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Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

MEF2C is a member of the highly conserved MEF2 family of transcription factors and is a key regulator of cardiovascular development. In mice, Mef2c is expressed in the developing heart and vasculature, including the endothelium. Loss of Mef2c function in germline knockout mice leads to early embryonic demise and profound developmental abnormalities in the cardiovascular system. Previous attempts to uncover the cause of embryonic lethality by specifically disrupting Mef2c function in the heart or vasculature failed to recapitulate the global Mef2c knockout phenotype and instead resulted in relatively minor defects that did not compromise viability or result in significant cardiovascular defects. However, previous studies examined the requirement of Mef2c in the myocardial and endothelial lineages using Cre lines that begin to be expressed after the expression of Mef2c has already commenced. Here, we tested the requirement of Mef2c in the myocardial and endothelial lineages using conditional knockout approaches in mice with Cre lines that deleted Mef2c prior to onset of its expression in embryonic development. We found that deletion of Mef2c in the early myocardial lineage using Nkx2-5Cre resulted in cardiac and vascular abnormalities that were indistinguishable from the defects in the global Mef2c knockout. In contrast, early deletion of Mef2c in the vascular endothelium using an Etv2::Cre line active prior to the onset of Mef2c expression resulted in viable offspring that were indistinguishable from wild type controls with no overt defects in vascular development, despite nearly complete early deletion of Mef2c in the vascular endothelium. Thus, these studies support the idea that the requirement of MEF2C for vascular development is secondary to its requirement in the heart and suggest that the observed failure in vascular remodeling in Mef2c knockout mice results from defective heart function.

Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo. The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on a conserved FOX:ETS motif, a composite binding site for Forkhead transcription factors and the Ets transcription factor Etv2, for activity in vivo. The ECE1 FOX:ETS motif is bound and cooperatively activated by FoxC2 and Etv2, but unlike other described FOX:ETS-dependent enhancers, ECE1 enhancer activity becomes restricted to arterial endothelium and endocardium by embryonic day 9.5 in transgenic mouse embryos. The ECE1 endothelial enhancer also contains an evolutionarily-conserved, consensus SOX binding site, which is required for activity in transgenic mouse embryos. Importantly, the ECE1 SOX site is bound and activated by Sox17, a transcription factor involved in endothelial cell differentiation and an important regulator of arterial identity. Moreover, the ECE1 enhancer is cooperatively activated by the combinatorial action of FoxC2, Etv2, and Sox17. Although Sox17 is required for arterial identity, few direct transcriptional targets have been identified in endothelial cells. Thus, this work has important implications for our understanding of endothelial specification and arterial subspecification.

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2008.04.037 Byline: Joshua W. Vincentz, Ralston M. Barnes, Rhonda Rodgers, Beth A. Firulli, Simon J. Conway, Anthony B. Firulli Keywords: Twist1; Heart development; Neural crest; Outflow track; Mouse Abstract: The basic helix-loop-helix transcription factor Twist1 plays an essential role in mesenchymal cell populations during embryonic development and in pathological disease. Remodeling of the cardiac outflow tract (OFT) into the functionally separate aortic arch and pulmonary trunk is dependent upon the dynamic, coordinated contribution of multiple mesenchymal cell populations. Here, we report that Twist1.sup.-/- mice exhibit OFTs that contain amorphic cellular nodules within their OFT endocardial cushions. The nodular mesenchyme expresses the related bHLH factors Hand1 and Hand2, but reduced levels of the normal cushion marker Periostin. Lineage mapping confirms that nodule cells are exclusively of cardiac neural crest origin (cNCC), and are not ectopic cardiomyocytes or smooth muscle cells. These studies also reveal a delay in cNCC colonization of the OFT cushions. Furthermore, these mapping studies uncover nodules in the pharyngeal arches, and identify Twist1.sup.-/- neural crest cell defects within the dorsal neural tube, which exhibits an expanded domain of Wnt1-Cre-lineage marked cells. Together, these data support a model where Twist1 is required both for proper cNCC delamination, and for emigration from the dorsal neural tube and along cNCC migration pathways. Within the Twist1.sup.-/- neural crest cell populations that do emigrate to the OFT, a Hand-expressing subpopulation displays defective maturation, tracking, and, presumably, cell-cell adhesion, further compromising cNCC morphogenesis. Author Affiliation: Riley Heart Research Center, Herman B Wells Center for Pediatric Research Riley Hospital for Children Departments of Pediatrics (Pediatric Cardiology), Anatomy and Medical and Molecular Genetics, Indiana Medical School, 1044 W. Walnut St., Indianapolis, IN 46202-5225, USA Article History: Received 8 February 2008; Revised 23 April 2008; Accepted 29 April 2008

The interactions of diverse transcription factors mediate the molecular programs that regulate mammalian heart development. Among these, Nkx2.5 and the Mef2c regulate common downstream targets and exhibit striking phenotypic similarities when disrupted, suggesting a potential interaction during heart development. Co-immunoprecipitation and mammalian two-hybrid experiments revealed a direct molecular interaction between Nkx2.5 and Mef2c. Assessment of mRNA expression verified spatiotemporal cardiac coexpression. Finally, genetic interaction studies employing histological and molecular analyses showed that, although Nkx2.5−/− and Mef2c−/− individual mutants both have identifiable ventricles, Nkx2.5−/−; Mef2c−/− double mutants do not, and that mutant cardiomyocytes express only atrial and second heart field markers. Molecular marker and cell death and proliferation analyses provide evidence that ventricular hypoplasia is the result of defective ventricular cell differentiation. Collectively, these data support a hypothesis where physical, functional, and genetic interactions between Nkx2.5 and Mef2c are necessary for ventricle formation. Developmental Dynamics 237:3809-3819, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

Endothelin-converting enzyme-1 (Ece-1), a crucial component of the Endothelin signaling pathway, is required for embryonic development and is an important regulator of vascular tone, yet the transcriptional regulation of the ECE1 gene has remained largely unknown. Here, we define the activity and regulation of an enhancer from the human ECE1 locus in vivo . The enhancer identified here becomes active in endothelial progenitor cells shortly after their initial specification and is dependent on a conserved FOX:ETS motif, a composite binding site for Forkhead transcription factors and the Ets transcription factor Etv2, for activity in vivo . The ECE1 FOX:ETS motif is bound and cooperatively activated by FoxC2 and Etv2, but unlike other described FOX:ETS-dependent enhancers, ECE1 enhancer activity becomes restricted to arterial endothelium and endocardium by embryonic day 9.5 in transgenic mouse embryos. The ECE1 endothelial enhancer also contains an evolutionarily-conserved, consensus SOX binding site, which is required for activity in transgenic mouse embryos. Importantly, the ECE1 SOX site is bound and activated by Sox17, a transcription factor involved in endothelial cell differentiation and an important regulator of arterial identity. Moreover, the ECE1 enhancer is cooperatively activated by the combinatorial action of FoxC2, Etv2, and Sox17. Although Sox17 is required for arterial identity, few direct transcriptional targets have been identified in endothelial cells. Thus, this work has important implications for our understanding of endothelial specification and arterial subspecification. [ABSTRACT FROM AUTHOR]

B cell receptors (BCRs) display a combination of variable (V)-gene-encoded complementarity determining regions (CDRs) and adaptive/hypervariable CDR3 loops to engage antigens. It has long been proposed that the former tune for recognition of pathogens or groups of pathogens. To experimentally evaluate this within the human antibody repertoire, we perform immune challenges in transgenic mice that bear diverse human CDR3 and light chains but are constrained to different human V H - genes. We find that, of six commonly deployed V H sequences, only those CDRs encoded by IGHV1-2 ∗ 02 enable polyclonal antibody responses against bacterial lipopolysaccharide (LPS) when introduced to the bloodstream. The LPS is from diverse strains of gram-negative bacteria, and the V H -gene-dependent responses are directed against the non-variable and universal saccrolipid substructure of this antigen. This reveals a broad-spectrum anti-LPS response in which germline-encoded CDRs naturally hardwire the human antibody repertoire for recognition of a conserved microbial target., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)