Lars Wittler

Disruptions of Topological Chromatin Domains Cause Pathogenic Rewiring of Gene-Enhancer Interactions

Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory architecture and result in pathogenic phenotypes. We show that distinct human limb malformations are caused by deletions, inversions, or duplications altering the structure of the TAD-spanning WNT6/IHH/EPHA4/PAX3 locus. Using CRISPR/Cas genome editing, we generated mice with corresponding rearrangements. Both in mouse limb tissue and patient-derived fibroblasts, disease-relevant structural changes cause ectopic interactions between promoters and non-coding DNA, and a cluster of limb enhancers normally associated with Epha4 is misplaced relative to TAD boundaries and drives ectopic limb expression of another gene in the locus. This rewiring occurred only if the variant disrupted a CTCF-associated boundary domain. Our results demonstrate the functional importance of TADs for orchestrating gene expression via genome architecture and indicate criteria for predicting the pathogenicity of human structural variants, particularly in non-coding regions of the human genome.

FGF8 functions in the specification of the right body side of the chick

Left-right asymmetry in vertebrate embryos is first recognisable using molecular markers that encode secreted proteins or transcription factors. The asymmetry becomes morphologically obvious in the turning of the embryo and in the development of the heart, the gut and other visceral organs. In the chick embryo, a signalling pathway for the specification of the left body side was demonstrated. Here, Sonic hedgehog (Shh) protein is the first asymmetric signal identified in the node [1] [2]. Further downstream in this pathway are the left-specific genes nodal, lefty-1, lefty-2 and Pitx2 [1] [3] [4] [5]. On the right body side, a function of the activin pathway is indicated by the right-sided expression of cActRIIa [1] [6]. We detected that another key molecule in vertebrate development, fibroblast growth factor 8 (FGF8) [7] [8], is expressed asymmetrically on the right side of the posterior node. We demonstrate that transcription of FGF8 is induced by activin and the FGF8 protein inhibits the expression of nodal and Pitx2 and leads to expression of the chicken snail related gene (cSnR) [9]. Left-sided application of FGF8 randomises the direction of heart looping.

Formation of new chromatin domains determines pathogenicity of genomic duplications

Chromosome conformation capture methods have identified subchromosomal structures of higher-order chromatin interactions called topologically associated domains (TADs) that are separated from each other by boundary regions. By subdividing the genome into discrete regulatory units, TADs restrict the contacts that enhancers establish with their target genes. However, the mechanisms that underlie partitioning of the genome into TADs remain poorly understood. Here we show by chromosome conformation capture (capture Hi-C and 4C-seq methods) that genomic duplications in patient cells and genetically modified mice can result in the formation of new chromatin domains (neo-TADs) and that this process determines their molecular pathology. Duplications of non-coding DNA within the mouse Sox9 TAD (intra-TAD) that cause female to male sex reversal in humans, showed increased contact of the duplicated regions within the TAD, but no change in the overall TAD structure. In contrast, overlapping duplications that extended over the next boundary into the neighbouring TAD (inter-TAD), resulted in the formation of a new chromatin domain (neo-TAD) that was isolated from the rest of the genome. As a consequence of this insulation, inter-TAD duplications had no phenotypic effect. However, incorporation of the next flanking gene, Kcnj2, in the neo-TAD resulted in ectopic contacts of Kcnj2 with the duplicated part of the Sox9 regulatory region, consecutive misexpression of Kcnj2, and a limb malformation phenotype. Our findings provide evidence that TADs are genomic regulatory units with a high degree of internal stability that can be sculptured by structural genomic variations. This process is important for the interpretation of copy number variations, as these variations are routinely detected in diagnostic tests for genetic disease and cancer. This finding also has relevance in an evolutionary setting because copy-number differences are thought to have a crucial role in the evolution of genome complexity.

Expression of Msgn1 in the presomitic mesoderm is controlled by synergism of WNT signalling and Tbx6

The vertebral column and skeletal muscles of vertebrates are derived from the paraxial mesoderm, which is laid down initially as two stripes of mesenchymal cells alongside the neural tube and subsequently segmented. Previous work has shown that the wingless‐type MMTV integration site family (WNT), fibroblast growth factor‐ and Delta–Notch signalling pathways control presomitic mesoderm (psm) formation and segmentation. Here, we show that the expression of mesogenin 1, a basic helix–loop–helix transcription factor, which is essential for psm maturation, is regulated by synergism between WNT signalling and the T‐box 6 transcription factor, involving a feed‐forward control mechanism. These findings emphasize the crucial role of WNT signalling in the control of psm formation, maturation and segmentation.

De novo microduplications at 1q41, 2q37.3, and 8q24.3 in patients with VATER/VACTERL association

The acronym VATER/VACTERL association describes the combination of at least three of the following congenital anomalies: vertebral defects (V), anorectal malformations (A), cardiac defects (C), tracheoesophageal fistula with or without esophageal atresia (TE), renal malformations (R), and limb defects (L). We aimed to identify highly penetrant de novo copy number variations (CNVs) that contribute to VATER/VACTERL association. Array-based molecular karyotyping was performed in a cohort of 41 patients with VATER/VACTERL association and 6 patients with VATER/VACTERL-like phenotype including all of the patients’ parents. Three de novo CNVs were identified involving chromosomal regions 1q41, 2q37.3, and 8q24.3 comprising one (SPATA17), two (CAPN10, GPR35), and three (EPPK1, PLEC, PARP10) genes, respectively. Pre-existing data from the literature prompted us to choose GPR35 and EPPK1 for mouse expression studies. Based on these studies, we prioritized GPR35 for sequencing analysis in an extended cohort of 192 patients with VATER/VACTERL association and VATER/VACTERL-like phenotype. Although no disease-causing mutation was identified, our mouse expression studies suggest GPR35 to be involved in the development of the VATER/VACTERL phenotype. Follow-up of GPR35 and the other genes comprising the identified duplications is warranted.

An inducible RNA interference system for the functional dissection of mouse embryogenesis

Functional analysis of multiple genes is key to understanding gene regulatory networks controlling embryonic development. We have developed an integrated vector system for inducible gene silencing by shRNAmir-mediated RNA interference in mouse embryos, as a fast method for dissecting mammalian gene function. For validation of the vector system, we generated mutant phenotypes for Brachyury, Foxa2 and Noto, transcription factors which play pivotal roles in embryonic development. Using a series of Brachyury shRNAmir vectors of various strengths we generated hypomorphic and loss of function phenotypes allowing the identification of Brachyury target genes involved in trunk development. We also demonstrate temporal control of gene silencing, thus bypassing early embryonic lethality. Importantly, off-target effects of shRNAmir expression were not detectable. Taken together, the system allows the dissection of gene function at unprecedented detail and speed, and provides tight control of the genetic background minimizing intrinsic variation.

In vivo knockdown of Brachyury results in skeletal defects and urorectal malformations resembling caudal regression syndrome

The T-box transcription factor BRACHYURY (T) is a key regulator of mesoderm formation during early development. Complete loss of T has been shown to lead to embryonic lethality around E10.0. Here we characterize an inducible miRNA-based in vivo knockdown mouse model of T, termed KD3-T, which exhibits a hypomorphic phenotype. KD3-T embryos display axial skeletal defects caused by apoptosis of paraxial mesoderm, which is accompanied by urorectal malformations resembling the murine uro-recto-caudal syndrome and human caudal regression syndrome phenotypes. We show that there is a reduction of T in the notochord of KD3-T embryos which results in impaired notochord differentiation and its subsequent loss, whereas levels of T in the tailbud are sufficient for axis extension and patterning. Furthermore, the notochord in KD3-T embryos adopts a neural character and loses its ability to act as a signaling center. Since KD3-T animals survive until birth, they are useful for examining later roles for T in the development of urorectal tissues.

Dlk1 Promotes a Fast Motor Neuron Biophysical Signature Required for Peak Force Execution

Quick, Quick, Slow The slow muscles of postural stability and the fast muscles of running and jumping are driven by motor neurons that are differentiated by fast and slow biophysical properties. By retrograde labeling of mouse and chick muscle fibers, Müller et al. (p. 1264) characterized the developmental distinctions between fast and slow motor neurons. A transmembrane protein, when over- or underexpressed, was discovered to drive specification of the motor neurons and a downstream effector specified some, but not all, of the biophysical attributes. The fast versus slow profile of motor neurons is controlled by expression of a membrane protein. Motor neurons, which relay neural commands to drive skeletal muscle movements, encompass types ranging from “slow” to “fast,” whose biophysical properties govern the timing, gradation, and amplitude of muscle force. Here we identify the noncanonical Notch ligand Delta-like homolog 1 (Dlk1) as a determinant of motor neuron functional diversification. Dlk1, expressed by ~30% of motor neurons, is necessary and sufficient to promote a fast biophysical signature in the mouse and chick. Dlk1 suppresses Notch signaling and activates expression of the K+ channel subunit Kcng4 to modulate delayed-rectifier currents. Dlk1 inactivation comprehensively shifts motor neurons toward slow biophysical and transcriptome signatures, while abolishing peak force outputs. Our findings provide insights into the development of motor neuron functional diversity and its contribution to the execution of movements.

The acquisition of neural fate in the chick

Neural development in the chick embryo is now understood in great detail on a cellular and a molecular level. It begins already before gastrulation, when a separation of neural and epidermal cell fates occurs under the control of FGF and BMP/Wnt signalling, respectively. This early specification becomes further refined around the tip of the primitive streak, until finally the anterior-posterior level of the neuroectoderm becomes established through progressive caudalization. In this review we focus on processes in the chick embryo and put classical and more recent molecular data into a coherent scenario.

Exome sequencing and CRISPR/Cas genome editing identify mutations of ZAK as a cause of limb defects in humans and mice

The CRISPR/Cas technology enables targeted genome editing and the rapid generation of transgenic animal models for the study of human genetic disorders. Here we describe an autosomal recessive human disease in two unrelated families characterized by a split-foot defect, nail abnormalities of the hands, and hearing loss, due to mutations disrupting the SAM domain of the protein kinase ZAK. ZAK is a member of the MAPKKK family with no known role in limb development. We show that Zak is expressed in the developing limbs and that a CRISPR/Cas-mediated knockout of the two Zak isoforms is embryonically lethal in mice. In contrast, a deletion of the SAM domain induces a complex hindlimb defect associated with down-regulation of Trp63, a known split-hand/split-foot malformation disease gene. Our results identify ZAK as a key player in mammalian limb patterning and demonstrate the rapid utility of CRISPR/Cas genome editing to assign causality to human mutations in the mouse in <10 wk.