Conditional Creation and Rescue of Nipbl-Deficiency in Mice Reveals Multiple Determinants of Risk for Congenital Heart Defects

Elucidating the causes of congenital heart defects is made difficult by the complex morphogenesis of the mammalian heart, which takes place early in development, involves contributions from multiple germ layers, and is controlled by many genes. Here, we use a conditional/invertible genetic strategy to identify the cell lineage(s) responsible for the development of heart defects in a Nipbl-deficient mouse model of Cornelia de Lange Syndrome, in which global yet subtle transcriptional dysregulation leads to development of atrial septal defects (ASDs) at high frequency. Using an approach that allows for recombinase-mediated creation or rescue of Nipbl deficiency in different lineages, we uncover complex interactions between the cardiac mesoderm, endoderm, and the rest of the embryo, whereby the risk conferred by genetic abnormality in any one lineage is modified, in a surprisingly non-additive way, by the status of others. We argue that these results are best understood in the context of a model in which the risk of heart defects is associated with the adequacy of early progenitor cell populations relative to the sizes of the structures they must eventually form

Multiple Organ System Defects and Transcriptional Dysregulation in the Nipbl+/− Mouse, a Model of Cornelia de Lange Syndrome

Cornelia de Lange Syndrome (CdLS) is a multi-organ system birth defects disorder linked, in at least half of cases, to heterozygous mutations in the NIPBL gene. In animals and fungi, orthologs of NIPBL regulate cohesin, a complex of proteins that is essential for chromosome cohesion and is also implicated in DNA repair and transcriptional regulation. Mice heterozygous for a gene-trap mutation in Nipbl were produced and exhibited defects characteristic of CdLS, including small size, craniofacial anomalies, microbrachycephaly, heart defects, hearing abnormalities, delayed bone maturation, reduced body fat, behavioral disturbances, and high mortality (75–80%) during the first weeks of life. These phenotypes arose despite a decrease in Nipbl transcript levels of only ∼30%, implying extreme sensitivity of development to small changes in Nipbl activity. Gene expression profiling demonstrated that Nipbl deficiency leads to modest but significant transcriptional dysregulation of many genes. Expression changes at the protocadherin beta (Pcdhb) locus, as well as at other loci, support the view that NIPBL influences long-range chromosomal regulatory interactions. In addition, evidence is presented that reduced expression of genes involved in adipogenic differentiation may underlie the low amounts of body fat observed both in Nipbl+/− mice and in individuals with CdLS

Genetic enhancement of limb defects in a mouse model of Cornelia de Lange syndrome.

Cornelia de Lange Syndrome (CdLS) is characterized by a wide variety of structural and functional abnormalities in almost every organ system of the body. CdLS is now known to be caused by mutations that disrupt the function of the cohesin complex or its regulators, and studies of animal models and cell lines tell us that the effect of these mutations is to produce subtle yet pervasive dysregulation of gene expression. With many hundreds of mostly small gene expression changes occurring in every cell type and tissue, identifying the etiology of any particular birth defect is very challenging. Here we focus on limb abnormalities, which are commonly seen in CdLS. In the limb buds of the Nipbl-haploinsufficient mouse (Nipbl(+/-) mouse), a model for the most common form of CdLS, modest gene expression changes are observed in several candidate pathways whose disruption is known to cause limb abnormalities, yet the limbs of Nipbl(+/-) mice develop relatively normally. We hypothesized that further impairment of candidate pathways might produce limb defects similar to those seen in CdLS, and performed genetic experiments to test this. Focusing on Sonic hedgehog (Shh), Bone morphogenetic protein (Bmp), and Hox gene pathways, we show that decreasing Bmp or Hox function (but not Shh function) enhances polydactyly in Nipbl(+/-) mice, and in some cases produces novel skeletal phenotypes. However, frank limb reductions, as are seen in a subset of individuals with CdLS, do not occur, suggesting that additional signaling and/or gene regulatory pathways are involved in producing such dramatic changes. © 2016 Wiley Periodicals, Inc

Using mouse and zebrafish models to understand the etiology of developmental defects in Cornelia de Lange Syndrome.

Cornelia de Lange Syndrome (CdLS) is a multisystem birth defects disorder that affects every tissue and organ system in the body. Understanding the factors that contribute to the origins, prevalence, and severity of these developmental defects provides the most direct approach for developing screens and potential treatments for individuals with CdLS. Since the majority of cases of CdLS are caused by haploinsufficiency for NIPBL (Nipped-B-like, which encodes a cohesin-associated protein), we have developed mouse and zebrafish models of CdLS by using molecular genetic tools to create Nipbl-deficient mice and zebrafish (Nipbl(+/-) mice, zebrafish nipbl morphants). Studies of these vertebrate animal models have yielded novel insights into the developmental etiology and genes/gene pathways that contribute to CdLS-associated birth defects, particularly defects of the gut, heart, craniofacial structures, nervous system, and limbs. Studies of these mouse and zebrafish CdLS models have helped clarify how deficiency for NIPBL, a protein that associates with cohesin and other transcriptional regulators in the nucleus, affects processes important to the emergence of the structural and physiological birth defects observed in CdLS: NIPBL exerts chromosome position-specific effects on gene expression; it influences long-range interactions between different regulatory elements of genes; and it regulates combinatorial and synergistic actions of genes in developing tissues. Our current understanding is that CdLS should be considered as not only a cohesinopathy, but also a "transcriptomopathy," that is, a disease whose underlying etiology is the global dysregulation of gene expression throughout the organism. © 2016 Wiley Periodicals, Inc

Nipbl and Mediator Cooperatively Regulate Gene Expression to Control Limb Development

<div><p>Haploinsufficiency for Nipbl, a cohesin loading protein, causes Cornelia de Lange Syndrome (CdLS), the most common “cohesinopathy”. It has been proposed that the effects of Nipbl-haploinsufficiency result from disruption of long-range communication between DNA elements. Here we use zebrafish and mouse models of CdLS to examine how transcriptional changes caused by Nipbl deficiency give rise to limb defects, a common condition in individuals with CdLS. In the zebrafish pectoral fin (forelimb), knockdown of Nipbl expression led to size reductions and patterning defects that were preceded by dysregulated expression of key early limb development genes, including <i>fgfs</i>, <i>shha</i>, <i>hand2</i> and multiple <i>hox</i> genes. In limb buds of <i>Nipbl</i>-haploinsufficient mice, transcriptome analysis revealed many similar gene expression changes, as well as altered expression of additional classes of genes that play roles in limb development. In both species, the pattern of dysregulation of <i>hox</i>-gene expression depended on genomic location within the <i>Hox</i> clusters. In view of studies suggesting that Nipbl colocalizes with the mediator complex, which facilitates enhancer-promoter communication, we also examined zebrafish deficient for the Med12 Mediator subunit, and found they resembled Nipbl-deficient fish in both morphology and gene expression. Moreover, combined partial reduction of both Nipbl and Med12 had a strongly synergistic effect, consistent with both molecules acting in a common pathway. In addition, three-dimensional fluorescent in situ hybridization revealed that Nipbl and Med12 are required to bring regions containing long-range enhancers into close proximity with the zebrafish <i>hoxda</i> cluster. These data demonstrate a crucial role for Nipbl in limb development, and support the view that its actions on multiple gene pathways result from its influence, together with Mediator, on regulation of long-range chromosomal interactions.</p></div

Gene expression changes in <i>Nipbl^+/−</i> mouse limb buds.

<p>Relative gene expression levels in limb buds of stage-matched, E10.5 wildtype and <i>Nipbl^+/−</i> mice were determined as described (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#s4" target="_blank">Materials and Methods</a>), and in certain cases confirmed by Q-RT-PCR. Selected transcripts are shown.</p>i<p>Involved in control of <i>Shh</i> expression in limb bud <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Itou1" target="_blank">[101]</a>.</p>ii<p>Also controls <i>Hox</i> gene expression in the limb bud <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Capellini1" target="_blank">[102]</a>.</p>iii<p>Also controls <i>Hox</i> gene expression in the limb bud <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Zakany2" target="_blank">[69]</a>.</p>iv<p>Direct target of Hoxd13 in limb buds <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Salsi2" target="_blank">[103]</a>.</p>v<p>Directs the position of the <i>Shh</i> expression boundary delineating the experimentally defined ZPA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Lettice1" target="_blank">[104]</a>.</p>vi<p>Mesenchymal, involved in chondrocyte proliferation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Liu3" target="_blank">[105]</a>.</p>vii<p>AER-<i>Fgf</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Hung1" target="_blank">[106]</a>.</p>viii<p>Strongly activated by HOXA13 <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Williams1" target="_blank">[67]</a>.</p>ix<p>HOXA13 target in limb buds <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Salsi1" target="_blank">[68]</a>.</p>x<p>Functions as a HOX cofactor during development; complexes with HOXA9; also controls <i>Hox</i> and <i>Shh</i> expression <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Capellini1" target="_blank">[102]</a>.</p>xi<p>Non-canonical Wnt <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Pandur1" target="_blank">[107]</a>.</p>xii<p>Canonical Wnt <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Darken1" target="_blank">[108]</a>.</p>xiii<p>Interacts with some Wnts and Frizzleds and supports Wnt-Fz-Ror2 complex formation, and at the same reduces Wnt-Fz-LRP complex formation, thus favoring non-canonical Wnt signaling <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Yamamoto1" target="_blank">[109]</a>.</p>xiv<p>Wnt regulator; required for maintenance of AER and Shh signaling <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004671#pgen.1004671-Nam1" target="_blank">[110]</a>.</p><p>Gene expression changes in <i>Nipbl^+/−</i> mouse limb buds.</p

Reduced ZPA expression of <i>Shh</i> in <i>Nipbl</i>^+/− mouse limb buds.

<p>(A–B) Whole mount ISH for <i>Shh</i> in the hindlimb buds of E10.5 wild type (A) and <i>Nipbl</i>^+/− (B) mice. In these dorsal views, anterior to the top, the left and right ZPA are seen as localized patches of staining on the posteriolateral edge of each bud (arrowheads). The ZPA of the right forelimb bud is also visible in the background (arrows). Scale bar = 0.5 mm. (C) Quantification of ISH patterns from 5 wild type and 5 mutant embryos. Limb bud and ZPA size were estimated from image areas. Hybridization intensity was measured as mean pixel intensity in the ZPA multiplied by ZPA area. Data are normalized to wild type values. * = p<0.05, ** = p<0.01.</p

Nipbl knockdown disrupts pectoral fin development.

<p>(A–D) Reduced pectoral fins in live Nipbl-deficient embryos at 76 hpf. Dorsal views, anterior to the left. Uninjected control (A), Nipbl-deficient (<i>nipbla/b</i>-MO) (B), injected with 400 pg of <i>nipbla</i> mRNA alone (C) and co-injected with <i>nipbla/b</i>-MO+<i>nipbla</i> mRNA (D). (E) Whisker plots of fin length at 76 hpf; medians: 431.8 µm, n = 50 (control), 258.5 µm, n = 88 (<i>nipbla/b</i>-MOs), 423.0 µm, n = 22 (<i>nipbla</i> mRNA alone), and 319.5 µm, n = 70 (<i>nipbla/b</i>-MOs+<i>nipbla</i> mRNA). p-values are indicated in the graph. (F, F′, G, G′) Alcian blue staining of cartilages in pectoral fins in control (F, F′) and Nipbl-deficient embryos (G, G′) at 120 hpf. F′ and G′ are higher magnification pictures of boxed areas of endoskeletal discs in F and G, respectively. ac, actinotrichs; cl, cleithrum; ed, endoskeletal disc; sco, scapulocoracoid. (H) Numbers of endoskeletal cells in pectoral fins along proximodistal (PD) and anteroposterior (AP) axes (control; n = 13, Nipbl-deficient embryos; n = 16). PD (Ave ± S.D.): 33.2±1.5 (control) and 22.4±4.2 (Nipbl-deficient embryos), p<10⁻⁸. AP (Ave ± S.D.): 27.9±1.5 (control) and 17.7±2.2 (Nipbl-deficient embryos), p<10⁻¹³. (I-X) Morphology of developing pectoral fin buds in live embryos. Lateral views, anterior and dorsal to the left and top, respectively.</p