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Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

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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.
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pgen-1000240-g001: 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.

Mentions: Zebrafish flotte lotte (floti262c; hereafter flo) is a recessive lethal mutation that was identified as part of a large scale ENU mutagenesis screen [23]. Small eyes, optic tectum degeneration and digestive organ defects are prominent features of the flo mutant phenotype that are first evident in live fish on the fourth day post-fertilization (Figure 1A–1D). In previous work, the flo mutation was shown to disrupt differentiation and survival of 3 of the 4 principal intestinal epithelial cell lineages [24],[26], suggesting an effect on organ progenitor cells. Here, we show a comparable though less pronounced effect on retinal development (Figure 1E–1H). Histological analyses revealed that the cellular layers of the flo retinal epithelium were disorganized and contained numerous cells with condensed nuclei typical of apoptotic cells. Acridine orange and TUNEL staining revealed that apoptotic retinal (Figure 1I–1J) and intestinal epithelial cells (Figure 1K–1L) were already evident before flo mutants were morphologically distinguishable from wild type larvae. Immunohistochemical studies showed that differentiated retinal ganglion and photoreceptor cells were present in flo mutants, albeit at reduced levels compared with wild types, despite retinal disorganization and apoptosis (Figure 1M–1P). These studies suggested that early stages of retinal development proceed normally in flo mutants, most likely as a result of maternally derived mRNA and protein encoded by the affected gene (discussed below). To further assess the effect of the flo mutation on organ progenitors, we also measured cell proliferation in the intestinal and retinal epithelium. Both sets of data were consistent with G1 arrest of rapidly proliferating cells in these tissues (Tables S1 and S2). In summary, these descriptive analyses suggested a fundamental role for the flo gene in tissue progenitor cells. For this reason, a positional strategy was used to identify the responsible gene.


Mutation of the zebrafish nucleoporin elys sensitizes tissue progenitors to replication stress.

Davuluri G, Gong W, Yusuff S, Lorent K, Muthumani M, Dolan AC, Pack M - PLoS Genet. (2008)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2570612&req=5

pgen-1000240-g001: 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.
Mentions: Zebrafish flotte lotte (floti262c; hereafter flo) is a recessive lethal mutation that was identified as part of a large scale ENU mutagenesis screen [23]. Small eyes, optic tectum degeneration and digestive organ defects are prominent features of the flo mutant phenotype that are first evident in live fish on the fourth day post-fertilization (Figure 1A–1D). In previous work, the flo mutation was shown to disrupt differentiation and survival of 3 of the 4 principal intestinal epithelial cell lineages [24],[26], suggesting an effect on organ progenitor cells. Here, we show a comparable though less pronounced effect on retinal development (Figure 1E–1H). Histological analyses revealed that the cellular layers of the flo retinal epithelium were disorganized and contained numerous cells with condensed nuclei typical of apoptotic cells. Acridine orange and TUNEL staining revealed that apoptotic retinal (Figure 1I–1J) and intestinal epithelial cells (Figure 1K–1L) were already evident before flo mutants were morphologically distinguishable from wild type larvae. Immunohistochemical studies showed that differentiated retinal ganglion and photoreceptor cells were present in flo mutants, albeit at reduced levels compared with wild types, despite retinal disorganization and apoptosis (Figure 1M–1P). These studies suggested that early stages of retinal development proceed normally in flo mutants, most likely as a result of maternally derived mRNA and protein encoded by the affected gene (discussed below). To further assess the effect of the flo mutation on organ progenitors, we also measured cell proliferation in the intestinal and retinal epithelium. Both sets of data were consistent with G1 arrest of rapidly proliferating cells in these tissues (Tables S1 and S2). In summary, these descriptive analyses suggested a fundamental role for the flo gene in tissue progenitor cells. For this reason, a positional strategy was used to identify the responsible gene.

Bottom Line: Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine.These in vivo data indicate a role for Elys in Mcm2-chromatin interactions.Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.

ABSTRACT
The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress.

Show MeSH
Related in: MedlinePlus