Title Multifactorial Origins of Heart and Gut Defects in nipbl-Deficient Zebrafish , a Model of Cornelia de Lange Syndrome Permalink

Cornelia de Lange Syndrome (CdLS) is the founding member of a class of multi-organ system birth defect syndromes termed cohesinopathies, named for the chromatin-associated protein complex cohesin, which mediates sister chromatid cohesion. Most cases of CdLS are caused by haploinsufficiency for Nipped-B-like (Nipbl), a highly conserved protein that facilitates cohesin loading. Consistent with recent evidence implicating cohesin and Nipbl in transcriptional regulation, both CdLS cell lines and tissues of Nipbl-deficient mice show changes in the expression of hundreds of genes. Nearly all such changes are modest, however—usually less than 1.5-fold—raising the intriguing possibility that, in CdLS, severe developmental defects result from the collective action of many otherwise innocuous perturbations. As a step toward testing this hypothesis, we developed a model of nipbl-deficiency in zebrafish, an organism in which we can quantitatively investigate the combinatorial effects of gene expression changes. After characterizing the structure and embryonic expression of the two zebrafish nipbl genes, we showed that morpholino knockdown of these genes produces a spectrum of specific heart and gut/visceral organ defects with similarities to those in CdLS. Analysis of nipbl morphants further revealed that, as early as gastrulation, expression of genes involved in endodermal differentiation (sox32, sox17, foxa2, and gata5) and left-right patterning (spaw, lefty2, and dnah9) is altered. Experimental manipulation of the levels of several such genes—using RNA injection or morpholino knockdown—implicated both additive and synergistic interactions in causing observed developmental defects. These findings support the view that birth defects in CdLS arise from collective effects of quantitative changes in gene expression. Interestingly, both the phenotypes and gene expression changes in nipbl morphants differed from those in mutants or morphants for genes encoding cohesin subunits, suggesting that the transcriptional functions of Nipbl cannot be ascribed simply to its role in cohesin loading. Citation: Muto A, Calof AL, Lander AD, Schilling TF (2011) Multifactorial Origins of Heart and Gut Defects in nipbl-Deficient Zebrafish, a Model of Cornelia de Lange Syndrome. PLoS Biol 9(10): e1001181. doi:10.1371/journal.pbio. 1001181 Academic Editor: Derek L. Stemple, The Wellcome Trust Sanger Institute, United Kingdom Received December 15, 2010; Accepted September 13, 2011; Published October 25, 2011 Copyright: 2011 Muto et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the US National Institute of Health (P01-HD052860) and the Cornelia de Lange Syndrome Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: CdLS, Cornelia de Lange Syndrome; DIG, digoxigenin; EGFP, enhanced green fluorescent protein; ISH, in situ hybridization; KV, Kupffer’s vesicle; L/R, left-right; MO, morpholino; Nipbl, Nipped-B-like; NLS, nuclear localization signal; Q-PCR, quantitative RT-PCR; RT-PCR, reverse transcription polymerase chain reaction; UTR, untranslated region * E-mail: adlander@uci.edu . These authors contributed equally to this work.