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Cell May 2015Mammalian genomes are organized into megabase-scale topologically associated domains (TADs). We demonstrate that disruption of TADs can rewire long-range regulatory...
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.
Topics: Animals; Disease Models, Animal; Enhancer Elements, Genetic; Extremities; Gene Expression Regulation; Humans; Limb Deformities, Congenital; Mice; Promoter Regions, Genetic; RNA, Untranslated; Receptor, EphA4
PubMed: 25959774
DOI: 10.1016/j.cell.2015.04.004 -
Cell Oct 2016The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological...
The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.
Topics: Animals; Base Sequence; Biological Evolution; Enhancer Elements, Genetic; Evolution, Molecular; Extremities; Gene Knock-In Techniques; Hedgehog Proteins; Mice; Mice, Transgenic; Mutation; Phylogeny; Snakes
PubMed: 27768887
DOI: 10.1016/j.cell.2016.09.028 -
Cell Feb 2024Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory...
Transcription factors (TFs) can define distinct cellular identities despite nearly identical DNA-binding specificities. One mechanism for achieving regulatory specificity is DNA-guided TF cooperativity. Although in vitro studies suggest that it may be common, examples of such cooperativity remain scarce in cellular contexts. Here, we demonstrate how "Coordinator," a long DNA motif composed of common motifs bound by many basic helix-loop-helix (bHLH) and homeodomain (HD) TFs, uniquely defines the regulatory regions of embryonic face and limb mesenchyme. Coordinator guides cooperative and selective binding between the bHLH family mesenchymal regulator TWIST1 and a collective of HD factors associated with regional identities in the face and limb. TWIST1 is required for HD binding and open chromatin at Coordinator sites, whereas HD factors stabilize TWIST1 occupancy at Coordinator and titrate it away from HD-independent sites. This cooperativity results in the shared regulation of genes involved in cell-type and positional identities and ultimately shapes facial morphology and evolution.
Topics: Basic Helix-Loop-Helix Transcription Factors; Binding Sites; DNA; DNA-Binding Proteins; Gene Expression Regulation; Mesoderm; Transcription Factors; Humans; Animals; Mice; Extremities; Embryonic Development
PubMed: 38262408
DOI: 10.1016/j.cell.2023.12.032 -
ELife Feb 2021How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish...
How genetic changes are linked to morphological novelties and developmental constraints remains elusive. Here, we investigate genetic apparatuses that distinguish fish fins from tetrapod limbs by analyzing transcriptomes and open-chromatin regions (OCRs). Specifically, we compared mouse forelimb buds with the pectoral fin buds of an elasmobranch, the brown-banded bamboo shark (). A transcriptomic comparison with an accurate orthology map revealed both a mass heterochrony and hourglass-shaped conservation of gene expression between fins and limbs. Furthermore, open-chromatin analysis suggested that access to conserved regulatory sequences is transiently increased during mid-stage limb development. During this stage, stage-specific and tissue-specific OCRs were also enriched. Together, early and late stages of fin/limb development are more permissive to mutations than middle stages, which may have contributed to major morphological changes during the fin-to-limb evolution. We hypothesize that the middle stages are constrained by regulatory complexity that results from dynamic and tissue-specific transcriptional controls.
Topics: Animal Fins; Animals; Biological Evolution; Embryo, Mammalian; Embryo, Nonmammalian; Extremities; Limb Buds; Mice; Phylogeny; Sharks
PubMed: 33560225
DOI: 10.7554/eLife.62865 -
Developmental Dynamics : An Official... May 2011The development of the tetrapod limb during skeletogenesis follows a highly conservative pattern characterized by a general proximo-distal progression in the... (Review)
Review
The development of the tetrapod limb during skeletogenesis follows a highly conservative pattern characterized by a general proximo-distal progression in the establishment of skeletal elements and a postaxial polarity in digit development. Salamanders represent the only exception to this pattern and display an early establishment of distal autopodial structures, specifically the basale commune, an amalgamation of distal carpal and tarsal 1 and 2, and a distinct preaxial polarity in digit development. This deviance from the conserved tetrapod pattern has resulted in a number of hypotheses to explain its developmental basis and evolutionary history. Here we summarize the current knowledge of salamander limb development under consideration of the fossil record to provide a deep time perspective of this evolutionary pathway and highlight what data will be needed in the future to gain a better understanding of salamander limb development specifically and tetrapod limb development and evolution more broadly.
Topics: Animals; Biological Evolution; Extremities; Fossils; Regeneration; Urodela
PubMed: 21465623
DOI: 10.1002/dvdy.22629 -
Developmental Biology Sep 2017Vertebrate limb development relies on the activity of signaling centers that promote growth and control patterning along three orthogonal axes of the limb bud. The... (Review)
Review
Vertebrate limb development relies on the activity of signaling centers that promote growth and control patterning along three orthogonal axes of the limb bud. The apical ectodermal ridge, at the distal rim of the limb bud ectoderm, produces WNT and FGF signals, which promote limb bud growth and progressive distalization. The zone of polarizing activity, a discrete postero-distal mesenchymal domain, produces SHH, which stimulates growth and organizes patterning along the antero-posterior axis. The dorsal and ventral ectoderms produce, respectively, WNT7A and BMPs, which induce dorso-ventral limb fates. Interestingly, these signaling centers and the mechanisms they instruct interact with each other to coordinate events along the three axes. We review here the main interactions described between the three axial systems of the developing limb and discuss their relevance to proper limb growth and patterning.
Topics: Animals; Body Patterning; Extremities; Models, Biological; Signal Transduction
PubMed: 28283405
DOI: 10.1016/j.ydbio.2017.03.006 -
Current Osteoporosis Reports Dec 2014In the musculoskeletal system, muscle, tendon, and bone tissues develop in a spatially and temporally coordinated manner, and integrate into a cohesive functional unit... (Review)
Review
In the musculoskeletal system, muscle, tendon, and bone tissues develop in a spatially and temporally coordinated manner, and integrate into a cohesive functional unit by forming specific connections unique to each region of the musculoskeletal system. The mechanisms of these patterning and integration events are an area of great interest in musculoskeletal biology. Hox genes are a family of important developmental regulators and play critical roles in skeletal patterning throughout the axial and appendicular skeleton. Unexpectedly, Hox genes are not expressed in the differentiated cartilage or other skeletal cells, but rather are highly expressed in the tightly associated stromal connective tissues as well as regionally expressed in tendons and muscle connective tissue. Recent work has revealed a previously unappreciated role for Hox in patterning all the musculoskeletal tissues of the limb. These observations suggest that integration of the musculoskeletal system is regulated, at least in part, by Hox function in the stromal connective tissue. This review will outline our current understanding of Hox function in patterning and integrating the musculoskeletal tissues.
Topics: Animals; Cell Differentiation; Cell Proliferation; Extremities; Genes, Homeobox; Humans; Models, Animal; Musculoskeletal Development
PubMed: 25266923
DOI: 10.1007/s11914-014-0241-0 -
Current Opinion in Genetics &... Aug 2021Naturalists leading up to the early 20th century were captivated by the diversity of limb form and function and described its development in a variety of species. The... (Review)
Review
Naturalists leading up to the early 20th century were captivated by the diversity of limb form and function and described its development in a variety of species. The advent of discoveries in genetics followed by molecular biology led to focused efforts in few 'model' species, namely mouse and chicken, to understand conserved mechanisms of limb axis specification and development of the musculoskeletal system. 'Non-traditional' species largely fell by the wayside until their recent resurgence into the spotlight with advances in next-generation sequencing technologies (NGS). In this review, we focus on how the use of NGS has provided insights into the development, loss, and diversification of amniote limbs. Coupled with advances in chromatin interrogation techniques and functional tests in vivo, NGS is opening possibilities to understand the genetic mechanisms that govern the remarkable radiation of vertebrate limb form and function.
Topics: Animals; Chickens; Extremities; Genetic Variation; High-Throughput Nucleotide Sequencing; Mice; Musculoskeletal Development; Musculoskeletal System; Phenotype; Vertebrates
PubMed: 33647833
DOI: 10.1016/j.gde.2021.02.005 -
Developmental Dynamics : An Official... May 2011While the paired forelimb and hindlimb buds of vertebrates are initially morphologically homogeneous, as the limb progenitors differentiate, each individual tissue... (Review)
Review
While the paired forelimb and hindlimb buds of vertebrates are initially morphologically homogeneous, as the limb progenitors differentiate, each individual tissue element attains a characteristic limb-type morphology that ultimately defines the constitution of the forelimb or hindlimb. This review focuses on contemporary understanding of the regulation of limb bud initiation and formation of limb-type specific morphologies and how these regulatory mechanisms evolved in vertebrates. We also attempt to clarify the definition of the terms limb-type identity and limb-type morphology that have frequently been used interchangeably. Over the last decade, three genes, Tbx4, Tbx5, and Pitx1, have been extensively studied for their roles in limb initiation and determining limb-type morphologies. The role of Tbx4 and Tbx5 in limb initiation is clearly established. However, their putative role in the generation of limb-type morphologies remains controversial. In contrast, all evidence supports a function for Pitx1 in determination of hindlimb morphologies.
Topics: Animals; Extremities; Fibroblast Growth Factors; Gene Expression Regulation, Developmental; Humans; Limb Buds; Morphogenesis; T-Box Domain Proteins
PubMed: 21360788
DOI: 10.1002/dvdy.22582 -
Developmental Dynamics : An Official... Jun 2021Unlike axolotls, the urodele Notophthalmus viridescens completes two metamorphoses and emerges from its aquatic environment to mate as a fully terrestrial adult. Larval...
BACKGROUND
Unlike axolotls, the urodele Notophthalmus viridescens completes two metamorphoses and emerges from its aquatic environment to mate as a fully terrestrial adult. Larval and adult limb regeneration are commonly treated as roughly equivalent processes and, at least in part, as a recapitulation of embryonic development.
RESULTS
We compared larval limb development to regeneration of both larval and adult forelimbs and found that there are substantial differences in developmental pattern among larvae and adults. The larval pattern of preaxial dominance is absent in adult regenerates: adult regenerates instead develop digits synchronously, and they do so before proximal autopodial elements have formed discrete aggregation zones. By contrast, larval regenerates follow a pattern of sequential digit formation from anterior to posterior, like their embryonic limb buds.
CONCLUSIONS
Based upon these morphological clues, we conclude that larval regenerates are unlikely to exhibit features of epimorphic regeneration seen in adults, but are more likely to represent a form of developmental regulation. Furthermore, we confirm that post-metamorphic limb regeneration is not a simple recapitulation of ontology at the morphological level. These distinctions may help to explain and interpret some experiments and observations of regeneration in neotenic or paedomorphic urodeles.
Topics: Animals; Extremities; Larva; Limb Buds; Metamorphosis, Biological; Notophthalmus viridescens; Regeneration
PubMed: 33205502
DOI: 10.1002/dvdy.272