Target-Selected Gene Inactivation in Zebrafish

Hubrecht Laboratory, Center for Biomedical Genetics, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands.
Methods in cell biology (Impact Factor: 1.42). 02/2004; 77(77):69-90. DOI: 10.1016/S0091-679X(04)77004-1
Source: PubMed
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Available from: Erno Wienholds, Apr 30, 2014
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    • "Zebrafish embryos were raised and staged as previously described (Westerfield, 1995) in accordance with all Dutch regulations and guidelines under DEC protocol #08.2011. The gnl2 bw41c mutation was recovered in a forward genetic screen, whereas the ns hu3259 mutation was generated in a reverse genetic screen using the TILLING method (Wienholds and Plasterk, 2004). The gnl3l mutant was uncovered in a viral insertion mutagenesis screen (Golling et al., 2002). "
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    ABSTRACT: Nucleostemin (NS) is an essential protein for the growth and viability of developmental stem cells. Its functions are multi-faceted, including important roles in ribosome biogenesis and in the p53-induced apoptosis pathway. While NS has been well studied, the functions of its family members GNL2 and GNL3-like (GNL3L) remain relatively obscure despite a high degree of sequence and domain homology. Here, we use zebrafish lines carrying mutations in the ns family to compare and contrast their functions in vertebrates. We find the loss of zebrafish ns or gnl2 has a major impact on 60S large ribosomal subunit formation and/or function due to cleavage impairments at distinct sites of pre-rRNA transcript. In both cases this leads to a reduction of total protein synthesis. In contrast, gnl3l loss shows relatively minor rRNA processing delays that ultimately have no appreciable effects on ribosome biogenesis or protein synthesis. However, the loss of gnl3l still results in p53 stabilization, apoptosis, and lethality similarly to ns and gnl2 loss. The depletion of p53 in all three of the mutants led to partial rescues of the morphological phenotypes and surprisingly, a rescue of the 60S subunit collapse in the ns mutants. We show that this rescue is due to an unexpected effect of p53 loss that even in wild type embryos results in an increase of 60S subunits. Our study presents an in-depth description of the mechanisms through which ns and gnl2 function in vertebrate ribosome biogenesis and shows that despite the high degree of sequence and domain homology, gnl3l has critical functions in development that are unrelated to the ribosome.
    Developmental Biology 11/2013; 385(2). DOI:10.1016/j.ydbio.2013.10.029 · 3.55 Impact Factor
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    • "Zebrafish embryos were raised and staged as previously described (Westerfield, 1995). The gnl2 bw41c mutation was recovered in a forward genetic screen, whereas the ns hu3259 mutation was generated in a reverse genetic screen using the TILLING method (Wienholds and Plasterk, 2004). All mutant embryos were genotyped by nested PCR, followed by Taqa1 restriction and fragment length electrophoresis for gnl2 and DNA sequencing for ns mutants, using the following primers: PCR1 gnl2-1 5′-ggtgcctgtctgttaatcaag-3′, gnl2-4 5′-gcgcta tacacgcatt- taac-3′; PCR2 gnl2-2 5′-ttacacatttgccacagacc-3′, gnl2-3 5′-tgcagcaaa- gacattggtag-3′ and PCR1 ns-1 5′-taggt caacattgcccaac-3′, ns-4 5′- gagagtatcaaacacatgcagag-3′; PCR2 ns-2 5′-tttctca ctattccaggttcg-3′, ns-3 5′-tgttgagttcttggcatt tg-3′. "
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    ABSTRACT: Nucleostemin (NS), a member of a family of nucleolar GTP-binding proteins, is highly expressed in proliferating cells such as stem and cancer cells and is involved in the control of cell cycle progression. Both depletion and overexpression of NS result in stabilization of the tumor suppressor p53 protein in vitro. Although it has been previously suggested that NS has p53-independent functions, these to date remain unknown. Here, we report two zebrafish mutants recovered from forward and reverse genetic screens that carry loss of function mutations in two members of this nucleolar protein family, Guanine nucleotide binding-protein-like 2 (Gnl2) and Gnl3/NS. We demonstrate that these proteins are required for correct timing of cell cycle exit and subsequent neural differentiation in the brain and retina. Concomitantly, we observe aberrant expression of the cell cycle regulators cyclinD1 and p57kip2. Our models demonstrate that the loss of Gnl2 or NS induces p53 stabilization and p53-mediated apoptosis. However, the retinal differentiation defects are independent of p53 activation. Furthermore, this work demonstrates that Gnl2 and NS have both non-cell autonomously and cell-autonomous function in correct timing of cell cycle exit and neural differentiation. Finally, the data suggest that Gnl2 and NS affect cell cycle exit of neural progenitors by regulating the expression of cell cycle regulators independently of p53.
    Developmental Biology 07/2011; 355(2):286-301. DOI:10.1016/j.ydbio.2011.04.028 · 3.55 Impact Factor
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    • "Fish strains and screening F1 ENU-mutation library Embryos and adult fish were raised and maintained under standard laboratory conditions. An ENU-induced mutation library was screened for mutations in exon 4 of the nesprin-3 gene as described (Wienholds and Plasterk, 2004). The mutant allele (nesprin-3 hu6448 ) was outcrossed against the TL strain. "
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    ABSTRACT: The outer nuclear membrane protein nesprin-3 binds the cytoskeletal linker protein plectin, which are proposed to anchor the intermediate filaments to the nuclear envelope. To investigate the function of nesprin-3 in vivo, we used the zebrafish as a vertebrate model system. Zebrafish nesprin-3 is expressed at the nuclear envelope of epidermal and skeletal muscle cells during development. Unexpectedly, loss of nesprin-3 did not affect embryonic development, viability or fertility. However, nesprin-3-deficient zebrafish embryos showed a reduced concentration of intermediate filaments around the nucleus. Additional analysis revealed the presence of two nesprin-3 isoforms in zebrafish, nesprin-3α and nesprin-3β. Nesprin-3β is only expressed during early development and lacks seven amino acids in its first spectrin repeat that are crucial for plectin binding and recruitment to the nuclear envelope. These seven amino acids are highly conserved and we showed that residues R43 and L44 within this motif are required for plectin binding. Furthermore, several residues in the actin-binding domain of plectin that are crucial for binding to the integrin β4 subunit are also important for the binding to nesprin-3α, indicating partial overlapping binding sequences for nesprin-3α and integrin β4. All this shows that nesprin-3 is dispensable for normal development in zebrafish, but important for mediating the association of the intermediate filament system with the nucleus in vivo.
    Journal of Cell Science 03/2011; 124(Pt 5):755-64. DOI:10.1242/jcs.081174 · 5.43 Impact Factor
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