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A zebrafish model of Roberts syndrome reveals that Esco2 depletion interferes with development by disrupting the cell cycle.

Mönnich M, Kuriger Z, Print CG, Horsfield JA - PLoS ONE (2011)

Bottom Line: A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21.Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis.We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Dunedin School of Medicine, The University of Otago, Dunedin, New Zealand.

ABSTRACT
The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as "cohesinopathies". However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases.

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Caspase activity is increased in esco2 morphants and rad21 mutants.A, Numbers of S phase cells detected by BrdU labeling were similar in esco2 morphants and wild type at 1 and 2 dpf. Additional depletion of p53 significantly increased S phase entry in esco2 morphants at 2 dpf (asterisk). B, Apoptotic cells labeled with TUNEL were significantly increased in esco2 morphants compared with wild type at 1 and 2 dpf. Additional depletion of p53 did not rescue the observed apoptosis. Asterisks indicate where a significant difference in cell numbers was observed in esco2 morphants compared with wild type and the p53 mutant. C, Caspase 8 activity in esco2 morphants was increased at 1 dpf, but not at 2 dpf compared to wild type control. D, Caspase 8 activity was severely increased in rad21 mutants (rad21 mut) at 1 dpf compared to siblings (rad21 sibs). E, Caspase 3/7 activity was increased in esco2 morphants at 1 dpf and 2 dpf. F, Caspase 3/7 activity was severely increased in rad21 mutants at 1 dpf.
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pone-0020051-g005: Caspase activity is increased in esco2 morphants and rad21 mutants.A, Numbers of S phase cells detected by BrdU labeling were similar in esco2 morphants and wild type at 1 and 2 dpf. Additional depletion of p53 significantly increased S phase entry in esco2 morphants at 2 dpf (asterisk). B, Apoptotic cells labeled with TUNEL were significantly increased in esco2 morphants compared with wild type at 1 and 2 dpf. Additional depletion of p53 did not rescue the observed apoptosis. Asterisks indicate where a significant difference in cell numbers was observed in esco2 morphants compared with wild type and the p53 mutant. C, Caspase 8 activity in esco2 morphants was increased at 1 dpf, but not at 2 dpf compared to wild type control. D, Caspase 8 activity was severely increased in rad21 mutants (rad21 mut) at 1 dpf compared to siblings (rad21 sibs). E, Caspase 3/7 activity was increased in esco2 morphants at 1 dpf and 2 dpf. F, Caspase 3/7 activity was severely increased in rad21 mutants at 1 dpf.

Mentions: To determine if apoptosis in esco2 morphants is p53-dependent, we injected the esco2 MO into embryos homozygous for p53M214K, a genetic background that leads to compromized p53 function. In the p53M214K line, activation of p53 response genes is absent, therefore p53-dependent apoptosis should not be induced [51], [52]. In this p53-deficient background, cells in S phase were similar between esco2-depleted and wild type embryos at 24 hpf (Figs. 5A and S4A–D). However, there was a slight increase of S phases in esco2 morphants in the p53-deficient background at day 2 (Figs. 5B and S4E–H). This may indicate that intact p53 retards re-entry of damaged esco2-depleted cells into the cell cycle. Apoptosis in the p53 mutant background was just as prevalent as that observed on a wild type background (Figs. 5B and S4I–N). Thus, apoptosis in esco2 morphants appears to be independent of p53.


A zebrafish model of Roberts syndrome reveals that Esco2 depletion interferes with development by disrupting the cell cycle.

Mönnich M, Kuriger Z, Print CG, Horsfield JA - PLoS ONE (2011)

Caspase activity is increased in esco2 morphants and rad21 mutants.A, Numbers of S phase cells detected by BrdU labeling were similar in esco2 morphants and wild type at 1 and 2 dpf. Additional depletion of p53 significantly increased S phase entry in esco2 morphants at 2 dpf (asterisk). B, Apoptotic cells labeled with TUNEL were significantly increased in esco2 morphants compared with wild type at 1 and 2 dpf. Additional depletion of p53 did not rescue the observed apoptosis. Asterisks indicate where a significant difference in cell numbers was observed in esco2 morphants compared with wild type and the p53 mutant. C, Caspase 8 activity in esco2 morphants was increased at 1 dpf, but not at 2 dpf compared to wild type control. D, Caspase 8 activity was severely increased in rad21 mutants (rad21 mut) at 1 dpf compared to siblings (rad21 sibs). E, Caspase 3/7 activity was increased in esco2 morphants at 1 dpf and 2 dpf. F, Caspase 3/7 activity was severely increased in rad21 mutants at 1 dpf.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102698&req=5

pone-0020051-g005: Caspase activity is increased in esco2 morphants and rad21 mutants.A, Numbers of S phase cells detected by BrdU labeling were similar in esco2 morphants and wild type at 1 and 2 dpf. Additional depletion of p53 significantly increased S phase entry in esco2 morphants at 2 dpf (asterisk). B, Apoptotic cells labeled with TUNEL were significantly increased in esco2 morphants compared with wild type at 1 and 2 dpf. Additional depletion of p53 did not rescue the observed apoptosis. Asterisks indicate where a significant difference in cell numbers was observed in esco2 morphants compared with wild type and the p53 mutant. C, Caspase 8 activity in esco2 morphants was increased at 1 dpf, but not at 2 dpf compared to wild type control. D, Caspase 8 activity was severely increased in rad21 mutants (rad21 mut) at 1 dpf compared to siblings (rad21 sibs). E, Caspase 3/7 activity was increased in esco2 morphants at 1 dpf and 2 dpf. F, Caspase 3/7 activity was severely increased in rad21 mutants at 1 dpf.
Mentions: To determine if apoptosis in esco2 morphants is p53-dependent, we injected the esco2 MO into embryos homozygous for p53M214K, a genetic background that leads to compromized p53 function. In the p53M214K line, activation of p53 response genes is absent, therefore p53-dependent apoptosis should not be induced [51], [52]. In this p53-deficient background, cells in S phase were similar between esco2-depleted and wild type embryos at 24 hpf (Figs. 5A and S4A–D). However, there was a slight increase of S phases in esco2 morphants in the p53-deficient background at day 2 (Figs. 5B and S4E–H). This may indicate that intact p53 retards re-entry of damaged esco2-depleted cells into the cell cycle. Apoptosis in the p53 mutant background was just as prevalent as that observed on a wild type background (Figs. 5B and S4I–N). Thus, apoptosis in esco2 morphants appears to be independent of p53.

Bottom Line: A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21.Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis.We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Dunedin School of Medicine, The University of Otago, Dunedin, New Zealand.

ABSTRACT
The human developmental diseases Cornelia de Lange Syndrome (CdLS) and Roberts Syndrome (RBS) are both caused by mutations in proteins responsible for sister chromatid cohesion. Cohesion is mediated by a multi-subunit complex called cohesin, which is loaded onto chromosomes by NIPBL. Once on chromosomes, cohesin binding is stabilized in S phase upon acetylation by ESCO2. CdLS is caused by heterozygous mutations in NIPBL or cohesin subunits SMC1A and SMC3, and RBS is caused by homozygous mutations in ESCO2. The genetic cause of both CdLS and RBS reside within the chromosome cohesion apparatus, and therefore they are collectively known as "cohesinopathies". However, the two syndromes have distinct phenotypes, with differences not explained by their shared ontology. In this study, we have used the zebrafish model to distinguish between developmental pathways downstream of cohesin itself, or its acetylase ESCO2. Esco2 depleted zebrafish embryos exhibit features that resemble RBS, including mitotic defects, craniofacial abnormalities and limb truncations. A microarray analysis of Esco2-depleted embryos revealed that different subsets of genes are regulated downstream of Esco2 when compared with cohesin subunit Rad21. Genes downstream of Rad21 showed significant enrichment for transcriptional regulators, while Esco2-regulated genes were more likely to be involved the cell cycle or apoptosis. RNA in situ hybridization showed that runx1, which is spatiotemporally regulated by cohesin, is expressed normally in Esco2-depleted embryos. Furthermore, myca, which is downregulated in rad21 mutants, is upregulated in Esco2-depleted embryos. High levels of cell death contributed to the morphology of Esco2-depleted embryos without affecting specific developmental pathways. We propose that cell proliferation defects and apoptosis could be the primary cause of the features of RBS. Our results show that mutations in different elements of the cohesion apparatus have distinct developmental outcomes, and provide insight into why CdLS and RBS are distinct diseases.

Show MeSH
Related in: MedlinePlus