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Vertebrate genomes organize into topologically associating domains, delimited by boundaries that insulate regulatory elements from nontarget genes. However, how boundary function is established is not well understood. Here, we combine genome-wide analyses and transgenic mouse assays to dissect the regulatory logic of clustered-CCCTC-binding factor (CTCF) boundaries in vivo, interrogating their function at multiple levels: chromatin interactions, transcription and phenotypes. Individual CTCF binding site (CBS) deletions revealed that the characteristics of specific sites can outweigh other factors such as CBS number and orientation. Combined deletions demonstrated that CBSs cooperate redundantly and provide boundary robustness. We show that divergent CBS signatures are not strictly required for effective insulation and that chromatin loops formed by nonconvergently oriented sites could be mediated by a loop interference mechanism. Further, we observe that insulation strength constitutes a quantitative modulator of gene expression and phenotypes. Our results highlight the modular nature of boundaries and their control over developmental processes.

© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Copy number variations (CNVs) often include noncoding sequences and putative enhancers, but how these rearrangements induce disease is poorly understood. Here we investigate CNVs involving the regulatory landscape of IHH (encoding Indian hedgehog), which cause multiple, highly localized phenotypes including craniosynostosis and synpolydactyly1,2. We show through transgenic reporter and genome-editing studies in mice that Ihh is regulated by a constellation of at least nine enhancers with individual tissue specificities in the digit anlagen, growth plates, skull sutures and fingertips. Consecutive deletions, resulting in growth defects of the skull and long bones, showed that these enhancers function in an additive manner. Duplications, in contrast, caused not only dose-dependent upregulation but also misexpression of Ihh, leading to abnormal phalanges, fusion of sutures and syndactyly. Thus, precise spatiotemporal control of developmental gene expression is achieved by complex multipartite enhancer ensembles. Alterations in the composition of such clusters can result in gene misexpression and disease.

Runx2 is an essential factor for skeletogenesis and heterozygous loss causes cleidocranial dysplasia in humans and a corresponding phenotype in the mouse. Homozygous Runx2-deficient mice lack hypertrophic cartilage and bone. We compared the expression profiles of E14.5 wildtype and Runx2−/− murine embryonal humeri to identify new transcripts potentially involved in cartilage and bone development. Seventy-one differentially expressed genes were identified by two independent oligonucleotide-microarray hybridizations and quantitative RT-PCR experiments. Gene Ontology analysis demonstrated an enrichment of the differentially regulated genes in annotations to terms such as extracellular, skeletal development, and ossification. In situ hybridization on E15.5 limb sections was performed for all 71 differentially regulated genes. For 54 genes conclusive in situ hybridization results were obtained and all of them showed skeletal expression. Co-expression with Runx2 was demonstrated for 44 genes. While 41 of the 71 differentially expressed genes have a known role in bone and cartilage, we identified 21 known genes that have not yet been implicated in skeletal development and 9 entirely new transcripts. Expression in the developing skeleton was demonstrated for 21 of these genes.

Ror2 is a receptor tyrosine kinase mutated in the human syndromes Brachydactyly type B (BDB) and recessive Robinow syndrome (RS). In this study, we used the chick as a model to investigate the role of Ror2 in skeletogenesis and to elucidate the functional consequences of Ror2 mutations. For this purpose, we cloned chicken Ror2 and analyzed its expression pattern at various embryonic stages by in situ hybridization and immunolabeling. We document expression of cRor2 in several organs, including mesonephros, heart, nervous system, intestine and cartilage. The high conservation of expression when compared with the mouse underlines the validity of the chick as a model system. Using replication-competent retroviral vector-mediated overexpression, we analyzed the functional consequences of truncating BDB and RS mutations in the developing chick limb. Overexpression of Ror2 mutants led to a disturbance of growth plate architecture and a severe block of chondrocyte differentiation, demonstrating the functional importance of Ror2 in skeletogenesis.

The manuscript describes the “digital transcriptome atlas” of the developing mouse embryo, a powerful resource to determine co-expression of genes, to identify cell populations and lineages and to identify functional associations between genes relevant to development and disease., Ascertaining when and where genes are expressed is of crucial importance to understanding or predicting the physiological role of genes and proteins and how they interact to form the complex networks that underlie organ development and function. It is, therefore, crucial to determine on a genome-wide level, the spatio-temporal gene expression profiles at cellular resolution. This information is provided by colorimetric RNA in situ hybridization that can elucidate expression of genes in their native context and does so at cellular resolution. We generated what is to our knowledge the first genome-wide transcriptome atlas by RNA in situ hybridization of an entire mammalian organism, the developing mouse at embryonic day 14.5. This digital transcriptome atlas, the Eurexpress atlas (http://www.eurexpress.org), consists of a searchable database of annotated images that can be interactively viewed. We generated anatomy-based expression profiles for over 18,000 coding genes and over 400 microRNAs. We identified 1,002 tissue-specific genes that are a source of novel tissue-specific markers for 37 different anatomical structures. The quality and the resolution of the data revealed novel molecular domains for several developing structures, such as the telencephalon, a novel organization for the hypothalamus, and insight on the Wnt network involved in renal epithelial differentiation during kidney development. The digital transcriptome atlas is a powerful resource to determine co-expression of genes, to identify cell populations and lineages, and to identify functional associations between genes relevant to development and disease., Author Summary In situ hybridization (ISH) can be used to visualize gene expression in cells and tissues in their native context. High-throughput ISH using nonradioactive RNA probes allowed the Eurexpress consortium to generate a comprehensive, interactive, and freely accessible digital gene expression atlas, the Eurexpress transcriptome atlas (http://www.eurexpress.org), of the E14.5 mouse embryo. Expression data for over 15,000 genes were annotated for hundreds of anatomical structures, thus allowing us to systematically identify tissue-specific and tissue-overlapping gene networks. We illustrate the value of the Eurexpress atlas by finding novel regional subdivisions in the developing brain. We also use the transcriptome atlas to allocate specific components of the complex Wnt signaling pathway to kidney development, and we identify regionally expressed genes in liver that may be markers of hematopoietic stem cell differentiation.

Odd-skipped genes encode zinc-finger transcription factors with widespread roles in embryonic development. In Drosophila, odd-skipped acts as a pair-rule gene, while its orthologous gene in Caenorhabditis elegans is involved in gut development. In mammals two paralogs exist, Osr1 and Osr2, with functions described in heart and urogenital, and in secondary palate development, respectively. As the chicken embryo is a widely used system for analysing gene function in vivo, we determined the expression pattern of the two chicken orthologues, cOsr1 and cOsr2, during embryonic development. We demonstrate expression of both genes in a variety of organs and structures, such as kidney, eye, branchial arches and dermis. Both genes show a highly dynamic expression pattern with partially overlapping, but mostly distinct domains of expression. Special emphasis in this study was laid on the investigation of cOsr1 and cOsr2 in limb development, where we compared their expression pattern with the expression of Osr1 and Osr2 in the mouse.