Southard Smith et al., 1998). Zebrafish screens have also identified other mutations in genes not previously associated with ENS development (Chen et al., 1996;Schilling et al., 1996), for example, the DNA polymerase delta 1 mutant flathead (pold1), the elys nucleopore assembly protein mutant flotte lotte (ahctfi) and the rpc2, RNA polymerase III subunit mutant slim jim (polr3b) (Davuluri et al., 2008;de Jong-Curtain et al., 2008;Plaster et al., 2006;Wallace et al., 2005;Yee et al., 2007). All of these mutants have pleiotropic phenotypes and thus they were not identified based on ENS defects. ...
Article
The enteric nervous system (ENS) is composed of neurons and glia that modulate many aspects of intestinal function. The ability to use both forward and reverse genetic approaches and to visualize development in living embryos and larvae has made zebrafish an attractive model in which to study mechanisms underlying ENS development. In this chapter, we review the recent work describing the development and organization of the zebrafish ENS and how this relates to intestinal motility. We also discuss the cellular, molecular, and genetic mechanisms that have been revealed by these studies and how they are providing new insights into human ENS diseases.
... Furthermore, mutations in at least four nucleoporins, Aladin, Nup62, Nup155 and Nup358 give rise to diseases with tissue-specific phenotypes (Basel-Vanagaite et al., 2006; Cronshaw and Matunis, 2003; Neilson et al., 2009; Zhang et al., 2008). Finally, studies in transgenic knock-out animals have shown that the depletion of several nucleoporins specifically affect the function of distinct tissues or cells (de Jong-Curtain et al., 2009; Faria et al., 2006; Lupu et al., 2008; Smitherman et al., 2000). The existence of cell-type specific nuclear pores was previously suggested in a study where the tissue-specific expression of the scaffold nucleoporin Nup133 was determined by in situ hybridization (Lupu et al., 2008). ...
Article
Nuclear pore complexes (NPCs) are built from ∼30 different proteins called nucleoporins or Nups. Previous studies have shown that several Nups exhibit cell-type-specific expression and that mutations in NPC components result in tissue-specific diseases. Here we show that a specific change in NPC composition is required for both myogenic and neuronal differentiation. The transmembrane nucleoporin Nup210 is absent in proliferating myoblasts and embryonic stem cells (ESCs) but becomes expressed and incorporated into NPCs during cell differentiation. Preventing Nup210 production by RNAi blocks myogenesis and the differentiation of ESCs into neuroprogenitors. We found that the addition of Nup210 to NPCs does not affect nuclear transport but is required for the induction of genes that are essential for cell differentiation. Our results identify a single change in NPC composition as an essential step in cell differentiation and establish a role for Nup210 in gene expression regulation and cell fate determination.
... Of note, these tissues with prominent gle1 expression are the same as those that show the most pronounced phenotypes in gle1 mutants at later stages. Furthermore, these gle1-enriched tissues are those that maintain populations of highly proliferative cells at early larval stages (e.g. 3 dpf) (de Jong-Curtain et al., 2009). Therefore, we propose that although Gle1 is likely to be required in all cells, highly proliferative cells demand a higher level of Gle1 to support the robust metabolic activity required during proliferation. ...
Article
In humans, GLE1 is mutated in lethal congenital contracture syndrome 1 (LCCS1) leading to prenatal death of all affected fetuses. Although the molecular roles of Gle1 in nuclear mRNA export and translation have been documented, no animal models for this disease have been reported. To elucidate the function of Gle1 in vertebrate development, we used the zebrafish (Danio rerio) model system. gle1 mRNA is maternally deposited and widely expressed. Altering Gle1 using an insertional mutant or antisense morpholinos results in multiple defects, including immobility, small eyes, diminished pharyngeal arches, curved body axis, edema, underdeveloped intestine and cell death in the central nervous system. These phenotypes parallel those observed in LCCS1 human fetuses. Gle1 depletion also results in reduction of motoneurons and aberrant arborization of motor axons. Unexpectedly, the motoneuron deficiency results from apoptosis of neural precursors, not of differentiated motoneurons. Mosaic analyses further indicate that Gle1 activity is required extrinsically in the environment for normal motor axon arborization. Importantly, the zebrafish phenotypes caused by Gle1 deficiency are only rescued by expressing wild-type human GLE1 and not by the disease-linked Fin(Major) mutant form of GLE1. Together, our studies provide the first functional characterization of Gle1 in vertebrate development and reveal its essential role in actively dividing cells. We propose that defective GLE1 function in human LCCS1 results in both neurogenic and non-neurogenic defects linked to the apoptosis of proliferative organ precursors.
... Sec13 may have a role in the differentiation of retinal cells, according to a study conducted on Sec13 sq198 mutant zebrafish (Niu et al., 2014). Surprisingly, another member of this complex, ELYS, influenced the proliferation and development of neuronal, retinal, and intestinal cells of zebrafish (Davuluri et al., 2008;de Jong-Curtain et al., 2009). FIGURE 1 | Structure of nuclear pore complex and Nups subcomplexes. ...
Article
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Bidirectional molecular movements between the nucleus and cytoplasm take place through nuclear pore complexes (NPCs) embedded in the nuclear membrane. These macromolecular structures are composed of several nucleoporins, which form seven different subcomplexes based on their biochemical affinity. These nucleoporins are integral components of the complex, not only allowing passive transport but also interacting with importin, exportin, and other molecules that are required for transport of protein in various cellular processes. Transport of different proteins is carried out either dependently or independently on transport receptors. As well as facilitating nucleocytoplasmic transport, nucleoporins also play an important role in cell differentiation, possibly by their direct gene interaction. This review will cover the general role of nucleoporins (whether its dependent or independent) and nucleocytoplasmic transport receptors in cell differentiation.
... Once the optimal cycle number is determined, all other TSP primer design parameters and reaction conditions remain the same. TSP has been successfully deployed for SNP geno-typing in zebrafish [24], human [25] and mouse (unpublished data) using allele-specific PCR. These assays were performed using the TSP cycling conditions described for barley, and between 20 and 50 ng of genomic DNA as starting template. ...
Article
Full-text available
Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays. We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method. Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.
... Mutations in nups important for NPC assembly can also impact differentiation programs (Lupu et al., 2008; de Jong­Curtain et al., 2009; D'Angelo et al., 2012). These studies cumulatively suggest that NPCs themselves might be important for cell fate determination and underscore the importance of identifying mechanisms that control NPC number. ...
Article
Full-text available
Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear-cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.
... Thus, the mia40a mutation could trigger functional abnormalities especially in Ca 2+ signalling-dependent acinar cells. Finally, several mutants identified in ENU mutagenesis screens showed a smaller pancreas phenotype [67][68][69]. Interestingly, the mitomess (mms) zebrafish mutant, which presents dileted mitochondria in acinar cells, was additionally characterised with decreased levels of acinar-specific markers, as is observed in our mia40a -/model [67]. ...
Article
Full-text available
Development and function of tissues and organs are powered by the activity of mitochondria. In humans, inherited genetic mutations that lead to progressive mitochondrial pathology often manifest during infancy and can lead to death, reflecting the indispensable nature of mitochondrial biogenesis and function. Here, we describe a zebrafish mutant for the gene mia40a (chchd4a), the life-essential homologue of the evolutionarily conserved Mia40 oxidoreductase which drives the biogenesis of cysteine-rich mitochondrial proteins. We report that mia40a mutant animals undergo progressive cellular respiration defects and develop enlarged mitochondria in skeletal muscles before their ultimate death at the larval stage. We generated a deep transcriptomic and proteomic resource that allowed us to identify abnormalities in the development and physiology of endodermal organs, in particular the liver and pancreas. We identify the acinar cells of the exocrine pancreas to be severely affected by mutations in the MIA pathway. Our data contribute to a better understanding of the molecular, cellular and organismal effects of mitochondrial deficiency, important for the accurate diagnosis and future treatment strategies of mitochondrial diseases.
... On closed 30 min 14°C nuclear intermediates (CNE), i.e., nuclei that did not stain with Alexa647-labeled anti-DNA antibody ( POM121 is an integral membrane nuclear pore protein (Hallberg et al., 1993) recruited to the chromatin with the membrane vesicles. Nup133 is a signature member of the Nup107-160 complex, known to be key to the initiation of NPC assembly, which is recruited to chromatin by the poretargeting chromatin-binding protein ELYS (Mishra et al., 2010;Li et al., 1995;Goldstein et al., 1996;Belgareh et al., 2001;Griffis et al., 2003;Walther et al., 2003a;Blower et al., 2005;Fernandez and Piano, 2006;Galy et al., 2006;Orjalo et al., 2006;Rasala et al., 2006;Franz et al., 2007;Ratner et al., 2007;Chakraborty et al., 2008;Davuluri et al., 2008;de Jong-Curtain et al., 2009;Lau et al., 2009;Rotem et al., 2009) Observation of Nup133 is taken as indicative of the presence of the entire Nup107-160 complex on the chromatin (Vasu et al., 2001;Boehmer and Schwartz, 2007;Rotem et al., 2009). A time course of the recruitment of POM121 compared to that of FG nucleoporins ( Figure 2B), and Nup133 compared to FG nucleoporins ( Figure 2C) was performed on non-intact nuclei. ...
Article
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Nuclear pore complexes (NPCs) are large proteinaceous channels embedded in double nuclear membranes, which carry out nucleocytoplasmic exchange. The mechanism of nuclear pore assembly involves a unique challenge, as it requires creation of a long-lived membrane-lined channel connecting the inner and outer nuclear membranes. This stabilized membrane channel has little evolutionary precedent. Here we mapped inner/outer nuclear membrane fusion in NPC assembly biochemically by using novel assembly intermediates and membrane fusion inhibitors. Incubation of a Xenopus in vitro nuclear assembly system at 14°C revealed an early pore intermediate where nucleoporin subunits POM121 and the Nup107-160 complex were organized in a punctate pattern on the inner nuclear membrane. With time, this intermediate progressed to diffusion channel formation and finally to complete nuclear pore assembly. Correct channel formation was blocked by the hemifusion inhibitor lysophosphatidylcholine (LPC), but not if a complementary-shaped lipid, oleic acid (OA), was simultaneously added, as determined with a novel fluorescent dextran-quenching assay. Importantly, recruitment of the bulk of FG nucleoporins, characteristic of mature nuclear pores, was not observed before diffusion channel formation and was prevented by LPC or OA, but not by LPC+OA. These results map the crucial inner/outer nuclear membrane fusion event of NPC assembly downstream of POM121/Nup107-160 complex interaction and upstream or at the time of FG nucleoporin recruitment.
... Mouse Nup133 is essential for differentiation of the neural lineage (11). Genetic mutation of zebrafish elys, encoding a component of the Nup107-160 complex, perturbs proper development and proliferation of the retina and intestine, with massive apoptosis in these affected tissues (12,13). Zebrafish nup107 mutants exhibit defects in the pharyngeal arches, eyes, and intestine due to the disruption of nuclear pores and mRNA transportation (14). ...
Article
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Nucleoporin p62 (Nup62) localizes in the central channel of nuclear pore complexes (NPCs) and regulates nuclear pore permeability and nucleocytoplasmic transport. However, developmental roles of Nup62 in vertebrates remain largely unclear. Zebrafish Nup62-like protein (Nup62l) is a homolog of mammalian Nup62. The nup62l gene is maternally expressed, but its transcripts are ubiquitously distributed during early embryogenesis and enriched in the head, pharynx and intestine of developing embryos. Activation of Wnt/β-catenin pathway positively modulates the nup62l transcription, while Bmp signaling acts downstream of Wnt/β-catenin signaling to negatively regulate the nup62l expression. Overexpression of nup62l dorsalized embryos and enhanced gastrula convergence and extension (CE) movements. Contrarily, knockdown of Nup62l leads to ventralized embryos, an impediment to CE movements and defects in specification of midline organ progenitors. Mechanistically, Nup62l acts as an activator of Wnt/β-catenin signaling through interaction with and facilitating nuclear import of β-catenin-1/2 in zebrafish. Thus, Nup62l regulates dorsoventral patterning, gastrula CE movements and proper specification of midline organ precursors through mediating the nuclear import of β-catenins in zebrafish. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
... Furthermore, the recruitment of ELYS and the Nup107-160 complex to chromatin was found to be a prerequisite for the recruitment of the integral membrane pore proteins POM121 and NDC1, followed by the bulk of the soluble nucleoporins, including the FG repeat nucleoporins (Rasala et al., 2008), to form the final mature nuclear pore perforating the double nuclear membranes. Interestingly, a fraction of ELYS moves with the Nup107-160 complex to kinetochores at mitosis, and mutation or RNAi of either causes cell cycle defects in yeast, C. elegans, zebrafish, and humans (Bai et al., 2004; Fernandez and Piano, 2006; Rasala et al., 2006; Davuluri et al., 2008; de Jong-Curtain et al., 2008). Clearly, the formation of large cellular structures such as the mitotic spindle, nuclear membranes, and nuclear pores would be predicted to be the subject of careful and coordinated regulation. ...
Article
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Mitosis in higher eukaryotes is marked by the sequential assembly of two massive structures: the mitotic spindle and the nucleus. Nuclear assembly itself requires the precise formation of both nuclear membranes and nuclear pore complexes. Previously, importin alpha/beta and RanGTP were shown to act as dueling regulators to ensure that these assembly processes occur only in the vicinity of the mitotic chromosomes. We now find that the distantly related karyopherin, transportin, negatively regulates nuclear envelope fusion and nuclear pore assembly in Xenopus egg extracts. We show that transportin-and importin beta-initiate their regulation as early as the first known step of nuclear pore assembly: recruitment of the critical pore-targeting nucleoporin ELYS/MEL-28 to chromatin. Indeed, each karyopherin can interact directly with ELYS. We further define the nucleoporin subunit targets for transportin and importin beta and find them to be largely the same: ELYS, the Nup107/160 complex, Nup53, and the FG nucleoporins. Equally importantly, we find that transportin negatively regulates mitotic spindle assembly. These negative regulatory events are counteracted by RanGTP. We conclude that the interplay of the two negative regulators, transportin and importin beta, along with the positive regulator RanGTP, allows precise choreography of multiple cell cycle assembly events.
... Mitochondrial homeostasis plays an important role in the maintenance of hepatocyte survival as illustrated by the rapid liver degeneration observed in zebrafish embryos with defects in mitochondrial transport (tom22) [43], mitochondrial RNA helicase activity (supv3L1) [44] and mitochondrial antioxidant activity (trx2) [45]. Some zebrafish mutants such as def [46,47], elys [48,49], ssrp1a [50] and bms1l [51] exhibit liver hypoplasia due to nucleolar defects that culminate in replication stress or a DNA damage response. In similar work, Sadler and colleagues have determined that uhrf1 mutants developed a small-for-size liver during development due to DNA hypomethylation, which leads to cell cycle arrest and the induction of apoptosis [52][53][54][55]. ...
Article
The liver is an essential organ that plays a pivotal role in metabolism, digestion and nutrient storage. Major efforts have been made to develop zebrafish (Danio rerio) as a model system to study the pathways regulating hepatic growth during liver development and regeneration. Zebrafish offer unique advantages over other vertebrates including in vivo imaging at cellular resolution and the capacity for large-scale chemical and genetic screens. Here, we review the cellular and molecular mechanisms that regulate hepatic growth during liver development in zebrafish. We also highlight emerging evidence that developmental pathways are reactivated following liver injury to facilitate regeneration. Finally, we discuss how zebrafish have transformed drug discovery efforts and enabled the identification of drugs that stimulate hepatic growth and provide hepatoprotection in pre-clinical models of liver injury, with the ultimate goal of identifying novel therapeutic approaches to treat liver disease. Copyright © 2015 Elsevier Ltd. All rights reserved.
... Nup133, another stable Nup of the NPC scaffold, was found to be required for neuronal differentiation in the mouse embryo, and ESCs carrying a functionally null mutation in Nup133 are not able to undergo terminal differentiation into neurons (Lupu et al., 2008). Interestingly, a component of the same NPC scaffold sub-complex, ELYS, affects neuronal, retinal and intestinal development and proliferation in zebrafish (Davuluri et al., 2008;de Jong-Curtain et al., 2009). A large number of plant Nups, including Nup96, Nup160, ELYS and Tpr, have been reported to affect a diverse array of tissue specific processes, such as flowering, hormone signaling and immune function (Meier and Brkljacic, 2009). ...
Article
Full-text available
The eukaryotic cell nucleus houses an organism's genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate.
... Re-analysis of previously published genetic models of disrupted intestinal morphology (e.g. titania s450 and flotte lotte [49][50][51]) in the Tg(tnfa:GFP) and Tg(NFkB:EGFP) backgrounds or immune cell backgrounds will provide further insight into the onset of IEC integrity defects. Of particular interest is the titania s450 mutant, which displays an increase in IEC autophagosomes [49], as autophagy has been implicated in IBD onset [52]. ...
Article
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Inflammatory bowel diseases (IBD), which include Crohn’s disease and ulcerative colitis, contribute to significant morbidity and mortality globally. Despite an increase in incidence, IBD onset is still poorly understood. Mouse models of IBD recapitulate several aspects of human disease, but limited accessibility for live imaging and the lack of forward genetics highlight the need for new model systems for disease onset characterization. Zebrafish represent a powerful platform to model IBD using forward and reverse genetics, live imaging of transgenic lines, and physiological assays. In this review, we address current models of IBD in zebrafish and newly developed reagents available for future studies.
... Moreover, mutations in Nup133 are related to poor embryonic neural development (Lupu et al. 2008). ELYS (another NPC protein) mutation inhibits normal development in retina and intestine (de Jong Curtain et al. 2009). Furthermore, some authors link primary biliary cirrhosis with damage in Nup62 (Tartakovsky and Worman 1995). ...
Article
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Abstract The Nuclear Pore Complex (NPC) is a protein channel that communicates and transports molecules between the cytoplasm and the nucleoplasm. It has a complex structure composed of many structural proteins, mainly nucleoporins and transporter proteins, and its biosynthesis is an extremely regulated and cell cycle phase dependent process. Moreover, in the present review, it will be shown that this structure could play a decisive role in gene expression and in the pathogenesis of several diseases. On the one hand, NPC affects gene expression by regulating epigenetic enzymes and affecting the nucleocytoplasmic balance of transcription factors and small nuclear RNAs. On the other hand, diverse studies show that NPC is involved in some diseases and pathological processes like aging, neurodegenerative diseases and extrapyramidal syndromes, cancer, cardiovascular diseases, infectious diseases and genetic syndromes. In conclusion, NPC might be an important element in the control of gene transcription and cell phenotype, and perhaps, it will become a future pharmacologic target for several diseases in which it is involved in.
... Furthermore, the recruitment of ELYS and the Nup107-160 complex to chromatin was found to be a prerequisite for the recruitment of the integral membrane pore proteins POM121 and NDC1, followed by the bulk of the soluble nucleoporins, including the FG repeat nucleoporins , to form the final mature nuclear pore perforating the double nuclear membranes. Interestingly, a fraction of ELYS moves with the Nup107-160 complex to kinetochores at mitosis, and mutation or RNAi of either causes cell cycle defects in yeast, Caenorhabditis elegans, zebrafish, and humans (Bai et al., 2004;Fernandez and Piano, 2006;Rasala et al., 2006;Davuluri et al., 2008;de Jong-Curtain et al., 2009). ...
... Finally, the alteration of the total number of nuclear pores or transport rates also has profound effects on different tissue types during development. The reduction of zebrafish nucleoporin ELYS (Flo) leads to tissue defects and apoptosis during early development of highly proliferating intestinal epithelial cells [117]. ...
Article
Over the past few decades, it is increasingly evident that nucleoporins are multi-functional proteins that are not only pivotal for the formation of the nuclear pore complex. They also have key roles in mitosis, gene expression, development and disease. In this review, the versatility and functions of nucleoporins outside the NPC will be discussed.
... Scale bars represent 50 mm. zebrafish mutations in the nucleoporin gene elys trigger apoptosis in the gut but not fluid accumulation [23, 24]. Our data suggest a functional interaction between Cse1l and CFTR. ...
Article
Transport of chloride through the cystic fibrosis transmembrane conductance regulator (CFTR) channel is a key step in regulating fluid secretion in vertebrates [1, 2]. Loss of CFTR function leads to cystic fibrosis [1, 3, 4], a disease that affects the lungs, pancreas, liver, intestine, and vas deferens. Conversely, uncontrolled activation of the channel leads to increased fluid secretion and plays a major role in several diseases and conditions including cholera [5, 6] and other secretory diarrheas [7] as well as polycystic kidney disease [8-10]. Understanding how CFTR activity is regulated in vivo has been limited by the lack of a genetic model. Here, we used a forward genetic approach in zebrafish to uncover CFTR regulators. We report the identification, isolation, and characterization of a mutation in the zebrafish cse1l gene that leads to the sudden and dramatic expansion of the gut tube. We show that this phenotype results from a rapid accumulation of fluid due to the uncontrolled activation of the CFTR channel. Analyses in zebrafish larvae and mammalian cells indicate that Cse1l is a negative regulator of CFTR-dependent fluid secretion. This work demonstrates the importance of fluid homeostasis in development and establishes the zebrafish as a much-needed model system to study CFTR regulation in vivo.
Article
The zebrafish is a powerful vertebrate system with great advantages for both forward and reverse genetic screens and as a model for human disease conditions. Light microscopy has been used extensively to study zebrafish development but less frequently have these studies been combined with ultrastructural information. Zebrafish embryos are ideal for electron microscopy (EM) with a single transverse section containing many different cell types and tissues. However, conventional methods of EM do not provide optimal preservation of all tissues and are usually incompatible with immunolabelling and visualisation of expressed fluorescently tagged proteins. Here we examine methods that overcome these problems. We summarise a range of methods, applicable to the ultrastructural analysis of zebrafish embryos, including methods for fast freezing and processing of zebrafish embryos. These methods preserve antigenicity, ultrastructure and GFP fluorescence even after embedding in resin. In addition, they are compatible with electron tomography. These methods provide a new set of research tools that provide an additional level of information, complementing current methods for study of this widely used model system.
Article
Nuclear-pore complexes (NPCs) are large protein channels that span the nuclear envelope (NE), which is a double membrane that encloses the nuclear genome of eukaryotes. Each of the typically 2,000-4,000 pores in the NE of vertebrate cells is composed of multiple copies of 30 different proteins known as nucleoporins. The evolutionarily conserved NPC proteins have the well-characterized function of mediating the transport of molecules between the nucleoplasm and the cytoplasm. Mutations in nucleoporins are often linked to specific developmental defects and disease, and the resulting phenotypes are usually interpreted as the consequences of perturbed nuclear transport activity. However, recent evidence suggests that NPCs have additional functions in chromatin organization and gene regulation, some of which might be independent of nuclear transport. Here, we review the transport-dependent and transport-independent roles of NPCs in the regulation of nuclear function and gene expression.
Preprint
The nucleoporin ELYS, encoded by AHCTF1 , is a large multifunctional protein with essential roles in nuclear pore assembly and mitosis. Using a zebrafish model of hepatocellular carcinoma, in which the expression of an inducible mutant kras transgene (kras G12V ) drives hepatocyte-specific hyperplasia and liver enlargement, we show that reducing ahctf1 gene dosage by 50% markedly shrinks tumour burden, while non-hyperplastic tissues are unaffected. We demonstrate that ahctf1 heterozygosity impairs nuclear pore formation, mitotic spindle assembly and chromosome segregation, leading to DNA damage and activation of TP53-dependent and independent mechanisms of cell death and cell cycle arrest. This selective vulnerability of cancer cells to mild disruption of Elys function uncovers a novel synthetic lethal interaction between ahctf1 and oncogenic kras that could be exploited therapeutically. Heterozygous expression of both ahctf1 and ranbp2 , or treatment of heterozygous ahctf1 larvae with the nucleocytoplasmic transport inhibitor, Selinexor, completely blocked kras G12V -driven hepatocyte hyperplasia, revealing promising avenues for combinatorial treatments.
Article
The intestinal epithelium arises from undifferentiated endoderm via a developmental program known as the endoderm-intestine transition (EIT). Previously we found that the target of rapamycin complex 1 (TORC1) regulates intestinal growth and differentiation during the EIT in zebrafish. Here we address a possible role for the tumor-suppressor kinase Lkb1 in regulating TORC1 in this context. We find that TORC1 activity is transiently upregulated during the EIT in both zebrafish and mouse. Concomitantly, Lkb1 becomes transiently localized to the nucleus, suggesting that these two phenomena may be linked. Morpholino-mediated knockdown of lkb1 stimulated intestinal growth via upregulation of TORC1, and also induced precocious intestine-specific gene expression in the zebrafish gut epithelium. Knockdown of tsc2, which acts downstream of lkb1, likewise induced early expression of intestine-specific genes. These data suggest that programmed localization of Lkb1 could represent a novel mechanism for regulating the EIT during intestinal development in vertebrates. Developmental Dynamics 239:3000–3012, 2010. © 2010 Wiley-Liss, Inc.
Article
The nuclear envelope compartmentalizes chromatin within eukaryotic cells and influences diverse cellular functions by controlling nucleocytoplasmic trafficking. Recent evidence has revealed the importance of interactions between chromatin and nuclear envelope components in the maintenance of genome integrity. Nuclear pore complexes (NPCs), traditionally regarded as transport gateways, have emerged as specialized hubs involved in organizing genome architecture, influencing DNA topology, and modulating DNA repair. Here, we review the interplay between the nuclear envelope, chromatin and DNA damage checkpoint pathways, and discuss the physiological and pathological implications of these associations.
Article
Nuclear pore complexes are multiprotein channels that span the nuclear envelope, which connects the nucleus to the cytoplasm. In addition to their main role in the regulation of nucleocytoplasmic molecule exchange, it has become evident that nuclear pore complexes and their components also have multiple transport-independent functions. In recent years, an increasing number of studies have reported the involvement of nuclear pore complex components in embryogenesis, cell differentiation and tissue-specific processes. Here, we review the findings that highlight the dynamic nature of nuclear pore complexes and their roles in many cell type-specific functions during development and tissue homeostasis.
Article
Nuclear pore complexes (NPCs) are multiprotein aqueous channels that penetrate the nuclear envelope connecting the nucleus and the cytoplasm. NPCs consist of multiple copies of roughly 30 different proteins known as nucleoporins (NUPs). Due to their essential role in controlling nucleocytoplasmic transport, NPCs have traditionally been considered as structures of ubiquitous composition. The overall structure of the NPC is indeed conserved in all cells, but new evidence suggests that the protein composition of NPCs varies among cell types and tissues. Moreover, mutations in various nucleoporins result in tissue-specific diseases. These findings point towards a heterogeneity in NPC composition and function. This unexpected heterogeneity suggests that cells use a combination of different nucleoporins to assemble NPCs with distinct properties and specialized functions.
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Both the intestine and liver develop from the endoderm, yet little is known how these two digestive organs share and differ in their developmental programs, at the molecular level. A classical forward genetic screen, with no gene bias, is an effective way to address this question by examining the defects of the intestine and liver in obtained mutants to assess mutated genes responsible for the development of either organ or both. We report here such a screen in zebrafish. ENU was used as the mutagen because of its high mutagenic efficiency and no site preference. Embryos were collected at 3.5dpf for RNA whole mount in situ hybridization with a cocktail probe of the intestine marker ifabp and the liver marker lfabp to check phenotypes and determine their parental heterozygosis. A total of 52 F2 putative mutants were identified, and those with general developmental defects were aborted. To rule out non-inheritable phenotypes caused by high mutation background, F2 putative mutants were outcrossed with wild type fish and a re-screen in F3 generations was performed. After complementation tests between F3 mutants with similar phenotypes originating from the same F2 families, a total of 37 F3 mutant lines originated from 22 F2 families were identified after screening 78 mutagenized genomes. Classification of mutant phenotypes indicated that 31 out of the 37 mutants showed defects in both the intestine and liver. In addition, four "intestine specific mutants" and two "liver specific mutants" showed selectively more severe phenotype in the intestine and liver respectively. These results suggested that the intestine and liver share a substantial number of essential genes during both organs development in zebrafish. Further studies of the mutants are likely to shed more insights into the molecular basis of the digestive system development in the zebrafish and vertebrate. Copyright © 2015. Published by Elsevier Inc.
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Nuclear pores span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm, from individual proteins and RNAs to entire viral genomes. By far the largest subunit of the nuclear pore is the Nup107–160 complex, which consists of nine proteins and is critical for nuclear pore assembly. At mitosis, the Nup107–160 complex localizes to kinetochores, suggesting that it may also function in chromosome segregation. To investigate the dual roles of the Nup107–160 complex at the pore and during mitosis, we set out to identify binding partners by immunoprecipitation from both interphase and mitotic Xenopus egg extracts and mass spectrometry. ELYS, a putative transcription factor, was discovered to copurify with the Nup107–160 complex in Xenopus interphase extracts, Xenopus mitotic extracts, and human cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells. Importantly, depletion of ELYS by RNAi leads to severe disruption of nuclear pores in the nuclear envelope, whereas lamin, Ran, and tubulin staining appear normal. At mitosis, ELYS targets to kinetochores, and RNAi depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexpected member of the nuclear pore and kinetochore that functions in both pore assembly at the nucleus and faithful cell division. • Nup107–160 complex • MEL-28 • Nup133 • mitosis
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The metazoan nuclear envelope (NE) breaks down and re-forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re-forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL-28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL-28 interacts with the Nup107-160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107-160 complex to chromatin. We suggest that MEL-28 acts as a seeding point for NPC assembly.
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Xenopus egg extract supports all the major cell-cycle transitions in vitro. We have used a proteomics approach to identify proteins whose abundance on chromatin changes during the course of an in vitro cell cycle. One of the proteins we identified was ELYS/MEL-28, which has recently been described as the earliest-acting factor known to be required for nuclear pore complex (NPC) assembly [1-4]. ELYS interacts with the Nup107-160 complex and is required for its association with chromatin. ELYS contains an AT-hook domain, which we show binds to chromatin with a high affinity. This domain can compete with full-length ELYS for chromatin association, thereby blocking NPC assembly. This provides evidence that ELYS interacts directly with chromatin and that this interaction is essential for NPC assembly and compartmentalization of chromosomal DNA within the cell. Furthermore, we detected a physical association on chromatin between ELYS and the Mcm2-7 replication-licensing proteins. ELYS chromatin loading, NPC assembly, and nuclear growth were delayed when Mcm2-7 was prevented from loading onto chromatin. Because nuclear assembly is required to shut down licensing prior to entry into S phase, our results suggest a mechanism by which these two early cell-cycle events are coordinated with one another.
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The formation of the nuclear envelope (NE) around chromatin is a major membrane-remodelling event that occurs during cell division of metazoa. It is unclear whether the nuclear membrane reforms by the fusion of NE fragments or if it re-emerges from an intact tubular network of the endoplasmic reticulum (ER). Here, we show that NE formation and expansion requires a tubular ER network and occurs efficiently in the presence of the membrane fusion inhibitor GTPgammaS. Chromatin recruitment of membranes, which is initiated by tubule-end binding, followed by the formation, expansion and sealing of flat membrane sheets, is mediated by DNA-binding proteins residing in the ER. Thus, chromatin plays an active role in reshaping of the ER during NE formation.
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Author Summary The transmission of genetic information from DNA to messenger RNA to protein depends on the function of a large number of small noncoding RNA molecules. The genes encoding these RNAs are transcribed by RNA polymerase III (Pol III), a 17-subunit protein complex whose structure is closely related to that of RNA polymerases I and II. Here, we report the effect of a mutation in a gene encoding one Pol III subunit, Polr3b, which disrupts proliferation and growth of tissue progenitor cells in the zebrafish digestive system. Analyses of a nearly identical mutation in the yeast S. pombe gene encoding Polr3b, also known as Rpc2, suggested that the zebrafish mutation disrupted the mutant Polr3b protein's interaction with another Pol III subunit, Polr3k, also known as Rpc11. Overexpression of the gene encoding Polr3k in the Polr3b mutants partially rescued (reversed) the mutant phenotype. These findings extend our knowledge of the mechanism of Pol III function, which appears to have been highly conserved during eukaryotic evolution. Furthermore, these data also suggest that assembly of the 17-subunit Pol III enzyme is a dynamic process, since Polr3k overexpression can partially rescue the mutant phenotype. Understanding how Pol III is assembled has implications for human disease, since Pol III activity is markedly increased in most cancers.
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Assembly of the nuclear pore, gateway to the genome, from its component subunits is a complex process. In higher eukaryotes, nuclear pore assembly begins with the binding of ELYS/MEL-28 to chromatin and recruitment of the large critical Nup107-160 pore subunit. The choreography of steps that follow is largely speculative. Here, we set out to molecularly define early steps in nuclear pore assembly, beginning with chromatin binding. Point mutation analysis indicates that pore assembly is exquisitely sensitive to the change of only two amino acids in the AT-hook motif of ELYS. The dependence on AT-rich chromatin for ELYS binding is borne out by the use of two DNA-binding antibiotics. AT-binding Distamycin A largely blocks nuclear pore assembly, whereas GC-binding Chromomycin A(3) does not. Next, we find that recruitment of vesicles containing the key integral membrane pore proteins POM121 and NDC1 to the forming nucleus is dependent on chromatin-bound ELYS/Nup107-160 complex, whereas recruitment of gp210 vesicles is not. Indeed, we reveal an interaction between the cytoplasmic domain of POM121 and the Nup107-160 complex. Our data thus suggest an order for nuclear pore assembly of 1) AT-rich chromatin sites, 2) ELYS, 3) the Nup107-160 complex, and 4) POM121- and NDC1-containing membrane vesicles and/or sheets, followed by (5) assembly of the bulk of the remaining soluble pore subunits.
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We describe a series of stages for development of the embryo of the zebrafish, Danio (Brachydanio) rerio. We define seven broad periods of embryogenesis—the zygote, cleavage, blastula, gastrula, segmentation, pharyngula, and hatching periods. These divisions highlight the changing spectrum of major developmental processes that occur during the first 3 days after fertilization, and we review some of what is known about morphogenesis and other significant events that occur during each of the periods. Stages subdivide the periods. Stages are named, not numbered as in most other series, providing for flexibility and continued evolution of the staging series as we learn more about development in this species. The stages, and their names, are based on morphological features, generally readily identified by examination of the live embryo with the dissecting stereomicroscope. The descriptions also fully utilize the optical transparancy of the live embryo, which provides for visibility of even very deep structures when the embryo is examined with the compound microscope and Nomarski interference contrast illumination. Photomicrographs and composite camera lucida line drawings characterize the stages pictorially. Other figures chart the development of distinctive characters used as staging aid signposts. ©1995 Wiley-Liss, Inc.
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To automate examination of massive amounts of sequence data for biological function, it is important to computerize interpretation based on empirical knowledge of sequence-function relationships. For this purpose, we have been constructing a knowledge base by organizing various experimental and computational observations as a collection of if-then rules. Here we report an expert system, which utilizes this knowledge base, for predicting localization sites of proteins only from the information on the amino acid sequence and the source origin. We collected data for 401 eukaryotic proteins with known localization sites (subcellular and extracellular) and divided them into training data and testing data. Fourteen localization sites were distinguished for animal cells and 17 for plant cells. When sorting signals were not well characterized experimentally, various sequence features were computationally derived from the training data. It was found that 66% of the training data and 59% of the testing data were correctly predicted by our expert system. This artificial intelligence approach is powerful and flexible enough to be used in genome analyses.
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The precise mechanism governing the generation of haematopoietic stem cells still remains to be understood, partly because the molecules required for early haematopoiesis have not fully been identified. We have identified a novel gene expressed in embryonic haematopoietic tissues, designated ELYS (for embryonic large molecule derived from yolk sac), which has no significant homology with any other known molecules. Based on the cDNA sequence, mouse ELYS protein is composed of 2243 amino acid residues and contains an AT-hook DNA-binding domain, eight nuclear localization signals (NLSs) at the C-terminal region, three nuclear export signals (NESs) and two WD repeats at the N-terminal region. ELYS has a potential to shuttle between the cytoplasm and nucleus. When in the nucleus, ELYS is present in the nuclear matrix. Fusions of the yeast GAL4 DNA-binding domain and various ELYS mutants reveal the presence of transcriptional activation and inhibitory domains. The ELYS gene is predominantly expressed in embryonic haematopoietic tissues, i.e. foetal liver, spleen, and thymus, whereas the expression is down-regulated in the adult. In the aorta-gonad-mesonephros (AGM) region of an 11.5 dpc mouse embryo, ELYS is expressed in the endothelium lining the dorsal aorta. In the adult bone marrow, ELYS is notably expressed in the Lin-/c-kit+/Sca-1+ population. We have reported the isolation and characterization of a novel molecule, ELYS. ELYS seems to be a nuclear transcription factor associated with both early and mature haematopoietic events.
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Despite the essential functions of the digestive system, much remains to be learned about the cellular and molecular mechanisms responsible for digestive organ morphogenesis and patterning. We introduce a novel zebrafish transgenic line, the gutGFP line, that expresses GFP throughout the digestive system, and use this tool to analyze the development of the liver. Our studies reveal two phases of liver morphogenesis: budding and growth. The budding period, which can be further subdivided into three stages, starts when hepatocytes first aggregate, shortly after 24 h postfertilization (hpf), and ends with the formation of a hepatic duct at 50 hpf. The growth phase immediately follows and is responsible for a dramatic alteration of liver size and shape. We also analyze gene expression in the developing liver and find a correlation between the expression of certain transcription factor genes and the morphologically defined stages of liver budding. To further expand our understanding of budding morphogenesis, we use loss-of-function analyses to investigate factors potentially involved in this process. It had been reported that no tail mutant embryos appear to lack a liver primordium, as assessed by gata6 expression. However, analysis of gutGFP embryos lacking Ntl show that the liver is in fact present. We also find that, in these embryos, the direction of liver budding does not correlate with the direction of intestinal looping, indicating that the left/right behavior of these tissues can be uncoupled. In addition, we use the cloche mutation to analyze the role of endothelial cells in liver morphogenesis, and find that in zebrafish, unlike what has been reported in mouse, endothelial cells do not appear to be necessary for the budding of this organ.
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Digestive organ development occurs through a sequence of morphologically distinct stages, from overtly featureless endoderm, through organ primordia to, ultimately, adult form. The developmental controls that govern progression from one stage to the next are not well understood. To identify genes required for the formation of vertebrate digestive organs we performed a genetic screen in zebrafish. We isolated the nil per os (npo) mutation, which arrests morphogenesis and cytodifferentiation of the gut and exocrine pancreas in a primordial state. We identified the npo gene by positional cloning. It encodes a conserved protein, with multiple RNA recognition motifs, that is related to the yeast protein Mrd1p. During development npo is expressed in a dynamic fashion, functioning cell autonomously to promote organ cytodifferentiation. Antisense-mediated knockdown of npo results in organ hypoplasia, and overexpression of npo causes an overgrowth of gastrointestinal organs. Thus, npo is a gene essential for a key step in the gut morphogenetic sequence.
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Early mouse development is a complicated process that is controlled by various proteins including transcription factors. Recently, we identified a putative transcription factor, ELYS (embryonic large molecule derived from yolk sac), using a subtraction strategy. To elucidate the role of ELYS in vivo, we generated ELYS-deficient mice by homologous recombination. Although heterozygous mice appeared to be healthy, fertile and normal, embryos homozygous for the ELYS mutation died between embryonic day (E) 3.5 and 5.5. Null mutant blastocysts collected from the uterus at E3.5 were viable and indistinguishable from wild-type littermates. However, when cultured in vitro, they showed impaired proliferation of the inner cells, because of apoptosis. The expression of ELYS mRNA was detected in both the inner cell mass (ICM) and the trophectoderm at the blastocyst stage, and persisted throughout the developing embryo during E4.5 to 6.5. These results indicate that ELYS is a critical factor for early mouse development and is essential for the survival of the inner cells.
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Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.
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Current optical methods to collect Nomarski differential interference contrast (DIC) or phase images with a transmitted light detector (TLD) in conjunction with confocal laser scanning microscopy (CLSM) can be technically challenging and inefficient. We describe for the first time a simple method that combines the use of the commercial product QPm (Iatia, Melbourne Australia) with brightfield images collected with the TLD of a CLSM, generating DIC, phase, Zernike phase, dark-field or Hoffman modulation contrast images. The brightfield images may be collected at the same time as the confocal images. This method also allows the calculation of contrast-enhanced images from archival data. The technique described here allows for the creation of contrast-enhanced images such as DIC or phase, without compromising the intensity or quality of confocal images collected simultaneously. Provided the confocal microscope is equipped with a motorized z-drive and a TLD, no hardware or optical modifications are required. The contrast-enhanced images are calculated with software using the quantitative phase-amplitude microscopy technique (Barone-Nugent et al., 2002). This technique, being far simpler during image collection, allows the microscopist to concentrate on their confocal imaging and experimental procedures. Unlike conventional DIC, this technique may be used to calculate DIC images when cells are imaged through plastic, and without the use of expensive strain-free objective lenses.
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Zebrafish meltdown (mlt) mutants develop cystic expansion of the posterior intestine as a result of stromal invasion of nontransformed epithelial cells. Positional cloning identified zebrafish smooth muscle myosin heavy chain (myh11) as the responsible gene. The mlt mutation constitutively activates the Myh11 ATPase, which disrupts smooth muscle cells surrounding the posterior intestine. Adjacent epithelial cells ectopically express metalloproteinases, integrins, and other genes implicated in human cancer cell invasion. Knockdown and pharmacological inhibition of these genes restores intestinal structure in mlt mutants despite persistent smooth muscle defects. These data identify an essential role for smooth muscle signaling in the maintenance of epithelial architecture and support gene expression analyses and other studies that identify a role for stromal genes in cancer cell invasion. Furthermore, they suggest that high-throughput screens to identify regulators of cancer cell invasion may be feasible in zebrafish.
Article
Recent analysis of a novel strain of transgenic zebrafish (gutGFP) has provided a detailed description of the early morphological events that occur during the development of the liver and pancreas. In this paper, we aim to complement these studies by providing an analysis of the morphological events that shape the zebrafish intestinal epithelium. One of our goals is to provide a framework for the future characterization of zebrafish mutant phenotypes in which intestinal epithelial morphogenesis has been disrupted. Our analysis encompasses the period between 26 and 126 h post-fertilization (hpf) and follows the growth, lumen formation and differentiation of a continuous layer of endoderm into a functional intestinal epithelium with three morphologically distinct segments: the intestinal bulb, mid-intestine and posterior intestine. Between 26 hpf and 76 hpf, the entire intestinal endoderm is a highly proliferative organ. To make a lumen, the zebrafish endoderm cells undergo apical membrane biogenesis, adopt a bilayer configuration and form small cavities that coalesce without cell death. Thereafter, the endoderm cells polarize and differentiate into distinct cell lineages. Enteroendocrine cells are distinguished first at 52 hpf in the caudal region of the intestine in a new stable transgenic line, Tg[nkx2.2a:mEGFP]. The differentiation of mucin-containing goblet cells is first evident at 100 hpf and is tightly restricted to a middle segment of the intestine, designated the mid-intestine, that is also demarcated by the presence of enterocytes with large supranuclear vacuoles. Meanwhile, striking expansion of the lumen in the rostral intestine forms the intestinal bulb. Here the epithelium elaborates folds and proliferating cells become progressively restricted to a basal compartment analogous to the crypts of Lieberkühn in mammals. At 126 hpf, the posterior intestine remains an unfolded monolayer of simple columnar epithelium.
Article
The nuclear envelope (NE) is a highly specialized membrane that delineates the eukaryotic cell nucleus. It is composed of the inner and outer nuclear membranes, nuclear pore complexes (NPCs) and, in metazoa, the lamina. The NE not only regulates the trafficking of macromolecules between nucleoplasm and cytosol but also provides anchoring sites for chromatin and the cytoskeleton. Through these interactions, the NE helps position the nucleus within the cell and chromosomes within the nucleus, thereby regulating the expression of certain genes. The NE is not static, rather it is continuously remodeled during cell division. The most dramatic example of NE reorganization occurs during mitosis in metazoa when the NE undergoes a complete cycle of disassembly and reformation. Despite the importance of the NE for eukaryotic cell life, relatively little is known about its biogenesis or many of its functions. We thus are far from understanding the molecular etiology of a diverse group of NE-associated diseases.
Article
Nup98 and Nup96 are components of the nuclear transport machinery and are induced by interferons (IFN). Nup98 is a constituent of an mRNA export pathway that is targeted by viruses and regulated by IFN. However, the role of Nup96 in IFN-related mechanisms has not been established. To investigate the function of Nup96 in vivo, we generated Nup96(+/-) mice that express low levels of Nup96, as Nup96(-/-) mice are lethal. The Nup96(+/-) mice presented selective alterations of the immune system, which resulted in downregulation and impaired IFN alpha- and gamma-mediated induction of MHC I and IFNgamma induction of MHC II, ICAM-1, and other proteins. Frequency of TCRalphabeta+ and CD4+ T cells, which depends on MHC function, is reduced in NUP96(+/-) mice. Upon immunization, Nup96(+/-) mice showed impaired antigen presentation and T cell proliferation. Nup96(+/-) cells and mice were highly susceptible to viral infection, demonstrating a role for Nup96 in innate and adaptive immunity.
Article
Early embryonic development depends on the faithful execution of basic cell biological processes whose coordination remains largely unknown. With a global network analysis, we found MEL-28 to be associated with two types of complexes, one implicated in nuclear-envelope function and the other in chromatin organization. Here, we show that MEL-28, a protein that shuttles between the nucleus and the kinetochore during the cell cycle, is required for the structural and functional integrity of the nuclear envelope. In addition, mel-28(RNAi) embryos exhibit defects in chromosome condensation, pronuclear migration, kinetochore assembly, and spindle assembly. This combination of mel-28(RNAi) phenotypes resemble those caused by depleting members of the Ran cycle in C. elegans, a conserved cellular signaling pathway that is required for mitotic spindle assembly, nuclear-envelope reformation after mitosis, and nucleocytoplasmic exchange (reviewed in). Although MEL-28 localization to the nuclear periphery is not dependent on nuclear pore components, it is dependent on RAN-1 and other key components of the Ran cycle. Thus, MEL-28 is downstream of the Ran cycle and is required for both proper nuclear-envelope function and chromatin maintenance.
Article
The nuclear envelope (NE) of eukaryotic cells separates nucleoplasm from cytoplasm, mediates nucleo-cytoplasmic transport, and contributes to the control of gene expression [1 • Prunuske A.J. • Ullman K.S. The nuclear envelope: Form and reformation.Curr. Opin. Cell Biol. 2006; 18: 108-116 • Crossref • PubMed • Scopus (68) • Google Scholar , 2 • Hetzer M. • Walther T.C. • Mattaj I.W. Pushing the envelope: Structure, function, and dynamics of the nuclear periphery.Annu. Rev. Cell Dev. Biol. 2005; 21: 347-380 • Crossref • PubMed • Scopus (231) • Google Scholar ]. The NE consists of three major components: the nuclear membranes, the nuclear pore complexes (NPCs), and the nuclear lamina. The list of identified NE proteins has increased considerably during recent years but is most likely not complete. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. The assembly of NPCs and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes [3 • Antonin W. • Franz C. • Haselmann U. • Antony C. • Mattaj I.W. The integral membrane nucleoporin pom121 functionally links nuclear pore complex assembly and nuclear envelope formation.Mol. Cell. 2005; 17: 83-92 • Abstract • Full Text • Full Text PDF • PubMed • Scopus (113) • Google Scholar ]. Here, we report the identification and characterization of MEL-28, a large protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. The structural defects of the NE were associated with functional defects and lack of nuclear exclusion of soluble proteins. MEL-28 localizes to NPCs during interphase, to kinetochores in early to middle mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early-assembling, stable NE component required for all aspects of NE assembly.
Article
The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.
Article
Serving as the primary conduit for communication between the nucleus and the cytoplasm, nuclear pore complexes (NPCs) impact nearly every cellular process. The extent to which NPC composition varies and the functional significance of such variation in mammalian development has not been investigated. Here we report that a null allele of mouse nucleoporin Nup133, a structural subunit of the NPC, disrupts neural differentiation. We find that expression of Nup133 is cell type and developmental stage restricted, with prominent expression in dividing progenitors. Nup133-deficient epiblast and ES cells abnormally maintain features of pluripotency and differentiate inefficiently along the neural lineage. Neural progenitors achieve correct spatial patterning in mutant embryos; however, they are impaired in generating terminally differentiated neurons, as are Nup133 null ES cells. Our results reveal a role for structural nucleoporins in coordinating cell differentiation events in the developing embryo.
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Formation of the digestive system in zebrafish: III. Intestinal epithelium morpho-genesis
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Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis
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Mutations affecting development of zebrafish digestive organs
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