DNA-guided transcription factor cooperativity shapes face and limb mesenchyme.

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.

A single-cell census of mouse limb development identifies complex spatiotemporal dynamics of skeleton formation.

Limb development has long served as a model system for coordinated spatial patterning of progenitor cells. Here, we identify a population of naive limb progenitors and show that they differentiate progressively to form the skeleton in a complex, non-consecutive, three-dimensional pattern. Single-cell RNA sequencing of the developing mouse forelimb identified three progenitor states: naive, proximal, and autopodial, as well as Msx1 as a marker for the naive progenitors. In vivo lineage tracing confirmed this role and localized the naive progenitors to the outer margin of the limb, along the anterior-posterior axis. Sequential pulse-chase experiments showed that the progressive transition of Msx1+ naive progenitors into proximal and autopodial progenitors coincides with their differentiation to Sox9+ chondroprogenitors, which occurs along all the forming skeletal segments. Indeed, tracking the spatiotemporal sequence of differentiation showed that the skeleton forms progressively in a complex pattern. These findings suggest an alternative model for limb skeleton development.

Butterfly eyespots evolved via cooption of an ancestral gene-regulatory network that also patterns antennae, legs, and wings.

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via cooption of parts of an ancestral appendage gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster most closely with antennae, relative to multiple other tissues. Furthermore, three genes essential for eyespot development, Distal-less (Dll), spalt (sal), and Antennapedia (Antp), share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots, antennae, legs, and also wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely reused an ancient GRN for their development, a network also previously implicated in the development of antennae, legs, and wings.

Developmental biology: A 5'Hoxd-Gli3 balance in tetrapod axial polarity.

Almost all living tetrapods exhibit postaxial dominance in digit formation, apart from urodele amphibians, which show preaxial dominance. Recent work shines light on the genetic differences between the two modes of limb development, suggesting that differences in 5'Hoxd expression, mediated by Gli3, may explain the switch in axial polarity.

The Shh/Gli3 gene regulatory network precedes the origin of paired fins and reveals the deep homology between distal fins and digits.

One of the central problems of vertebrate evolution is understanding the relationship among the distal portions of fins and limbs. Lacking comparable morphological markers of these regions in fish and tetrapods, these relationships have remained uncertain for the past century and a half. Here we show that Gli3 functions in controlling the proliferative expansion of distal progenitors are shared among dorsal and paired fins as well as tetrapod limbs. Mutant knockout gli3 fins in medaka (Oryzias latipes) form multiple radials and rays, in a pattern reminiscent of the polydactyly observed in Gli3-null mutant mice. In limbs, Gli3 controls both anterior-posterior patterning and cell proliferation, two processes that can be genetically uncoupled. In situ hybridization, quantification of proliferation markers, and analysis of regulatory regions reveal that in paired and dorsal fins, gli3 plays a main role in controlling proliferation but not in patterning. Moreover, gli3 down-regulation in shh mutant fins rescues fin loss in a manner similar to how Gli3 deficiency restores digits in the limbs of Shh mutant mouse embryos. We hypothesize that the Gli3/Shh gene pathway preceded the origin of paired appendages and was originally involved in modulating cell proliferation. Accordingly, the distal regions of dorsal fins, paired fins, and limbs retain a deep regulatory and functional homology that predates the origin of paired appendages.

Spatial waves and temporal oscillations in vertebrate limb development.

The mesenchymal tissue of the developing vertebrate limb bud is an excitable medium that sustains both spatial and temporal periodic phenomena. The first of these is the outcome of general Turing-type reaction-diffusion dynamics that generate spatial standing waves of cell condensations. These condensations are transformed into the nodules and rods of the cartilaginous, and eventually (in most species) the bony, endoskeleton. In the second, temporal periodicity results from intracellular regulatory dynamics that generate oscillations in the expression of one or more gene whose products modulate the spatial patterning system. Here we review experimental evidence from the chicken embryo, interpreted by a set of mathematical and computational models, that the spatial wave-forming system is based on two glycan-binding proteins, galectin-1A and galectin-8 in interaction with each other and the cells that produce them, and that the temporal oscillation occurs in the expression of the transcriptional coregulator Hes1. The multicellular synchronization of the Hes1 oscillation across the limb bud serves to coordinate the biochemical states of the mesenchymal cells globally, thereby refining and sharpening the spatial pattern. Significantly, the wave-forming reaction-diffusion-based mechanism itself, unlike most Turing-type systems, does not contain an oscillatory core, and may have evolved to this condition as it came to incorporate the cell-matrix adhesion module that enabled its pattern-forming capability.

Limb development in skeletally-immature large-sized dogs: A radiographic study.

Despite the extreme morphological variability of the canine species, data on limb development are limited and the time windows for the appearance of the limb ossification centres (OCs) reported in veterinary textbooks, considered universally valid for all dogs, are based on dated studies. The aim of this study was to acquire up-to-date information regarding the arm, forearm and leg bone development in skeletally-immature large-sized dogs from 6 weeks to 16 weeks of age. Nine litters of 5 large-sized breeds (Boxer, German Shepherd, Labrador Retriever, Saarloos Wolfdog, White Swiss Shepherd Dog) were included, for a total of 54 dogs, which were subject to radiographic examination on a bi-weekly basis. The appearance of 18 limb OCs was recorded and 14 radiographic measurements were performed; their relationship with age and body weight was investigated and any breed differences were analysed using different statistical non-parametric tests. The number of OCs present was significantly different at 6 and 8 weeks of age between the investigated breeds. The appearance of the OCs occurred earlier in the Saarloos Wolfdog, while the Labrador Retriever was the later breed. In Boxers and Labrador Retrievers, various OCs showed a delayed appearance compared to the data reported in the literature. The number of OCs was strongly and positively correlated to body weight. Breed differences were also observed in the relative increase of the measured OCs and were not limited to dogs of different morphotypes. Statistically significant differences were most frequently observed between Saarloos Wolfdogs and the other breeds. The OCs that showed a greater variability in their development were the olecranon tuber, the patella and the tibial tuberosity. Their increase was more strongly correlated with the dog's age and body weight. Our data strongly suggest that differences in limb development exist in dog breeds of similar size and morphotype.

Secreted inhibitors drive the loss of regeneration competence in Xenopus limbs.

Absence of a specialized wound epidermis is hypothesized to block limb regeneration in higher vertebrates. However, the factors preventing its formation in regeneration-incompetent animals are poorly understood. To characterize the endogenous molecular and cellular regulators of specialized wound epidermis formation in Xenopus laevis tadpoles, and the loss of their regeneration competency during development, we used single-cell transcriptomics and ex vivo regenerating limb cultures. Transcriptomic analysis revealed that the specialized wound epidermis is not a novel cell state, but a re-deployment of the apical-ectodermal-ridge (AER) programme underlying limb development. Enrichment of secreted inhibitory factors, including Noggin, a morphogen expressed in developing cartilage/bone progenitor cells, are identified as key inhibitors of AER cell formation in regeneration-incompetent tadpoles. These factors can be overridden by Fgf10, which operates upstream of Noggin and blocks chondrogenesis. These results indicate that manipulation of the extracellular environment and/or chondrogenesis may provide a strategy to restore regeneration potential in higher vertebrates.

FGF signalling plays similar roles in development and regeneration of the skeleton in the brittle star Amphiura filiformis.

Regeneration as an adult developmental process is in many aspects similar to embryonic development. Although many studies point out similarities and differences, no large-scale, direct and functional comparative analyses between development and regeneration of a specific cell type or structure in one animal exist. Here, we use the brittle star Amphiura filiformis to characterise the role of the FGF signalling pathway during skeletal development in embryos and arm regeneration. In both processes, we find ligands expressed in ectodermal cells that flank underlying skeletal mesenchymal cells, which express the receptors. Perturbation of FGF signalling showed inhibited skeleton formation in both embryogenesis and regeneration, without affecting other key developmental processes. Differential transcriptome analysis finds mostly differentiation genes rather than transcription factors to be downregulated in both contexts. Moreover, comparative gene analysis allowed us to discover brittle star-specific differentiation genes. In conclusion, our results show that the FGF pathway is crucial for skeletogenesis in the brittle star, as in other deuterostomes, and provide evidence for the re-deployment of a developmental gene regulatory module during regeneration.

Diversification of the vertebrate limb: sequencing the events.

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.

Common pathogenesis for sirenomelia, OEIS complex, limb-body wall defect, and other malformations of caudal structures.

Decades of clinical, pathological, and epidemiological study and the recent application of advanced microarray and gene sequencing technologies have led to an understanding of the causes and pathogenesis of most recognized patterns of malformation. Still, there remain a number of patterns of malformation whose pathogenesis has not been established. Six such patterns of malformation are sirenomelia, VACTERL association, OEIS complex, limb-body wall defect (LBWD), urorectal septum malformation (URSM) sequence, and MURCS association, all of which predominantly affect caudal structures. On the basis of the overlap of the component malformations, the co-occurrence in individual fetuses, and the findings on fetal examination, a common pathogenesis is proposed for these patterns of malformation. The presence of a single artery in the umbilical cord provides a visible clue to the pathogenesis of all cases of sirenomelia and 30%-50% of cases of VACTERL association, OEIS complex, URSM sequence, and LBWD. The single artery is formed by a coalescence of arteries that supply the yolk sac, arises from the descending aorta high in the abdominal cavity, and redirects blood flow from the developing caudal structures of the embryo to the placenta. This phenomenon during embryogenesis is termed vitelline vascular steal.

Hedgehog signaling regulates regenerative patterning and growth in Harmonia axyridis leg.

Appendage regeneration has been widely studied in many species. Compared to other animal models, Harmonia axyridis has the advantage of a short life cycle, is easily reared, has strong regeneration capacity and contains systemic RNAi, making it a model organism for research on appendage regeneration. Here, we performed transcriptome analysis, followed by gene functional assays to reveal the molecular mechanism of H. axyridis leg regenerative growth process. Signaling pathways including Decapentaplegic (Dpp), Wingless (Wg), Ds/Ft/Hippo, Notch, Egfr, and Hedgehog (Hh) were all upregulated during the leg regenerative patterning and growth. Among these, Hh and its auxiliary receptor Lrp2 were required for the proper patterning and growth of the regenerative leg. The targets of canonical Hh signaling were required for the regenerative growth which contributes to the leg length, but were not essential for the pattern formation of the regenerative leg. dpp, wg and leg developmental-related genes including rn, dac and Dll were all regulated by hh and lrp2 and may play an essential role in the regenerative patterning of the leg.

Limb regeneration in salamanders: the plethodontid tale.

Salamanders are the only vertebrates that can regenerate limbs as adults. This makes them ideal models to investigate the cellular and molecular mechanisms of tissue regeneration. Ambystoma mexicanum and Nothopthalmus viridescens have long served as primary salamander models of limb regeneration, and the recent sequencing of the axolotl genome now provides a blueprint to mine regeneration insights from other salamander species. In particular, there is a need to study South American plethodontid salamanders that present different patterns of limb development and regeneration. A broader sampling of species using next-generation sequencing approaches is needed to reveal shared and unique mechanisms of regeneration, and more generally, the evolutionary history of salamander limb regeneration.

Generation of Acute Hind Limb Ischemia in NOD/SCID Mice and Treatment with Nanofiber-Expanded CD34+ Hematopoietic Stem Cells.

Critical limb ischemia (CLI) is primarily associated with a high risk of major amputation, cardiovascular events, and death. The current therapy involves direct endovascular intervention and is associated with long-term recurrence. However, patients with significant comorbidities are not eligible for this therapy. Hind limb ischemia model via femoral artery excision has commonly been used to determine therapeutic potential and for investigating cellular and molecular mechanisms. This protocol describes the ischemic model development in NOD/SCID mice and the use of human umbilical cord blood-derived and nanofiber scaffold-expanded CD34+ stem cells to investigate the efficacy of regenerative therapy.

Regeneration and development. An amphibian call to arms.

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.

Murine Limb Explant Cultures to Assess Cartilage Development.

To investigate chondrocyte biology in an organized structure, limb explant cultures have been established that allow for the cultivation of the entire cartilaginous skeletal elements. In these organ cultures, the arrangement of chondrocytes in the cartilage elements and their interaction with the surrounding perichondrium and joint tissue are maintained. Chondrocyte proliferation and differentiation can thus be studied under nearly in vivo conditions. Growth factors and other soluble agents can be administered to the explants and their effect on limb morphogenesis, gene expression and cell-matrix interactions can be studied. Cotreatment with distinct growth factors and their inhibitors as well as the use of transgenic mice will allow one to decipher the epistatic relationship between different signaling systems and other regulators of chondrocyte differentiation. Here we describe the protocol to culture cartilage explants ex vivo and discuss the advantages and disadvantages of the culture system.

Chromatin topology and the timing of enhancer function at the HoxD locus.

The HoxD gene cluster is critical for proper limb formation in tetrapods. In the emerging limb buds, different subgroups of Hoxd genes respond first to a proximal regulatory signal, then to a distal signal that organizes digits. These two regulations are exclusive from one another and emanate from two distinct topologically associating domains (TADs) flanking HoxD, both containing a range of appropriate enhancer sequences. The telomeric TAD (T-DOM) contains several enhancers active in presumptive forearm cells and is divided into two sub-TADs separated by a CTCF-rich boundary, which defines two regulatory submodules. To understand the importance of this particular regulatory topology to control Hoxd gene transcription in time and space, we either deleted or inverted this sub-TAD boundary, eliminated the CTCF binding sites, or inverted the entire T-DOM to exchange the respective positions of the two sub-TADs. The effects of such perturbations on the transcriptional regulation of Hoxd genes illustrate the requirement of this regulatory topology for the precise timing of gene activation. However, the spatial distribution of transcripts was eventually resumed, showing that the presence of enhancer sequences, rather than either their exact topology or a particular chromatin architecture, is the key factor. We also show that the affinity of enhancers to find their natural target genes can overcome the presence of both a strong TAD border and an unfavorable orientation of CTCF sites.

Conservation analysis of core cell cycle regulators and their transcriptional behavior during limb regeneration in Ambystoma mexicanum.

Ambystoma mexicanum (axolotl) has been one of the major experimental models for the study of regeneration during the past 100 years. Axolotl limb regeneration takes place through a multi-stage and complex developmental process called epimorphosis that involves diverse events of cell reprogramming. Such events start with dedifferentiation of somatic cells and the proliferation of quiescent stem cells to generate a population of proliferative cells called blastema. Once the blastema reaches a mature stage, cells undergo progressive differentiation into the diverse cell lineages that will form the new limb. Such pivotal cell reprogramming phenomena depend on the fine-tuned regulation of the cell cycle in each regeneration stage, where cell populations display specific proliferative capacities and differentiation status. The axolotl genome has been fully sequenced and released recently, and diverse RNA-seq approaches have also been generated, enabling the identification and conservatory analysis of core cell cycle regulators in this species. We report here our results from such analyses and present the transcriptional behavior of key regulatory factors during axolotl limb regeneration. We also found conserved protein interactions between axolotl Cyclin Dependent Kinases 2, 4 and 6 and Cyclins type D and E. Canonical CYC-CDK interactions that play major roles in modulating cell cycle progression in eukaryotes.

Transcriptome and epigenome diversity and plasticity of muscle stem cells following transplantation.

Adult skeletal muscles are maintained during homeostasis and regenerated upon injury by muscle stem cells (MuSCs). A heterogeneity in self-renewal, differentiation and regeneration properties has been reported for MuSCs based on their anatomical location. Although MuSCs derived from extraocular muscles (EOM) have a higher regenerative capacity than those derived from limb muscles, the molecular determinants that govern these differences remain undefined. Here we show that EOM and limb MuSCs have distinct DNA methylation signatures associated with enhancers of location-specific genes, and that the EOM transcriptome is reprogrammed following transplantation into a limb muscle environment. Notably, EOM MuSCs expressed host-site specific positional Hox codes after engraftment and self-renewal within the host muscle. However, about 10% of EOM-specific genes showed engraftment-resistant expression, pointing to cell-intrinsic molecular determinants of the higher engraftment potential of EOM MuSCs. Our results underscore the molecular diversity of distinct MuSC populations and molecularly define their plasticity in response to microenvironmental cues. These findings provide insights into strategies designed to improve the functional capacity of MuSCs in the context of regenerative medicine.

Integrated Transcriptome and Proteome Analyses Reveal the Regulatory Role of miR-146a in Human Limbal Epithelium via Notch Signaling.

MiR-146a is upregulated in the stem cell-enriched limbal region vs. central human cornea and can mediate corneal epithelial wound healing. The aim of this study was to identify miR-146a targets in human primary limbal epithelial cells (LECs) using genomic and proteomic analyses. RNA-seq combined with quantitative proteomics based on multiplexed isobaric tandem mass tag labeling was performed in LECs transfected with miR-146a mimic vs. mimic control. Western blot and immunostaining were used to confirm the expression of some targeted genes/proteins. A total of 251 differentially expressed mRNAs and 163 proteins were identified. We found that miR-146a regulates the expression of multiple genes in different pathways, such as the Notch system. In LECs and organ-cultured corneas, miR-146a increased Notch-1 expression possibly by downregulating its inhibitor Numb, but decreased Notch-2. Integrated transcriptome and proteome analyses revealed the regulatory role of miR-146a in several other processes, including anchoring junctions, TNF-α, Hedgehog signaling, adherens junctions, TGF-ß, mTORC2, and epidermal growth factor receptor (EGFR) signaling, which mediate wound healing, inflammation, and stem cell maintenance and differentiation. Our results provide insights into the regulatory network of miR-146a and its role in fine-tuning of Notch-1 and Notch-2 expressions in limbal epithelium, which could be a balancing factor in stem cell maintenance and differentiation.