Bats are the only mammals capable of self-powered flight, an evolutionary innovation based on the transformation of forelimbs into wings. The bat wing is characterized by an extreme elongation of the second to fifth digits with a wing membrane called the chiropatagium connecting them. Here we investigated the developmental and cellular origin of this structure by comparing bat and mouse limbs using omics tools and single-cell analyses. Despite the substantial morphological differences between the species, we observed an overall conservation of cell populations and gene expression patterns including interdigital apoptosis. Single-cell analyses of micro-dissected embryonic chiropatagium identified a specific fibroblast population, independent of apoptosis-associated interdigital cells, as the origin of this tissue. These distal cells express a conserved gene programme including the transcription factors MEIS2 and TBX3, which are commonly known to specify and pattern the early proximal limb. Transgenic ectopic expression of MEIS2 and TBX3 in mouse distal limb cells resulted in the activation of genes expressed during wing development and phenotypic changes related to wing morphology, such as the fusion of digits. Our results elucidate fundamental molecular mechanisms of bat wing development and illustrate how drastic morphological changes can be achieved through repurposing of existing developmental programmes during evolution.

Erythropoietin (Epo) is the master regulator of erythropoiesis and oxygen homeostasis. Despite its physiological importance, the molecular and genomic contexts of the cells responsible for renal Epo production remain unclear, limiting more-effective therapies for anemia. Here, we performed single-cell RNA and transposase-accessible chromatin (ATAC) sequencing of an Epo reporter mouse to molecularly identify Epo-producing cells under hypoxic conditions. Our data indicate that a distinct population of kidney stroma, which we term Norn cells, is the major source of endocrine Epo production in mice. We use these datasets to identify the markers, signaling pathways and transcriptional circuits characteristic of Norn cells. Using single-cell RNA sequencing and RNA in situ hybridization in human kidney tissues, we further provide evidence that this cell population is conserved in humans. These preliminary findings open new avenues to functionally dissect EPO gene regulation in health and disease and may serve as groundwork to improve erythropoiesis-stimulating therapies.

Systemic sclerosis (scleroderma, SSc) is an incurable autoimmune disease with high morbidity and mortality rates. Here, we conducted a population-scale single-cell genomic analysis of skin and blood samples of 56 healthy controls and 97 SSc patients at different stages of the disease. We found immune compartment dysfunction only in a specific subtype of diffuse SSc patients but global dysregulation of the stromal compartment, particularly in a previously undefined subset of LGR5+-scleroderma-associated fibroblasts (ScAFs). ScAFs are perturbed morphologically and molecularly in SSc patients. Single-cell multiome profiling of stromal cells revealed ScAF-specific markers, pathways, regulatory elements, and transcription factors underlining disease development. Systematic analysis of these molecular features with clinical metadata associates specific ScAF targets with disease pathogenesis and SSc clinical traits. Our high-resolution atlas of the sclerodermatous skin spectrum will enable a paradigm shift in the understanding of SSc disease and facilitate the development of biomarkers and therapeutic strategies.

Chromatin undergoes extensive reprogramming during immune cell differentiation. Histone clipping, an underexplored epigenetic mechanism, ensures precise macrophage development and function and is now found to be dysregulated in autoinflammatory disease.

Copy-number variations (CNVs) are a common cause of congenital limb malformations and are interpreted primarily on the basis of their effect on gene dosage. However, recent studies show that CNVs also influence the 3D genome chromatin organization. The functional interpretation of whether a phenotype is the result of gene dosage or a regulatory position effect remains challenging. Here, we report on two unrelated families with individuals affected by bilateral hypoplasia of the femoral bones, both harboring de novo duplications on chromosome 10q24.32. The ∼0.5 Mb duplications include FGF8, a key regulator of limb development and several limb enhancer elements. To functionally characterize these variants, we analyzed the local chromatin architecture in the affected individuals' cells and re-engineered the duplications in mice by using CRISPR-Cas9 genome editing. We found that the duplications were associated with ectopic chromatin contacts and increased FGF8 expression. Transgenic mice carrying the heterozygous tandem duplication including Fgf8 exhibited proximal shortening of the limbs, resembling the human phenotype. To evaluate whether the phenotype was a result of gene dosage, we generated another transgenic mice line, carrying the duplication on one allele and a concurrent Fgf8 deletion on the other allele, as a control. Surprisingly, the same malformations were observed. Capture Hi-C experiments revealed ectopic interaction with the duplicated region and Fgf8, indicating a position effect. In summary, we show that duplications at the FGF8 locus are associated with femoral hypoplasia and that the phenotype is most likely the result of position effects altering FGF8 expression rather than gene dosage effects.

Postaxial polydactyly (PAP) is a common limb malformation that often leads to cosmetic and functional complications. Molecular evaluation of polydactyly can serve as a tool to elucidate genetic and signaling pathways that regulate limb development, specifically, the anterior-posterior specification of the limb. To date, only five genes have been identified for nonsyndromic PAP: FAM92A, GLI1, GLI3, IQCE and ZNF141. In this study, two Pakistani multiplex consanguineous families with autosomal recessive nonsyndromic PAP were clinically and molecularly evaluated. From both pedigrees, a DNA sample from an affected member underwent exome sequencing. In each family, we identified a segregating frameshift (c.591dupA [p.(Q198Tfs*21)]) and nonsense variant (c.2173A>T [p.(K725*)]) in KIAA0825 (also known as C5orf36). Although KIAA0825 encodes a protein of unknown function, it has been demonstrated that its murine ortholog is expressed during limb development. Our data contribute to the establishment of a catalog of genes important in limb patterning, which can aid in diagnosis and obtaining a better understanding of the biology of polydactyly.

Background Structural variants (SVs) affecting non-coding cis-regulatory elements are a common cause of congenital limb malformation. Yet, the functional interpretation of these non-coding variants remains challenging. The human Liebenberg syndrome is characterised by a partial transformation of the arms into legs and has been shown to be caused by SVs at the PITX1 locus leading to its misregulation in the forelimb by its native enhancer element Pen. This study aims to elucidate the genetic cause of an unsolved family with a mild form of Liebenberg syndrome and investigate the role of promoters in long-range gene regulation. Methods Here, we identify SVs by whole genome sequencing (WGS) and use CRISPR-Cas9 genome editing in transgenic mice to assign pathogenicity to the SVs. Results In this study, we used WGS in a family with three mildly affected individuals with Liebenberg syndrome and identified the smallest deletion described so far including the first non-coding exon of H2AFY. To functionally characterise the variant, we re-engineered the 8.5 kb deletion using CRISPR-Cas9 technology in the mouse and showed that the promoter of the housekeeping gene H2afy insulates the Pen enhancer from Pitx1 in forelimbs; its loss leads to misexpression of Pitx1 by the pan-limb activity of the Pen enhancer causing Liebenberg syndrome. Conclusion Our data indicate that housekeeping promoters may titrate promiscuous enhancer activity to ensure normal morphogenesis. The deletion of the H2AFY promoter as a cause of Liebenberg syndrome highlights this new mutational mechanism and its role in congenital disease.

The regulatory specificity of enhancers and their interaction with gene promoters is thought to be controlled by their sequence and the binding of transcription factors. By studying Pitx1, a regulator of hindlimb development, we show that dynamic changes in chromatin conformation can restrict the activity of enhancers. Inconsistent with its hindlimb-restricted expression, Pitx1 is controlled by an enhancer (Pen) that shows activity in forelimbs and hindlimbs. By Capture Hi-C and three-dimensional modeling of the locus, we demonstrate that forelimbs and hindlimbs have fundamentally different chromatin configurations, whereby Pen and Pitx1 interact in hindlimbs and are physically separated in forelimbs. Structural variants can convert the inactive into the active conformation, thereby inducing Pitx1 misexpression in forelimbs, causing partial arm-to-leg transformation in mice and humans. Thus, tissue-specific three-dimensional chromatin conformation can contribute to enhancer activity and specificity in vivo and its disturbance can result in gene misexpression and disease.

To the Editor: We thank Peron et al.1.Peron A. Boito S. Rizzuti T. et al.Prenatal upper-limb mesomelia and 2q31.1 microdeletions affecting the regulatory genome.10.1038/gim.2018.19Genet Med. 2018; 20: 1-2Google Scholar for their interest and appreciation of our work2.Flöttmann R. Kragesteen B.K. Geuer S. et al.Noncoding copy-number variations are associated with congenital limb malformation.10.1038/gim.2017.154Genet Med. 2018; 20: 1-2Google Scholar and for their contribution to expanding the current limb copy-number-variation morbidity map. The authors describe a fetus with shortening and bowing of radius and ulna (mesomelic dysplasia). Using array comparative genomic hybridization the authors identified two de novo deletions flanking the 5′ and 3′ regions of the HOXD cluster (Supplementary Figure S1 online). The 5′ centromeric 998-kb deletion spans the region upstream of the 5′ HOXD genes including the genes ATF, ATP5G3, and KIAA1715 as well as the regulatory HOXD archipelago, a cluster of well-characterized limb enhancer elements (I-V, GCR, and Prox).3.Andrey G. Montavon T. Mascrez B. et al.A switch between topological domains underlies HoxD genes collinearity in mouse limbs.10.1126/science.1234167Science. 2013; 340: 1234167Google Scholar The telomeric deletion is located downstream 3′ of the HOXD cluster (153 kb including MTX2). The authors conclude that the latter telomeric deletion is likely the cause of the phenotype via the loss of cis-regulatory elements controlling early 3′ HOXD genes in the proximal limb resulting in forelimb-specific mesomelic dysplasia. While the larger deletion removes all known 5′ HOXD enhancers, we point out that the telomeric deletion does not include any of the previously characterized HOXD limb enhancers. Therefore, alternative explanations other than regulatory loss of function need to be considered. Extensive mouse studies have revealed that during limb development, a subset of genes belonging to the HoxD cluster (Hoxd8–Hoxd13) are transcribed in two waves that determine the proximal–distal axis of the limb. The early wave is regulated by the forelimb enhancers CNS39 and CNS65 positioned in the telomeric topologically associating domain (TAD) activating Hoxd8–Hoxd11 transcription that patterns radius/tibia and ulna/fibula (Supplementary Figure S1).3.Andrey G. Montavon T. Mascrez B. et al.A switch between topological domains underlies HoxD genes collinearity in mouse limbs.10.1126/science.1234167Science. 2013; 340: 1234167Google Scholar The succeeding wave is regulated by enhancers located in the centromeric TAD that activate Hoxd9–Hoxd13 expression to form the digits.4.Montavon T. Soshnikova N. Mascrez B. et al.A regulatory archipelago controls Hox genes transcription in digits.1:CAS:528:DC%2BC3MXhsFeisrjE10.1016/j.cell.2011.10.023Cell. 2011; 147: 1132-1145Google Scholar Due to its strong chromatin interaction with the centromeric TAD, Hoxd13 is only very weakly contacted by the telomeric enhancers, leading to its absence of transcription in the developing radius/ulna region. Several structural variants (SVs) at the locus in both mice and humans have been shown to disrupt this regulatory topography of the HOXD cluster causing limb malformation. The mouse mutant Ulnaless is caused by a 770-kb inversion of the HoxD cluster bringing Hoxd13 into the neighborhood of the telomeric enhancer elements and hijacking their regulatory potential. This results in the premature and ectopic onset of Hoxd13 transcription in proximal limb, where Hoxd13 is normally not expressed. This misexpression negatively interferes with the proliferation of mesenchymal cells in the zeugopod, which results in truncation of radius/tibia and ulna/fibula, with the forelimb being more severely affected. Tandem duplications of the HOXD cluster in humans cause Kantaputra mesomelic dysplasia, a severe mesomelic shortening of the upper limbs. These duplications place HOXD13 into the telomeric TAD 3′ regulatory landscape, thereby inducing misexpression of HOXD13 in the proximal limb, similar to the Ulnaless inversion. Based on the specific upper-limb phenotype and the location of the deletions, we strongly suspect that the limb malformation reported by Peron et al. is not a result of a loss of function of anterior 3′ HOXD genes, because the 153-kb deletion in the telomeric TAD does not include any known enhancer elements. Also, chromatin analyses revealed that the critical region for proximal limb enhancers in the telomeric TAD is not included into the reported deletion.3.Andrey G. Montavon T. Mascrez B. et al.A switch between topological domains underlies HoxD genes collinearity in mouse limbs.10.1126/science.1234167Science. 2013; 340: 1234167Google Scholar Finally, the function of HOXD genes in developing limbs is largely redundant with that of HOXA genes and even a homozygous deletion of the entire murine HoxD cluster did not elicit any substantial mesomelic phenotype. Instead, we consider it more likely that the phenotype results from a gain of function of HOXD13 in proximal cells, as already reported in various cases in the mouse. Several scenarios are possible to account for this gain of function (Supplementary Figure S1). First, the deletion of the centromeric regulatory landscape might allow ectopic interactions to be established between HOXD13 and the CNS39 and CNS65 enhancers located in the telomeric TAD. This particular mechanism of contact reallocation was previously observed in a mouse mutant where the centromeric regulatory landscape was displaced several Mb away from the HoxD cluster via a targeted inversion. Proximal limb bud cells showed ectopic expression of Hoxd13 and chromatin interactions between Hoxd13 and the telomeric landscape were increased significantly. These mice displayed a mild mesomelic dysplasia.3.Andrey G. Montavon T. Mascrez B. et al.A switch between topological domains underlies HoxD genes collinearity in mouse limbs.10.1126/science.1234167Science. 2013; 340: 1234167Google Scholar Second, the presence of two deletions flanking both sides of the HOXD cluster might illustrate that the region between the two deletions containing the HOXD cluster itself is inverted in the affected individual, a condition that would remain unnoticed with the technology used by Peron et al. Such an inversion would place HOXD13 on the side of the telomeric regulatory elements resulting in proximal enhancer adoption and misexpression of HOXD13 with consequent development of severe mesomelic dysplasia. This situation would be similar to the Ulnaless allele in mouse and the Kantaputra duplication. This scenario is only possible if the deletions are located in cis on the same chromosome. Only quantitative reverse-transcription polymerase chain reaction in combination with Sanger sequencing of the breakpoints will reveal the exact nature of this complex SV. Third, another possibility is that the centromeric deletion removes a boundary element at its centromeric end resulting in a fusion of the neighboring TAD with the 5′ HOXD genes leading to HOXD13 misexpression in the zeugopod. In conclusion, SVs at the HOXD locus, including the case reported by Peron et al., demonstrate how complex rearrangements can reshuffle noncoding regulatory landscapes of developmental genes causing disease. A major shortcoming of our study and the report by Peron et al. is that array comparative genomic hybridization does not provide positional information nor does it detect inversions, smaller deletions, and duplications (<20 kb). However, as highlighted above, mapping of the exact breakpoints is essential to determine the true pathogenic potential of complex SVs. Whole-genome sequencing promises to be a more appropriate method to detect the full spectrum of SVs at single-nucleotide resolution, which will help to predict their effect on gene regulation. Moreover, in vivo mouse models using CRISPR-Cas9 system provide an excellent tool to understand the effect of chromosomal rearrangements in the genomic and chromatin regulatory context. The authors declare no conflict of interest. We thank Guillaume Andrey for helpful discussion. This work was supported by a grant from the Deutsche Forschungsgemeinschaft to M.S. (SP1532/2-1). Download .jpg (.46 MB) Help with files Supplementary Figure 1

Insulin-resistance is the main cause of type 2 diabetes. Here we describe the identification and characterization of BMP2 and BMP6 as new insulin-sensitizing growth factors in mature adipocytes. We show that BMP2 and BMP6 lead to enhanced insulin-mediated glucose uptake in both insulin-sensitive and -insensitive adipocytes. We exclude a direct effect of BMP2 or BMP6 on translocation of GLUT4 to the plasma membrane and demonstrate that these BMPs increase GLUT4 protein levels equipotent to Rosiglitazone. BMPs induce expression of PPARγ as the crucial mediator for the insulin-sensitizing effect. A comprehensive RNA-Seq analysis in mature adipocytes revealed regulation of both BMP/Smad and PPARγ target genes. The effects of BMP2 and BMP6 are not completely redundant and include regulation of genes involved in glucose and fatty acid metabolism and adipokine expression. Collectively, these findings suggest the BMP2 and BMP6 pathway(s) as promising new drug targets to treat insulin resistance.

PurposeCopy-number variants (CNVs) are generally interpreted by linking the effects of gene dosage with phenotypes. The clinical interpretation of noncoding CNVs remains challenging. We investigated the percentage of disease-associated CNVs in patients with congenital limb malformations that affect noncoding cis-regulatory sequences versus genes sensitive to gene dosage effects.MethodsWe applied high-resolution copy-number analysis to 340 unrelated individuals with isolated limb malformation. To investigate novel candidate CNVs, we re-engineered human CNVs in mice using clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing.ResultsOf the individuals studied, 10% harbored CNVs segregating with the phenotype in the affected families. We identified 31 CNVs previously associated with congenital limb malformations and four novel candidate CNVs. Most of the disease-associated CNVs (57%) affected the noncoding cis-regulatory genome, while only 43% included a known disease gene and were likely to result from gene dosage effects. In transgenic mice harboring four novel candidate CNVs, we observed altered gene expression in all cases, indicating that the CNVs had a regulatory effect either by changing the enhancer dosage or altering the topological associating domain architecture of the genome.ConclusionOur findings suggest that CNVs affecting noncoding regulatory elements are a major cause of congenital limb malformations.

Background Congenital central hypoventilation syndrome (CCHS) is a rare life-threatening disorder of respiratory and autonomic regulation. It is classically caused by dominant mutations in the transcription factor PHOX2B. The objective of the present study was to identify the molecular cause of a recessive form of central hypoventilation with autonomic dysfunction. Methods Here, we used homozygosity mapping and whole-genome sequencing in a consanguineous family with CCHS in combination with functional analyses in CRISPR/Cas9 engineered mice. Results We report on a consanguineous family with three affected children, all tested PHOX2B mutation negative, presenting with alveolar hypoventilation and symptoms of autonomic dysregulation. Whole-genome sequencing revealed a homozygous frameshift mutation in exon 25 of the MYO1H gene (c.2524_2524delA) segregating with the phenotype in the family. MYO1H encodes for the unconventional myosin IH, which is thought to function as a motor protein in intracellular transport and vesicle trafficking. We show that Myo1h is broadly expressed in the mouse lower medulla, including the CO2-sensitive Phox2b+ retrotrapezoid neurons. To test the pathogenicity of the variant, we engineered two Myo1h mutant mouse strains: the first strain (Myo1h*) resembling the human mutation and the second being a full knock-out (Myo1hFS ). Whole-body plethysmography studies in Myo1h* newborns with the re-engineered human mutation revealed hypoventilation and a blunted response to CO2, recapitulating the breathing phenotype observed in the kindred. Conclusions Our results identify MYO1H as an important gene in CO2 sensitivity and respiratory control and as the cause of a rare recessive form of congenital central hypoventilation.

Structural variations (SVs) contribute to the variability of our genome and are often associated with disease. Their study in model systems was hampered until now by labor-intensive genetic targeting procedures and multiple mouse crossing steps. Here we present the use of CRISPR/Cas for the fast (10 weeks) and efficient generation of SVs in mice. We specifically produced deletions, inversions, and also duplications at six different genomic loci ranging from 1.1 kb to 1.6 Mb with efficiencies up to 42%. After PCR-based selection, clones were successfully used to create mice via aggregation. To test the practicability of the method, we reproduced a human 500 kb disease-associated deletion and were able to recapitulate the human phenotype in mice. Furthermore, we evaluated the regulatory potential of a large genomic interval by deleting a 1.5 Mb fragment. The method presented permits rapid in vivo modeling of genomic rearrangements.