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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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Pectoral fin development is impaired in esco2 morphants.A, B, E, F, The pectoral fin was shorter and abnormally shaped in esco2 morphants. Fins of wild type (A) and morphant (E) live embryos at 3 dpf (dorsal view). Flat-mounted pectoral fins at 2.5 dpf were smaller and less structured in esco2 morphants (F) compared with wild type (B). C, G, D, H, Alcian Blue staining revealed catch-up growth in pectoral fins at 6 dpf. The fins of esco2 morphants (G) were comparable to wild type (C) in size and overall structure. Closer views of cell structure in pectoral fins at 6 dpf showed that cells in esco2 morphant embryos (H) were smaller and more condensed compared to evenly shaped and similar sized cells in wild type (D), perhaps indicating rapid cell divisions in the morphants.
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pone-0020051-g003: Pectoral fin development is impaired in esco2 morphants.A, B, E, F, The pectoral fin was shorter and abnormally shaped in esco2 morphants. Fins of wild type (A) and morphant (E) live embryos at 3 dpf (dorsal view). Flat-mounted pectoral fins at 2.5 dpf were smaller and less structured in esco2 morphants (F) compared with wild type (B). C, G, D, H, Alcian Blue staining revealed catch-up growth in pectoral fins at 6 dpf. The fins of esco2 morphants (G) were comparable to wild type (C) in size and overall structure. Closer views of cell structure in pectoral fins at 6 dpf showed that cells in esco2 morphant embryos (H) were smaller and more condensed compared to evenly shaped and similar sized cells in wild type (D), perhaps indicating rapid cell divisions in the morphants.

Mentions: A defining characteristic of RBS is tetraphocomelia [26], therefore we were interested to determine the effects of Esco2 depletion on pectoral fin growth. In 72 hpf esco2 morphants, the pectoral fin was abnormally shaped and severely reduced in length when compared with wild type (Fig. 3A, E; B, F). Fin reduction in esco2 morphants was enhanced when the function of p53 was reduced (data not shown). Interestingly, in esco2 hypomorphants, pectoral fin growth was retarded at first, until around 3 dpf, when presumably, the esco2 MO becomes too diluted to retain its effect. At this stage, the fins appear to display catch-up growth (Fig. 3C, G). Strikingly, by 6 dpf, cells appeared to be dividing more actively in the developing fins of esco2 hypomorphants than in wild type embryos (Fig. 3D, H). Our interpretation is that the affected structures appear to compensate for an early paucity of cells by accelerating proliferation once Esco2 levels start to recover. The data suggest that fin stunting is not due to a patterning defect caused by loss of Esco2, but rather by inadequate cell numbers.


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)

Pectoral fin development is impaired in esco2 morphants.A, B, E, F, The pectoral fin was shorter and abnormally shaped in esco2 morphants. Fins of wild type (A) and morphant (E) live embryos at 3 dpf (dorsal view). Flat-mounted pectoral fins at 2.5 dpf were smaller and less structured in esco2 morphants (F) compared with wild type (B). C, G, D, H, Alcian Blue staining revealed catch-up growth in pectoral fins at 6 dpf. The fins of esco2 morphants (G) were comparable to wild type (C) in size and overall structure. Closer views of cell structure in pectoral fins at 6 dpf showed that cells in esco2 morphant embryos (H) were smaller and more condensed compared to evenly shaped and similar sized cells in wild type (D), perhaps indicating rapid cell divisions in the morphants.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020051-g003: Pectoral fin development is impaired in esco2 morphants.A, B, E, F, The pectoral fin was shorter and abnormally shaped in esco2 morphants. Fins of wild type (A) and morphant (E) live embryos at 3 dpf (dorsal view). Flat-mounted pectoral fins at 2.5 dpf were smaller and less structured in esco2 morphants (F) compared with wild type (B). C, G, D, H, Alcian Blue staining revealed catch-up growth in pectoral fins at 6 dpf. The fins of esco2 morphants (G) were comparable to wild type (C) in size and overall structure. Closer views of cell structure in pectoral fins at 6 dpf showed that cells in esco2 morphant embryos (H) were smaller and more condensed compared to evenly shaped and similar sized cells in wild type (D), perhaps indicating rapid cell divisions in the morphants.
Mentions: A defining characteristic of RBS is tetraphocomelia [26], therefore we were interested to determine the effects of Esco2 depletion on pectoral fin growth. In 72 hpf esco2 morphants, the pectoral fin was abnormally shaped and severely reduced in length when compared with wild type (Fig. 3A, E; B, F). Fin reduction in esco2 morphants was enhanced when the function of p53 was reduced (data not shown). Interestingly, in esco2 hypomorphants, pectoral fin growth was retarded at first, until around 3 dpf, when presumably, the esco2 MO becomes too diluted to retain its effect. At this stage, the fins appear to display catch-up growth (Fig. 3C, G). Strikingly, by 6 dpf, cells appeared to be dividing more actively in the developing fins of esco2 hypomorphants than in wild type embryos (Fig. 3D, H). Our interpretation is that the affected structures appear to compensate for an early paucity of cells by accelerating proliferation once Esco2 levels start to recover. The data suggest that fin stunting is not due to a patterning defect caused by loss of Esco2, but rather by inadequate cell numbers.

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