Limits...
Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair.

Larin M, Gallo D, Tamblyn L, Yang J, Liao H, Sabat N, Brown GW, McPherson JP - Nucleic Acids Res. (2014)

Bottom Line: Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair.This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity.Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada.

Show MeSH

Related in: MedlinePlus

Propensity for apoptosis and DNA damage in FkoMko embryos. (a) Representative TUNEL of head region, neuroepithelium and branchial arch from wild-type, Mko, Fko, and FkoMko E10.5 embryos. (b) Quantification of TUNEL-positive cell/mm2 neuroepithelium and branchial arch. Data represent the mean number of TUNEL-positive cells per genotype ±SD (n = 3). **P < 0.01 for FkoMko or Fko versus wt and Mko by one-way ANOVA followed by Holm–Sidak post hoc test. No differences in TUNEL-labeled cells were detected in the branchial arch. (c) Representative γH2AX staining in head, neuroepithelium and branchial arch from wild-type, Mko, Fko and FkoMko E10.5 embryos. (d) Quantification of γH2AX-positive cells/mm2 in neuroepithelium and branchial arch. Data represent mean number of γH2AX-positive cells ±SD. †††P < 0.001 for FkoMko versus wt and Mko, ††P < 0.01 for FkoMko versus wt or Mko, ***P < 0.001 for FkoMko versus all other genotypes assessed by one-way ANOVA followed by Holm–Sidak post hoc test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150781&req=5

Figure 4: Propensity for apoptosis and DNA damage in FkoMko embryos. (a) Representative TUNEL of head region, neuroepithelium and branchial arch from wild-type, Mko, Fko, and FkoMko E10.5 embryos. (b) Quantification of TUNEL-positive cell/mm2 neuroepithelium and branchial arch. Data represent the mean number of TUNEL-positive cells per genotype ±SD (n = 3). **P < 0.01 for FkoMko or Fko versus wt and Mko by one-way ANOVA followed by Holm–Sidak post hoc test. No differences in TUNEL-labeled cells were detected in the branchial arch. (c) Representative γH2AX staining in head, neuroepithelium and branchial arch from wild-type, Mko, Fko and FkoMko E10.5 embryos. (d) Quantification of γH2AX-positive cells/mm2 in neuroepithelium and branchial arch. Data represent mean number of γH2AX-positive cells ±SD. †††P < 0.001 for FkoMko versus wt and Mko, ††P < 0.01 for FkoMko versus wt or Mko, ***P < 0.001 for FkoMko versus all other genotypes assessed by one-way ANOVA followed by Holm–Sidak post hoc test.

Mentions: In order to understand the underlying cause of the developmental delay in utero, we examined embryonic cell proliferation, apoptosis and DNA damage in situ. Compared to sibling controls, viable FkoMko embryos at E10.5 and E11.5 showed a greatly reduced number of BrdU-positive cells, indicative of reduced proliferation (Figure 3A). In addition, the BrdU-positive cells that were present exhibited a lower level of BrdU incorporation per cell throughout the entire embryo (Figure 3A). When frequency of BrdU-positive cells in the branchial arch was quantified, FkoMko embryos at E10.5 showed a significant decrease in the number of cells synthesizing DNA compared to sibling FhetMhet (P = 0.006), FhetMko (P = 0.012) and FkoMhet (P = 0.018) embryos (Figure 3B). Compared to sibling controls, both Fko and FkoMko embryos at E10.5 showed a dramatic increase in the frequency of apoptotic cells specifically in the neuroepithelium of the developing embryonic brain (P < 0.01 versus wild-type and Mko; Figure 4A and B). Interestingly, the frequency of apoptotic cells was equivalent for all genotypes in the branchial arch, suggesting that loss of FancC renders embryos more susceptible to apoptosis in the forebrain and neuroepithelium, but this effect is specific for FancC loss only and is independent of Mus81 status. We examined the possibility that accumulated DNA damage might serve to trigger apoptosis or proliferation arrest in E10.5 embryos by comparing sibling embryos for the presence of γH2AX. Quantification of γH2AX-positive cells in both the neuroepithelium and branchial arch revealed that FkoMko embryos exhibited a higher frequency of γH2AX-positive cells in both regions (P < 0.001 versuswild-type and Mko, P = 0.002 versus Fko for neuroepithelium, P < 0.001 versus all genotypes assessed for branchial arch; Figure 4C-D), suggesting that the accumulated DNA damage in the absence of both FancC and Mus81 triggered checkpoints leading to either apoptosis or proliferation arrest.


Fanconi anemia signaling and Mus81 cooperate to safeguard development and crosslink repair.

Larin M, Gallo D, Tamblyn L, Yang J, Liao H, Sabat N, Brown GW, McPherson JP - Nucleic Acids Res. (2014)

Propensity for apoptosis and DNA damage in FkoMko embryos. (a) Representative TUNEL of head region, neuroepithelium and branchial arch from wild-type, Mko, Fko, and FkoMko E10.5 embryos. (b) Quantification of TUNEL-positive cell/mm2 neuroepithelium and branchial arch. Data represent the mean number of TUNEL-positive cells per genotype ±SD (n = 3). **P < 0.01 for FkoMko or Fko versus wt and Mko by one-way ANOVA followed by Holm–Sidak post hoc test. No differences in TUNEL-labeled cells were detected in the branchial arch. (c) Representative γH2AX staining in head, neuroepithelium and branchial arch from wild-type, Mko, Fko and FkoMko E10.5 embryos. (d) Quantification of γH2AX-positive cells/mm2 in neuroepithelium and branchial arch. Data represent mean number of γH2AX-positive cells ±SD. †††P < 0.001 for FkoMko versus wt and Mko, ††P < 0.01 for FkoMko versus wt or Mko, ***P < 0.001 for FkoMko versus all other genotypes assessed by one-way ANOVA followed by Holm–Sidak post hoc test.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150781&req=5

Figure 4: Propensity for apoptosis and DNA damage in FkoMko embryos. (a) Representative TUNEL of head region, neuroepithelium and branchial arch from wild-type, Mko, Fko, and FkoMko E10.5 embryos. (b) Quantification of TUNEL-positive cell/mm2 neuroepithelium and branchial arch. Data represent the mean number of TUNEL-positive cells per genotype ±SD (n = 3). **P < 0.01 for FkoMko or Fko versus wt and Mko by one-way ANOVA followed by Holm–Sidak post hoc test. No differences in TUNEL-labeled cells were detected in the branchial arch. (c) Representative γH2AX staining in head, neuroepithelium and branchial arch from wild-type, Mko, Fko and FkoMko E10.5 embryos. (d) Quantification of γH2AX-positive cells/mm2 in neuroepithelium and branchial arch. Data represent mean number of γH2AX-positive cells ±SD. †††P < 0.001 for FkoMko versus wt and Mko, ††P < 0.01 for FkoMko versus wt or Mko, ***P < 0.001 for FkoMko versus all other genotypes assessed by one-way ANOVA followed by Holm–Sidak post hoc test.
Mentions: In order to understand the underlying cause of the developmental delay in utero, we examined embryonic cell proliferation, apoptosis and DNA damage in situ. Compared to sibling controls, viable FkoMko embryos at E10.5 and E11.5 showed a greatly reduced number of BrdU-positive cells, indicative of reduced proliferation (Figure 3A). In addition, the BrdU-positive cells that were present exhibited a lower level of BrdU incorporation per cell throughout the entire embryo (Figure 3A). When frequency of BrdU-positive cells in the branchial arch was quantified, FkoMko embryos at E10.5 showed a significant decrease in the number of cells synthesizing DNA compared to sibling FhetMhet (P = 0.006), FhetMko (P = 0.012) and FkoMhet (P = 0.018) embryos (Figure 3B). Compared to sibling controls, both Fko and FkoMko embryos at E10.5 showed a dramatic increase in the frequency of apoptotic cells specifically in the neuroepithelium of the developing embryonic brain (P < 0.01 versus wild-type and Mko; Figure 4A and B). Interestingly, the frequency of apoptotic cells was equivalent for all genotypes in the branchial arch, suggesting that loss of FancC renders embryos more susceptible to apoptosis in the forebrain and neuroepithelium, but this effect is specific for FancC loss only and is independent of Mus81 status. We examined the possibility that accumulated DNA damage might serve to trigger apoptosis or proliferation arrest in E10.5 embryos by comparing sibling embryos for the presence of γH2AX. Quantification of γH2AX-positive cells in both the neuroepithelium and branchial arch revealed that FkoMko embryos exhibited a higher frequency of γH2AX-positive cells in both regions (P < 0.001 versuswild-type and Mko, P = 0.002 versus Fko for neuroepithelium, P < 0.001 versus all genotypes assessed for branchial arch; Figure 4C-D), suggesting that the accumulated DNA damage in the absence of both FancC and Mus81 triggered checkpoints leading to either apoptosis or proliferation arrest.

Bottom Line: Individuals with Fanconi anemia (FA) are susceptible to bone marrow failure, congenital abnormalities, cancer predisposition and exhibit defective DNA crosslink repair.This cooperativity of FancC and Mus81 in developmental outcome was also mirrored in response to crosslink damage and chromosomal integrity.Thus, our findings reveal that both pathways safeguard against DNA damage from exceeding a critical threshold that triggers proliferation arrest and apoptosis, leading to compromised in utero development.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Toxicology, University of Toronto, Toronto, M5S 1A8, Canada.

Show MeSH
Related in: MedlinePlus