ISL1 Directly Regulates FGF10 Transcription during Human Cardiac Outflow Formation

The LIM homeodomain gene Islet-1 (ISL1) encodes a transcription factor that has been associated with the multipotency of human cardiac progenitors, and in mice enables the correct deployment of second heart field (SHF) cells to become the myocardium of atria, right ventricle and outflow tract. Other markers have been identified that characterize subdomains of the SHF, such as the fibroblast growth factor Fgf10 in its anterior region. While functional evidence of its essential contribution has been demonstrated in many vertebrate species, SHF expression of Isl1 has been shown in only some models. We examined the relationship between human ISL1 and FGF10 within the embryonic time window during which the linear heart tube remodels into four chambers. ISL1 transcription demarcated an anatomical region supporting the conserved existence of a SHF in humans, and transcription factors of the GATA family were co-expressed therein. In conjunction, we identified a novel enhancer containing a highly conserved ISL1 consensus binding site within the FGF10 first intron. ChIP and EMSA demonstrated its direct occupation by ISL1. Transcription mediated by ISL1 from this FGF10 intronic element was enhanced by the presence of GATA4 and TBX20 cardiac transcription factors. Finally, transgenic mice confirmed that endogenous factors bound the human FGF10 intronic enhancer to drive reporter expression in the developing cardiac outflow tract. These findings highlight the interest of examining developmental regulatory networks directly in human tissues, when possible, to assess candidate non-coding regions that may be responsible for congenital malformations

Matthew-Wood Syndrome Is Caused by Truncating Mutations in the Retinol-Binding Protein Receptor Gene STRA6

Retinoic acid (RA) is a potent teratogen in all vertebrates when tight homeostatic controls on its endogenous dose, location, or timing are perturbed during early embryogenesis. STRA6 encodes an integral cell-membrane protein that favors RA uptake from soluble retinol-binding protein; its transcription is directly regulated by RA levels. Molecular analysis of STRA6 was undertaken in two human fetuses from consanguineous families we previously described with Matthew-Wood syndrome in a context of severe microphthalmia, pulmonary agenesis, bilateral diaphragmatic eventration, duodenal stenosis, pancreatic malformations, and intrauterine growth retardation. The fetuses had either a homozygous insertion/deletion in exon 2 or a homozygous insertion in exon 7 predicting a premature stop codon in STRA6 transcripts. Five other fetuses presenting at least one of the two major signs of clinical anophthalmia or pulmonary hypoplasia with at least one of the two associated signs of diaphragmatic closure defect or cardiopathy had no STRA6 mutations. These findings suggest a molecular basis for the prenatal manifestations of Matthew-Wood syndrome and suggest that phenotypic overlap with other associations may be due to genetic heterogeneity of elements common to the RA- and fibroblast growth factor–signaling cascades

Expression of <i>ISL1</i> and <i>GATA4</i> transcripts in the human heart between 26 and 38 days of gestation.

<p><b>A–H</b>: <i>ISL1 in situ</i> at Carnegie stages (CS)12 (26–28 days post fertilization [dpf]), CS13 (28–31 dpf), CS14 (32–33 dpf) and CS15 (34–36 dpf) respectively. <b>E–H</b> are magnifications of <b>A–D</b> respectively. <b>I–K</b> show <i>GATA4</i> expression in adjacent sections to <b>B–D</b>. <b>A</b>: <i>ISL1</i> is expressed at CS12 in foregut endoderm, splanchnic mesoderm, and early motoneurons. <b>B, F</b>: At CS13, <i>ISL1</i> is transcribed by mesenchyme around the cardiac OFT and pharyngeal arches. <i>ISL1</i> expression continues in the splanchnic mesoderm between the trachea and OFT, and is visible in dorsal root ganglia, at CS14 (<b>C, G</b>) and CS15 (<b>D, H</b>). <b>I–K</b>: <i>GATA4</i> is expressed in the endocardium and myocardium of the arterial pole at CS13, CS14 and CS15 (<b>I, J, K</b> respectively). <b>Inset</b>: RT-PCR of <i>ISL1</i>, <i>GATA4</i>, <i>GATA5</i>, <i>GATA6</i>, <i>FGF10</i> and positive control <i>ACTB</i> mRNAs in embryonic human hearts at stages CS13-16 (to 40 dpf). Abbreviations: drg, dorsal root ganglia; es, esophagus; fb, forebrain; fg, foregut; ph, pharynx; nt, neural tube; oft, OFT; ra, right atrium; t, trachea. Arrows, motoneurons. Bar: 110 µm (A–D, I) and 55 µm (E–H, J, K).</p

<i>In vitro</i> reporter assays support an additive combinatorial effect of transcription factors upon the FGF10 intronic enhancer.

<p>LUC-<i>FGF10</i>-Int1, which construct placed the luciferase gene under the control of the FGF10-Int1 element, was transfected alone or together with <i>ISL1</i>, <i>GATA4</i> and <i>TBX20</i> expression vectors into 10T1/2 cells. Each factor alone potentiated luciferase expression and these effects were additive in combination.</p

Sites of β-galactosidase activity in transgenic mouse embryos.

<p>All sites showed only selective cells positive for enhancer activation. DRGs = dorsal root ganglia; E = embryonic day of gestation; MN = motoneurons; OFT = cardiac outflow tract; PA = pharyngeal arch; PSM = pre-somitic mesoderm.</p

Bioinformatics analyses of the human <i>FGF10</i> locus surrounding the first exon.

<p><b>A</b>: Alignment of genomic regions around and within the human [hg18] <i>FGF10</i> locus to those of frog [xenTro2], chicken [galGal3], opossum [monDom4], mouse [mm9], dog [canFam2] and rhesus macaque [rheMac2] with colored regions >90% identical and the vertical scale ranging from 50% (bottom) to 100% (top). Color code for genomic features at <a href="http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html" target="_blank">http://ecrbrowser.dcode.org/ecrInstructions/ecrInstructions.html</a>. The <i>FGF10</i>-Pr1, <i>FGF10</i>-Pr2 and FGF10-Int1 regions examined in this study are boxed. <b>B</b>: A non-canonical predicted site for GATA-type transcription factors is 52 nucleotides 5′ to the ISL1 cognate sequence in <i>FGF10</i>-Int1 in the direction of transcription on the – strand in humans, mice and (not shown) macaque and opossum. <b>C</b>: Nucleotide sequence of the <i>FGF10</i>-Int1 enhancer module and position of conserved putative transcription factor binding sites as predicted by rVista (<a href="http://rvista.dcode.org" target="_blank">http://rvista.dcode.org</a>). All indicated human sites are identical to those of the macaque and mouse except for the SMAD prediction, only found in mouse; the ISL1, GATA and HOXA7 sites are also identical to the opossum, and the ISL1, NKX2-5 and TBX sites are also identical to the dog.</p

The Immune Signaling Adaptor LAT Contributes to the Neuroanatomical Phenotype of 16p11.2 BP2-BP3 CNVs

Copy-number changes in 16p11.2 contribute significantly to neuropsychiatric traits. Besides the 600 kb BP4-BP5 CNV found in 0.5%-1% of individuals with autism spectrum disorders and schizophrenia and whose rearrangement causes reciprocal defects in head size and body weight, a second distal 220 kb BP2-BP3 CNV is likewise a potent driver of neuropsychiatric, anatomical, and metabolic pathologies. These two CNVs are engaged in complex reciprocal chromatin looping, intimating a functional relationship between genes in these regions that might be relevant to pathomechanism. We assessed the drivers of the distal 16p11.2 duplication by overexpressing each of the nine encompassed genes in zebrafish. Only overexpression of LAT induced a reduction of brain proliferating cells and concomitant microcephaly. Consistently, suppression of the zebrafish ortholog induced an increase of proliferation and macrocephaly. These phenotypes were not unique to zebrafish; Lat knockout mice show brain volumetric changes. Consistent with the hypothesis that LAT dosage is relevant to the CNV pathology, we observed similar effects upon overexpression of CD247 and ZAP70, encoding members of the LAT signalosome. We also evaluated whether LAT was interacting with KCTD13, MVP, and MAPK3, major driver and modifiers of the proximal 16p11.2 600 kb BP4-BP5 syndromes, respectively. Co-injected embryos exhibited an increased microcephaly, suggesting the presence of genetic interaction. Correspondingly, carriers of 1.7 Mb BP1-BP5 rearrangements that encompass both the BP2-BP3 and BP4-BP5 loci showed more severe phenotypes. Taken together, our results suggest that LAT, besides its well-recognized function in T cell development, is a major contributor of the 16p11.2 220 kb BP2-BP3 CNV-associated neurodevelopmental phenotypes.PMC563023

A Potential Contributory Role for Ciliary Dysfunction in the 16p11.2 600 kb BP4-BP5 Pathology

The 16p11.2 600 kb copy-number variants (CNVs) are associated with mirror phenotypes on BMI, head circumference, and brain volume and represent frequent genetic lesions in autism spectrum disorders (ASDs) and schizophrenia. Here we interrogated the transcriptome of individuals carrying reciprocal 16p11.2 CNVs. Transcript perturbations correlated with clinical endophenotypes and were enriched for genes associated with ASDs, abnormalities of head size, and ciliopathies. Ciliary gene expression was also perturbed in orthologous mouse models, raising the possibility that ciliary dysfunction contributes to 16p11.2 pathologies. In support of this hypothesis, we found structural ciliary defects in the CA1 hippocampal region of 16p11.2 duplication mice. Moreover, by using an established zebrafish model, we show genetic interaction between KCTD13, a key driver of the mirrored neuroanatomical phenotypes of the 16p11.2 CNV, and ciliopathy-associated genes. Overexpression of BBS7 rescues head size and neuroanatomical defects of kctd13 morphants, whereas suppression or overexpression of CEP290 rescues phenotypes induced by KCTD13 under- or overexpression, respectively. Our data suggest that dysregulation of ciliopathy genes contributes to the clinical phenotypes of these CNVs.status: publishe