Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M.

DK61142 NIDDK NIH HHS , P30 DK50306 NIDDK NIH HHS , P30 DK050306 NIDDK NIH HHS , R01 DK061142 NIDDK NIH HHS

	Figure 1. flo intestinal and retinal defects.(A,B) Lateral view of live 5 dpf wild type (wt) and flo larvae. The flo intestine lacks folds (arrow) and the lumen contains detached epithelial cells (arrowheads). (C,D) Dorsal view showing reduced size of the 5 dpf flo eye. (E,F) Histological cross section showing cells with condensed nuclei typical of apoptotic cells in the 60 hpf flo retina, and disorganization of the flo photoreceptor (*) and outer plexiform (arrow) layers. (G,H) Histological cross section showing marked disorganization of the 4 dpf flo retina and cells with condensed nuclei typical of apoptotic cells (white arrow). (I,J) Acridine orange staining showing apoptotic cells in the 48 hpf flo retina but not sibling wild types. (K,L) TUNEL staining showing apoptotic cells in the 75 hpf flo intestine (arrowheads) but not sibling wild types (anterior–left, posterior–right). (M,N) Dorsal view showing mild reduction in the number of flo retinal ganglion cells (arrowheads) and optic nerve diameter (arrow) identified with the Zn5 antibody. (O,P) Confocal projection of immunostained wt and flo larvae showing reduced rod cells in the flo retina including the large ventral cluster of cells and in the periphery of the mid retina (arrow). onl, outer nuclear layer; inl, inner nuclear layer; ipl, inner plexiform layer; rgc, retinal ganglion cell layer; on, optic nerve; le, lens; y, yolk.
	Figure 2. The flo locus encodes zebrafish elys.(A) Schematic representation of the genomic region surrounding the flo locus. The names of the polymorphic markers with the corresponding number of recombinants are listed. (B) DNA sequence analysis showing the cytosine to thymidine transition encoding the premature stop codon in the elysti262c allele. (C) Schematic representation of the functional domains of the human (hs) and zebrafish (dr) Elys protein and the protein encoded by the elysti262c allele (flo-ELYS). (D–I) Acridine orange staining showing apoptotic cells in the retina and growth plate of the optic tectum of 48 hpf flo (E,H) and elys-morpholino injected (F,I) larvae but not wt (D,G). (J–R) Confocal projections through the posterior intestine of 96 hpf larvae showing wheat-germ agglutinin positive goblet cells in the epithelium of the posterior intestine of wt (J) but not flo (K) or elys-morpholino injected (L) larvae; secretory cells in wt (M) but not flo (N) or elys-morpholino (O) injected larvae; enterocytes in wt (P) but not flo (Q) or elys-morpholino (R) injected larvae. (S–T) Carboxy-peptidase A positive cells are abundant in the 5 dpf wt (S) but not in elys-morpholino injected (T) exocrine pancreas. (U) Histological cross section through the retina of a 4 dpf elys morpholino injected larva showing retinal disorganization that is comparable to the 4 dpf flo retina (Figure 1H).
	Figure 3. elys expression in developing zebrafish.Images (A–H) are whole mount RNA in situ hybridizations. (A) Maternal elys expression at 3 hpf. (B) Strong elys expression is evident in the midbrain (black arrow) and eye (white arrow) at 24 hpf. (C–F) Expression at 48 hpf in the retina (C,E), growth plate of the optic tectum (F), and digestive organs [(D): pancreas (arrow), anterior intestine (white arrowhead), posterior intestine (black arrowheads), liver (*)]. (G–H) Weak elys expression in the 5 dpf anterior (G) and posterior (H) intestine. (I) Graph showing elys expression in whole embryos (4 hpf–120 hpf) as determined via real-time quantitative PCR.
	Figure 4. Nuclear pore disruption in flo mutants.(A–C) Confocal projections through the posterior intestine of 75 hpf wild type (A), flo (B), and elys morpholino injected larvae (C), following anti-FG nucleoporin immunostainings with mAb414 (green; DAPI–blue). There is a dramatic reduction of nuclear pores in the flo and morpholino injected larvae. Inset shows higher magnification of localized regions of the DAPI-stained image. (D–F) Identical findings are evident in the retina of these larvae. Note apparent cytoplasmic accumulation of the immunoreactive FG-nucleoporins in flo and morpholino injected larvae. (G,H) Normal nuclear distribution of FG nucleoporins in wild type and flo skeletal muscle. (I) Western blot showing levels of FG nucleoporin proteins relative to beta-actin in nuclear (nucl) and cytoplasmic (cyto) extracts derived from the intestine of 75 hpf flo and sibling wild type larvae.
	Figure 5. Nuclear ultrastructure in flo mutants.Transmission electron micrographs of nuclei from representative 5 dpf wild type and flo intestinal epithelial cells. (A,B) Intact nuclear envelope in wild type (A) and flo (B). (C–F) Tangential sections through the nuclear envelope showing abundant nuclear pores (arrows) in the wild type larva (C,E) but few if any well defined pores in the flo larva (D,F). (E) and (F) are higher magnification views of (C) and (D), respectively.
	Figure 6. The flo mutation activates the DNA damage response.(A–C) Acridine orange staining showing apoptosis in the 50 hpf flo retina that is rescued by the tp53 morpholino (mo) knockdown. (D) Histological cross section showing rescue of flo retinal architecture defects by tp53 knockdown [compare (F) with Figure 1G and 1H]. (E) Intestinal defects persist in flo/tp53 double mutants. Arrow, thin intestinal wall; Arrowhead, apoptotic cells in the intestinal lumen. (F) Western blot showing elevated levels of phospho-Chk2 (Serine 33) in the intestine of flo larvae, compared with sibling wild type larvae, but not slim jim larvae (I). (G) Western blot showing comparable levels of phospho-Chk1 (Ser 345) in flo and sibling wild type larvae, before and after γ-radiation (30 Gy) and treatment with hydroxyurea (HU). (H) Western blot showing enhanced phospho-Chk2 activation in the intestine of flo and wild type larvae following γ-radiation (30 Gy). (I) γH2AX is not detected in the flo or wild type intestine (75 hpf), but is detected at this stage following γ-irradiation (30 Gy) or hydroxyurea treatment (HU).
	Figure 7. Reduced chromatin bound Mcm2 in the flo intestine.(A) Western analysis showing reduced levels of chromatin bound Mcm2 in 75 hpf and 96 hpf flo intestine compared with wild type siblings. By contrast, levels of chromatin bound Mcm3 (B) and Mcm4 (C) in the flo intestine are comparable to wild type. Multiple bands (*) corresponding to phospho-Mcm3 and phospho-Mcm4 are recognized by the antibodies directed against the native proteins in wt and flo samples.

Abud, Shaping developing tissues by apoptosis. 2004, Pubmed

Abud, Shaping developing tissues by apoptosis. 2004, Pubmed
Ahn, The Chk2 protein kinase. 2004, Pubmed
Bailis, Minichromosome maintenance proteins interact with checkpoint and recombination proteins to promote s-phase genome stability. 2008, Pubmed
Baker, Nonsense-mediated mRNA decay: terminating erroneous gene expression. 2004, Pubmed
Bennett, Genes required for ionizing radiation resistance in yeast. 2001, Pubmed
Berghmans, tp53 mutant zebrafish develop malignant peripheral nerve sheath tumors. 2005, Pubmed
Bochman, Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and MCM5 define a slow ATP-dependent step. 2007, Pubmed
Brown, ATR disruption leads to chromosomal fragmentation and early embryonic lethality. 2000, Pubmed
Burke, Remodelling the walls of the nucleus. 2002, Pubmed
Carlessi, Biochemical and cellular characterization of VRX0466617, a novel and selective inhibitor for the checkpoint kinase Chk2. 2007, Pubmed
Chang, A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage. 2002, Pubmed
Chen, Loss of function of def selectively up-regulates Delta113p53 expression to arrest expansion growth of digestive organs in zebrafish. 2005, Pubmed
Chen, Mutations affecting the cardiovascular system and other internal organs in zebrafish. 1996, Pubmed
Crosnier, Delta-Notch signalling controls commitment to a secretory fate in the zebrafish intestine. 2005, Pubmed
D'Arpa, Topoisomerase-targeting antitumor drugs. 1989, Pubmed
Davis, Nuclear pore complex contains a family of glycoproteins that includes p62: glycosylation through a previously unidentified cellular pathway. 1987, Pubmed
De Souza, A point mutation in the Aspergillus nidulans sonBNup98 nuclear pore complex gene causes conditional DNA damage sensitivity. 2006, Pubmed
Dobles, Chromosome missegregation and apoptosis in mice lacking the mitotic checkpoint protein Mad2. 2000, Pubmed
Fernandez, MEL-28 is downstream of the Ran cycle and is required for nuclear-envelope function and chromatin maintenance. 2006, Pubmed
Franz, MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly. 2007, Pubmed , Xenbase
Frappart, An essential function for NBS1 in the prevention of ataxia and cerebellar defects. 2005, Pubmed
Galy, MEL-28, a novel nuclear-envelope and kinetochore protein essential for zygotic nuclear-envelope assembly in C. elegans. 2006, Pubmed
Ge, Dormant origins licensed by excess Mcm2-7 are required for human cells to survive replicative stress. 2007, Pubmed
Geiger, Zebrafish as a "biosensor"? Effects of ionizing radiation and amifostine on embryonic viability and development. 2006, Pubmed
Gillespie, ELYS/MEL-28 chromatin association coordinates nuclear pore complex assembly and replication licensing. 2007, Pubmed , Xenbase
Hakem, The tumor suppressor gene Brca1 is required for embryonic cellular proliferation in the mouse. 1996, Pubmed
Hirano, ALADINI482S causes selective failure of nuclear protein import and hypersensitivity to oxidative stress in triple A syndrome. 2006, Pubmed
Ho, Stalled replication induces p53 accumulation through distinct mechanisms from DNA damage checkpoint pathways. 2006, Pubmed
Iizuka, Hbo1 Links p53-dependent stress signaling to DNA replication licensing. 2008, Pubmed
Inoue, Control of p53 nuclear accumulation in stressed cells. 2005, Pubmed
Ishimi, Identification of MCM4 as a target of the DNA replication block checkpoint system. 2003, Pubmed
Kimura, Identification of a novel transcription factor, ELYS, expressed predominantly in mouse foetal haematopoietic tissues. 2002, Pubmed
Lam, Chk1 is haploinsufficient for multiple functions critical to tumor suppression. 2004, Pubmed
Lambert, Arrested replication fork processing: interplay between checkpoints and recombination. 2007, Pubmed
Langheinrich, Zebrafish as a model organism for the identification and characterization of drugs and genes affecting p53 signaling. 2002, Pubmed
Li, Modulation of cell proliferation in the embryonic retina of zebrafish (Danio rerio). 2000, Pubmed
Lim, A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53. 1996, Pubmed
Liu, Chk1 is an essential kinase that is regulated by Atr and required for the G(2)/M DNA damage checkpoint. 2000, Pubmed
Liu, Inactivation of the mouse Brca1 gene leads to failure in the morphogenesis of the egg cylinder in early postimplantation development. 1996, Pubmed
Loeb-Hennard, Prominent transcription of zebrafish N-myc (nmyc1) in tectal and retinal growth zones during embryonic and early larval development. 2005, Pubmed
Loeillet, Genetic network interactions among replication, repair and nuclear pore deficiencies in yeast. 2005, Pubmed
Lowndes, DNA repair: the importance of phosphorylating histone H2AX. 2005, Pubmed
Luo, Disruption of mRad50 causes embryonic stem cell lethality, abnormal embryonic development, and sensitivity to ionizing radiation. 1999, Pubmed
Manova, Apoptosis in mouse embryos: elevated levels in pregastrulae and in the distal anterior region of gastrulae of normal and mutant mice. 1998, Pubmed
Matthews, The zebrafish onecut gene hnf-6 functions in an evolutionarily conserved genetic pathway that regulates vertebrate biliary development. 2004, Pubmed
Ng, Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis. 2005, Pubmed
O'Keefe, Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination. 2003, Pubmed
Okita, Targeted disruption of the mouse ELYS gene results in embryonic death at peri-implantation development. 2004, Pubmed
Paulsen, The ATR pathway: fine-tuning the fork. 2007, Pubmed
Plaster, p53 deficiency rescues apoptosis and differentiation of multiple cell types in zebrafish flathead mutants deficient for zygotic DNA polymerase delta1. 2006, Pubmed
Poelmann, Apoptosis as an instrument in cardiovascular development. 2005, Pubmed
Pommier, Chk2 molecular interaction map and rationale for Chk2 inhibitors. 2006, Pubmed
Pruitt, Reduced Mcm2 expression results in severe stem/progenitor cell deficiency and cancer. 2007, Pubmed
Rasala, ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division. 2006, Pubmed , Xenbase
Rusche, The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae. 2003, Pubmed
Ryu, Depletion of minichromosome maintenance protein 5 in the zebrafish retina causes cell-cycle defect and apoptosis. 2005, Pubmed
Sharan, Embryonic lethality and radiation hypersensitivity mediated by Rad51 in mice lacking Brca2. 1997, Pubmed
Stoeber, DNA replication licensing and human cell proliferation. 2001, Pubmed
Sun, The replication capacity of intact mammalian nuclei in Xenopus egg extracts declines with quiescence, but the residual DNA synthesis is independent of Xenopus MCM proteins. 2000, Pubmed , Xenbase
Suntharalingam, Peering through the pore: nuclear pore complex structure, assembly, and function. 2003, Pubmed
Takahashi, Does gammaH2AX foci formation depend on the presence of DNA double strand breaks? 2005, Pubmed
Tebbs, Requirement for the Xrcc1 DNA base excision repair gene during early mouse development. 1999, Pubmed
Therizols, Telomere tethering at the nuclear periphery is essential for efficient DNA double strand break repair in subtelomeric region. 2006, Pubmed
Tran, Dynamic nuclear pore complexes: life on the edge. 2006, Pubmed
Wallace, Intestinal growth and differentiation in zebrafish. 2005, Pubmed
Wallace, Mutation of smooth muscle myosin causes epithelial invasion and cystic expansion of the zebrafish intestine. 2005, Pubmed
Woodward, Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress. 2006, Pubmed , Xenbase
Yee, Mutation of RNA Pol III subunit rpc2/polr3b Leads to Deficiency of Subunit Rpc11 and disrupts zebrafish digestive development. 2007, Pubmed
Yee, Exocrine pancreas development in zebrafish. 2005, Pubmed
Zhu, Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice. 2001, Pubmed