Embryonic Limb Research Articles

A cost-effective set-up to demonstrate embryonic limb development in Aseel (Gallus gallus domesticus)

Limb development during embryogenesis is a classical model used to study pattern formation. Experiments with biological model systems currently re-quire expensive equipment to maintain optimal conditions, an impractical option for low-scale studies. Currently popular ex-vivo methods lead to stunted embryonic limb development, a high infection and fatality rate. As an alternative, the presented paper uses a novel, cost-effective set up to study the developing chick embryo as a biological model in order to visualize Chondrogenesis alongwith formation of ossification centers and apoptotic events occurring in the limb bud of the developing embryo.This cup-in-cup model constructed using polystyrene cups is instrumental in observing the developing embryos and correlating them to Hamilton-Hamburger (HH) de-velopmental stages without exposing them to the outside environment and approaching a near perfect embryo survival rate. Anatomical events during skeleton development such as chondrogenesis, osteogenesis, and apoptosis are studied using Alcian Blue, Alizarin Red and Nile Blue Sulphate staining protocols revealing successful formation and progression of ossification cen-ters and apoptotic regions in the limb bud. The chick embryo system is an excellent model that aids in understanding osteogenesis at both basic and clinical science level and enhance our knowledge about embryological devel-opment. The cup-in-cup system presented in this study proves to be a realis-tic addition to the subject of embryology and an ideal, sustainable experi-mental medium for low-scale research studies.

Extracellular matrix gene expression signatures as cell type and cell state identifiers

Transcriptomic signatures based on cellular mRNA expression profiles can be used to categorize cell types and states. Yet whether different functional groups of genes perform better or worse in this process remains largely unexplored. Here we test the core matrisome – that is, all genes coding for structural proteins of the extracellular matrix – for its ability to delineate distinct cell types in embryonic single-cell RNA-sequencing (scRNA-seq) data. We show that even though expressed core matrisome genes correspond to less than 2% of an entire cellular transcriptome, their RNA expression levels suffice to recapitulate essential aspects of cell type-specific clustering. Notably, using scRNA-seq data from the embryonic limb, we demonstrate that core matrisome gene expression outperforms random gene subsets of similar sizes and can match and exceed the predictive power of transcription factors. While transcription factor signatures generally perform better in predicting cell types at early stages of chicken and mouse limb development, i.e., when cells are less differentiated, the information content of the core matrisome signature increases in more differentiated cells. Moreover, using cross-species analyses, we show that these cell type-specific signatures are evolutionarily conserved. Our findings suggest that each cell type produces its own unique extracellular matrix, or matreotype, which becomes progressively more refined and cell type-specific as embryonic tissues mature.

Activation of the Melanocortin-4 receptor signaling by \u03b1-MSH stimulates nerve-dependent mouse digit regeneration

BackgroundExpression of Mc4r in peripheral organs indicates it has broader roles in organ homeostasis and regeneration. However, the expression and function of Mc4r in the mouse limb and digit has not been fully investigated. Our previous work showed that Mc4r−/− mice fail to regenerate the digit, but whether activation of MC4R signaling could rescue digit regeneration, or stimulate proximal digit regeneration is not clear.ResultsWe analyzed the expression dynamics of Mc4r in the embryonic and postnatal mouse limb and digit using the Mc4r-gfp mice. We found that Mc4r-GFP is mainly expressed in the limb nerves, and in the limb muscles that are undergoing secondary myogenesis. Expression of Mc4r-GFP in the adult mouse digit is restricted to the nail matrix. We also examined the effect of α-MSH on mouse digit regeneration. We found that administration of α-MSH in the Mc4r+/− mice rescue the delayed regeneration of distal digit tip. α-MSH could rescue distal digit regeneration in denervated hindlimbs. In addition, α-MSH could stimulate regeneration of the proximally amputated digit, which is non-regenerative.ConclusionsMc4r expression in the mouse limb and digit is closely related to nerve tissues, and α-MSH/MC4R signaling has a neurotrophic role in mouse digit tip regeneration.

In ovo electroporation of chicken limb bud ectoderm: Electroporation to chick limb ectoderm: Electroporation to chick limb ectoderm.

Deciphering how ectodermal tissues form, and how they maintain their integrity, is crucial for understanding epidermal development and pathogenesis. However, lack of simple and rapid gene manipulation techniques limits genetic studies to elucidate mechanisms underlying these events. Here we describe an easy method for electroporation of chick limb bud ectoderm enabling gene manipulation during ectoderm development and wound healing. Taking advantage of a small parafilm well that constrains DNA plasmids locally and the fact that the limb ectoderm arises from a defined site, we target the limb ectoderm forming region by in ovo electroporation. This approach results in focal and efficient transgenesis of the limb ectodermal cells. Further, using a previously described Msx2 promoter, gene manipulation can be specifically targeted to the apical ectodermal ridge (AER), a signaling center regulating limb development. Using the electroporation technique to deliver a fluorescent marker into the embryonic limb ectoderm, we show its utility in performing time-lapse imaging during wound healing. This analysis revealed previously unrecognized dynamic remodeling of the actin cytoskeleton and lamellipodia formation at the edges of the wound. We find that the lamellipodia formation requires activity of Rac1 GTPase, suggesting its necessity for wound closure. Our method is simple and easy. Thus, it would permit high throughput tests for gene function during limb ectodermal development and wound healing.

Hidden limbs in the "limbless skink" Brachymeles lukbani: Developmental observations.

Reduced limbs and limblessness have evolved independently in many lizard clades. Scincidae exhibit a wide range of limb-reduced morphologies, but only some species have been used to study the embryology of limb reduction (e.g., digit reduction in Chalcides and limb reduction in Scelotes). The genus Brachymeles, a Southeast Asian clade of skinks, includes species with a range of limb morphologies, from pentadactyl to functionally and structurally limbless species. Adults of the small, snake-like species Brachymeles lukbani show no sign of external limbs in the adult except for small depressions where they might be expected to occur. Here, we show that embryos of B.lukbani in early stages of development, on the other hand, show a truncated but well-developed limb with a stylopod and a zeugopod, but no signs of an autopod. As development proceeds, the limb's small size persists even while the embryo elongates. These observations are made based on external morphology. We used florescent whole-mount immunofluorescence to visualize the morphology of skeletal elements and muscles within the embryonic limb of B.lukabni. Early stages have a humerus and separated ulna and radius cartilages; associated with these structures are dorsal and ventral muscle masses as those found in the embryos of other limbed species. While the limb remains small, the pectoral girdle grows in proportion to the rest of the body, with well-developed skeletal elements and their associated muscles. In later stages of development, we find the small limb is still present under the skin, but there are few indications of its presence, save for the morphology of the scale covering it. By use of CT scanning, we find that the adult morphology consists of a well-developed pectoral girdle, small humerus, extremely reduced ulna and radius, and well-developed limb musculature connected to the pectoral girdle. These muscles form in association with a developing limb during embryonic stages, a hint that "limbless" lizards that possess these muscles may have or have had at least transient developing limbs, as we find in B.lukbani. Overall, this newly observed pattern of ontogenetic reduction leads to an externally limbless adult in which a limb rudiment is hidden and covered under the trunk skin, a situation called cryptomelia. The results of this work add to our growing understanding of clade-specific patterns of limb reduction and the convergent evolution of limbless phenotypes through different developmental processes.

Histone Epigenetic Signatures in Embryonic Limb Interdigital Cells Fated to Die.

During limb formation in vertebrates with free digits, the interdigital mesoderm is eliminated by a massive degeneration process that involves apoptosis and cell senescence. The degradation process is preceded by intense DNA damage in zones located close to methylated DNA, accompanied by the activation of the DNA repair response. In this study, we show that trimethylated histone 3 (H3K4me3, H3K9me3, and H3K27me3) overlaps with zones positive for 5mC in the nuclei of interdigital cells. This pattern contrasts with the widespread distribution of acetylated histones (H3K9ac and H4ac) and the histone variant H3.3 throughout the nucleoplasm. Consistent with the intense labeling of acetylated histones, the histone deacetylase genes Hdac1, Hdac2, Hdac3, and Hdac8, and at a more reduced level, Hdac10, are expressed in the interdigits. Furthermore, local treatments with the histone deacetylase inhibitor trichostatin A, which promotes an open chromatin state, induces massive cell death and transcriptional changes reminiscent of, but preceding, the physiological process of interdigit remodeling. Together, these findings suggest that the epigenetic profile of the interdigital mesoderm contributes to the sensitivity to DNA damage that precedes apoptosis during tissue regression.

RhoA/Rock activation represents a new mechanism for inactivating Wnt/β-catenin signaling in the aging-associated bone loss

The Wnt/β-catenin signaling pathway appears to be particularly important for bone homeostasis, whereas nuclear accumulation of β-catenin requires the activation of Rac1, a member of the Rho small GTPase family. The aim of the present study was to investigate the role of RhoA/Rho kinase (Rock)-mediated Wnt/β-catenin signaling in the regulation of aging-associated bone loss. We find that Lrp5/6-dependent and Lrp5/6-independent RhoA/Rock activation by Wnt3a activates Jak1/2 to directly phosphorylate Gsk3β at Tyr216, resulting in Gsk3β activation and subsequent β-catenin destabilization. In line with these molecular events, RhoA loss- or gain-of-function in mouse embryonic limb bud ectoderms interacts genetically with Dkk1 gain-of-function to rescue the severe limb truncation phenotypes or to phenocopy the deletion of β-catenin, respectively. Likewise, RhoA loss-of-function in pre-osteoblasts robustly increases bone formation while gain-of-function decreases it. Importantly, high RhoA/Rock activity closely correlates with Jak and Gsk3β activities but inversely correlates with β-catenin signaling activity in bone marrow mesenchymal stromal cells from elderly male humans and mice, whereas systemic inhibition of Rock therefore activates the β-catenin signaling to antagonize aging-associated bone loss. Taken together, these results identify RhoA/Rock-dependent Gsk3β activation and subsequent β-catenin destabilization as a hitherto uncharacterized mechanism controlling limb outgrowth and bone homeostasis.

Analysis of copy number variation in dogs implicates genomic structural variation in the development of anterior cruciate ligament rupture.

Anterior cruciate ligament (ACL) rupture is an important condition of the human knee. Second ruptures are common and societal costs are substantial. Canine cranial cruciate ligament (CCL) rupture closely models the human disease. CCL rupture is common in the Labrador Retriever (5.79% prevalence), ~100-fold more prevalent than in humans. Labrador Retriever CCL rupture is a polygenic complex disease, based on genome-wide association study (GWAS) of single nucleotide polymorphism (SNP) markers. Dissection of genetic variation in complex traits can be enhanced by studying structural variation, including copy number variants (CNVs). Dogs are an ideal model for CNV research because of reduced genetic variability within breeds and extensive phenotypic diversity across breeds. We studied the genetic etiology of CCL rupture by association analysis of CNV regions (CNVRs) using 110 case and 164 control Labrador Retrievers. CNVs were called from SNPs using three different programs (PennCNV, CNVPartition, and QuantiSNP). After quality control, CNV calls were combined to create CNVRs using ParseCNV and an association analysis was performed. We found no strong effect CNVRs but found 46 small effect (max(T) permutation P<0.05) CCL rupture associated CNVRs in 22 autosomes; 25 were deletions and 21 were duplications. Of the 46 CCL rupture associated CNVRs, we identified 39 unique regions. Thirty four were identified by a single calling algorithm, 3 were identified by two calling algorithms, and 2 were identified by all three algorithms. For 42 of the associated CNVRs, frequency in the population was <10% while 4 occurred at a frequency in the population ranging from 10–25%. Average CNVR length was 198,872bp and CNVRs covered 0.11 to 0.15% of the genome. All CNVRs were associated with case status. CNVRs did not overlap previous canine CCL rupture risk loci identified by GWAS. Associated CNVRs contained 152 annotated genes; 12 CNVRs did not have genes mapped to CanFam3.1. Using pathway analysis, a cluster of 19 homeobox domain transcript regulator genes was associated with CCL rupture (P = 6.6E-13). This gene cluster influences cranial-caudal body pattern formation during embryonic limb development. Clustered genes were found in 3 CNVRs on chromosome 14 (HoxA), 28 (NKX6-2), and 36 (HoxD). When analysis was limited to deletion CNVRs, the association was strengthened (P = 8.7E-16). This study suggests a component of the polygenic risk of CCL rupture in Labrador Retrievers is associated with small effect CNVs and may include aspects of stifle morphology regulated by homeobox domain transcript regulator genes.

The vascular origins of antero-medial tibial bowing in congenital fibular deficiency.

Anteromedial bowing and shortening of the tibia are intrinsic features of limbs with congenital fibular deficiency (CFD). Tibial bowing occurs more frequently when the fibula is radiographically absent rather than deficient. The bowing has been attributed to rapid longitudinal growth of the tibial anlage coupled with anteromedial tibial bending moments of the posterior crural and lateral peroneal musculature unopposed in the absence of a fibular strut. Eccentric mechanical loading results in asymmetric mineral deposition and thickening of the diaphyseal cortex. Skeletogenesis depends upon an intimate interplay between the normally prefigured tibial cartilage anlage and beginning muscular contractile actions during initial vascularization of the anlage, while the embryonic limb vasculature is undergoing a series of transitions. A diaphyseal periosteal collar normally forms at the site of nutrient artery invasion and stabilizes the growing anlage. In CFD however, arteriography consistently reveals anomalous tibial nutrient arterial branches, which originate from a primitive axial artery rather than from the usual posterior tibial artery. These anomalous nutrient arteries enter the tibial shaft at the posterior aspect of the proximal metaphysis, establishing an eccentric bone collar. The developing vasculature of the embryonic limb is responsive to the then most metabolically active tissues. Disruption of the reciprocal relationship between the transitioning vasculature and the developing long bones is pivotal in producing the diverse skeletal malformations of the congenital short limb (CSL). Embryonic vascular dysgenesis contributes not only to the well-recognized congenital tibial and fibular shortenings but also predisposes to congenital anteromedial bowing of the tibia.

Phylogenetic patterns and ontogenetic origins of limb length variation in ecologically diverse lacertine lizards

AbstractLimb length is intrinsically linked to function and, ultimately, fitness. Thus, it can co-evolve with habitat structure, as exemplified by tropical lizards in highly heterogeneous environments. But does lizard limb length respond in a similar manner during adaptive diversification in temperate zones? Here, we examine variation in habitat preference and limb length in lacertine lizards from the Palaearctic. We tested the following three hypotheses: (1) species of the Lacertini tribe descended from a generalist ancestor and subsequently underwent habitat specialization; (2) specialized ecological roles are associated with relative limb length in extant species; and (3) interspecific differences in limb length emerge in embryonic development. Our comparisons supported an ancestral ‘rocky’ or ‘generalist’ habitat preference, and phenotype–habitat associations were particularly supported when examining size-adjusted forelimb length in 69 species that represented all known Lacertini genera. Moreover, we revealed an elevated interlimb ratio in high-vegetation species, which might be linked to climbing performance in species with relatively longer forelimbs. Furthermore, embryonic limb variation was detected solely against an Eremiadini outgroup species. Instead, hind limb length differences within Lacertini originated in post-hatching ontogeny. The mechanisms that modulate limb growth are likely to be limited in Lacertini, because adaptive morphological change might mirror historical contingency and the ecological context wherein this clade diversified.

Regeneration and development. An amphibian call to arms.

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. 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. 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.

A histone H3.3K36M mutation in mice causes an imbalance of histone modifications and defects in chondrocyte differentiation

ABSTRACT Histone lysine-to-methionine (K-to-M) mutations have been identified as driver mutations in human cancers. Interestingly, these ‘oncohistone’ mutations inhibit the activity of histone methyltransferases. Therefore, they can potentially be used as versatile tools to investigate the roles of histone modifications. In this study, we generated a genetically engineered mouse line in which an H3.3K36M mutation could be induced in the endogenous H3f3b gene. Since H3.3K36M has been identified as a causative mutation of human chondroblastoma, we induced this mutation in the chondrocyte lineage in mouse embryonic limbs. We found that H3.3K36M causes a global reduction in H3K36me2 and defects in chondrocyte differentiation. Importantly, the reduction of H3K36me2 was accompanied by a collapse of normal H3K27me3 distribution. Furthermore, the changes in H3K27me3, especially the loss of H3K27me3 at gene regulatory elements, were associated with the mis-regulated expression of a set of genes important for limb development, including HoxA cluster genes. Thus, through the in vivo induction of the H3.3K36M mutation, we reveal the importance of maintaining the balance between H3K36me2 and H3K27me3 during chondrocyte differentiation and limb development.

Confluence of Cellular Degradation Pathways During Interdigital Tissue Remodeling in Embryonic Tetrapods.

Digits develop in the distal part of the embryonic limb primordium as radial prechondrogenic condensations separated by undifferentiated mesoderm. In a short time interval the interdigital mesoderm undergoes massive degeneration to determine the formation of free digits. This fascinating process has often been considered as an altruistic cell suicide that is evolutionarily-regulated in species with different degrees of digit webbing. Initial descriptions of interdigit remodeling considered lysosomes as the primary cause of the degenerative process. However, the functional significance of lysosomes lost interest among researcher and was displaced to a secondary role because the introduction of the term apoptosis. Accumulating evidence in recent decades has revealed that, far from being a unique method of embryonic cell death, apoptosis is only one among several redundant dying mechanisms accounting for the elimination of tissues during embryonic development. Developmental cell senescence has emerged in the last decade as a primary factor implicated in interdigit remodeling. Our review proposes that cell senescence is the biological process identified by vital staining in embryonic models and implicates lysosomes in programmed cell death. We review major structural changes associated with interdigit remodeling that may be driven by cell senescence. Furthermore, the identification of cell senescence lacking tissue degeneration, associated with the maturation of the digit tendons at the same stages of interdigital remodeling, allowed us to distinguish between two functionally distinct types of embryonic cell senescence, “constructive” and “destructive.”

Phosphorylation of Msx1 promotes cell proliferation through the Fgf9/18-MAPK signaling pathway during embryonic limb development.

Msh homeobox (Msx) is a subclass of homeobox transcriptional regulators that control cell lineage development, including the early stage of vertebrate limb development, although the underlying mechanisms are not clear. Here, we demonstrate that Msx1 promotes the proliferation of myoblasts and mesenchymal stem cells (MSCs) by enhancing mitogen-activated protein kinase (MAPK) signaling. Msx1 directly binds to and upregulates the expression of fibroblast growth factor 9 (Fgf9) and Fgf18. Accordingly, knockdown or antibody neutralization of Fgf9/18 inhibits Msx1-activated extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. Mechanistically, we determined that the phosphorylation of Msx1 at Ser136 is critical for enhancing Fgf9 and Fgf18 expression and cell proliferation, and cyclin-dependent kinase 1 (CDK1) is apparently responsible for Ser136 phosphorylation. Furthermore, mesenchymal deletion of Msx1/2 results in decreased Fgf9 and Fgf18 expression and Erk1/2 phosphorylation, which leads to serious defects in limb development in mice. Collectively, our findings established an important function of the Msx1-Fgf-MAPK signaling axis in promoting cell proliferation, thus providing a new mechanistic insight into limb development.

Inhibition of eNOS by L-NAME resulting in rat hind limb developmental defects through PFKFB3 mediated angiogenetic pathway

l-arginine/NOS/NO signaling pathway plays a critical role in controlling variety of vascular diseases. However, whether NOS inhibition by L-NAME suppresses late embryonic development is undefined. The aim of this study is to determine whether NOS inhibition by L-NAME is critical for late embryonic rat hind limb development. The pregnant rat at E13.5 administrated L-NAME by consecutive intraperitoneal injection. The embryos been harvested from E16.5 to E 20.5. Hematoxylin and Eosin Staining, Immunofluorescence and Immunohistochemistry performed to determine hind limb Vasculogenesis, HUVEC culture, Adenoviral PFKFB3 infection, Real time PCR and western blot were performed to determine whether l-arginine/NOS/NO pathway controlling late embryonic hind limb development through PFKFB3 mediated angiogenetic pathway. NOS inhibition by L-NAME resulting in late embryonic hind limb developmental defects characterized by severe hemorrhage. The in vivo studies showed that NOS inhibition strongly suppressed hind limb angiogenetic remodeling by impairing differentiation of endothelial cells and smooth muscle cells, and extracellular matrix synthesis. For underlie mechanism, our studies indicated that L-NAME treatment dramatically suppresses PFKFB3 expression in hematopoietic progenitor cells, tubulogenetic endothelial cells and smooth muscle cells. Knockdown of PFKFB3 dramatically inhibits the expression of angiogenetic genes, as well as tubulogenesis and extracellular matrix related genes. Taken together, our data in this study demonstrated that l-arginine-eNOS-NO pathway is important for rat hind limb development during late embryonic stage. This could be both a useful animal model and a promising therapeutic treatment for defects of late embryonic developmental hind limbs.

Molecular cloning, characterization and expression analysis of the ecdysone receptor from the spiny lobster Panulirus homarus (Linnaeus, 1758) (Decapoda, Palinuridae)

Abstract The ecdysone receptor (EcR) is a crucial nuclear transcription factor that participates in ecdysteroid signalling. In this study, cDNA of EcR in Panulirus homarus (Linnaeus, 1758) (PhEcR) was cloned and characterized. PhEcR cDNA was 3141 bp, in which a 12 bp insert/deletion splice variant region in the hinge domain was identified. PhEcR mRNA presented the most abundant expression in the ovary. PhEcR mRNA expression was rapidly upregulated in the premoult stage and downregulated in the ecdysis stage. PhEcR mRNA maintained a relatively high expression level from egg-nauplius stage to embryo stage with compound eye pigments formed. The transcript levels of PhEcR in muscle were up-regulated on the 5th and 10th day after antenna ablation. These data suggest that PhEcR may not only play a vital role in the ecdysteroid signalling, but also have relation to embryonic development and limb regeneration.

Expression of Fibroblast Growth Factor 4 in a Rat Model of Polydactyly of the Thumb Induced by Cytarabine.

BackgroundThe aim of this study was to assess the expression and mechanisms of fibroblast growth factor 4 in polydactyly of the thumb induced by cytarabine.Material/MethodsRats were intraperitoneally injected with cytarabine at different gestation periods (12.5 days, 13.5 days, and 14.5 days) to establish a polydactyly of the thumb model. Then, the expression of FGF4 in polydactyly was studied by whole-mount in situ hybridization. We used hematoxylin & eosin stain and cartilage stain to investigate the development of the skeleton and tissues in the embryo. Pictures were taken to determine the general shape of the deformity, then X-rays were taken to detect bone distortion of the rats born with a congenital malformation.ResultsIn the experimental group (11.5 days, 12.5 days, 13.5 days, and 14.5 days), whole-mount in situ hybridization showed that the FGF4 expression at the tip of the embryonic limb bud was significantly increased compared with the control group and FGF4 was distributed in a wider range and lasted longer than in the control group (P<0.01). HE staining and cartilage staining showed that there was an extra metacarpal bone and a phalanx in the rats with polydactyly of the thumb (P<0.01). Images of the deformed limbs showed polydactyly and syndactyly of the thumb in the rats. Further X-ray examination revealed 1 extra metacarpal bone and 1 extra phalanx.ConclusionsCytarabine can induce polydactyly and syndactyly of the thumb in rats. In this process, cytarabine can induce the expression of FGF4 on the tip of the embryonic limb bud, which further leads to abnormal development of the embryonic limb bud and eventually causes a congenital deformity.

Numerical Approach to a Nonlocal Advection-Reaction-Diffusion Model of Cartilage Pattern Formation

We propose a numerical approach that combines a radial basis function (RBF) meshless approximation with a finite difference discretization to solve a nonlinear system of integro-differential equations. The equations are of advection-reaction-diffusion type modeling the formation of pre-cartilage condensations in embryonic chicken limbs. The computational domain is four dimensional in the sense that the cell density depends continuously on two spatial variables as well as two structure variables, namely membrane-bound counterreceptor densities. The biologically proper Dirichlet boundary conditions imposed in the semi-infinite structure variable region is in favor of a meshless method with Gaussian basis functions. Coupled with WENO5 finite difference spatial discretization and the method of integrating factors, the time integration via method of lines achieves optimal complexity. In addition, the proposed scheme can be extended to similar models with more general boundary conditions. Numerical results are provided to showcase the validity of the scheme.

The methylation status of the embryonic limb skeletal progenitors determines their cell fate in chicken

Digits shape is sculpted by interdigital programmed cell death during limb development. Here, we show that DNA breakage in the periphery of 5-methylcytosine nuclei foci of interdigital precursors precedes cell death. These cells showed higher genome instability than the digit-forming precursors when exposed to X-ray irradiation or local bone morphogenetic protein (BMP) treatments. Regional but not global DNA methylation differences were found between both progenitors. DNA-Methyl-Transferases (DNMTs) including DNMT1, DNMT3B and, to a lesser extent, DNMT3A, exhibited well-defined expression patterns in regions destined to degenerate, as the interdigital tissue and the prospective joint regions. Dnmt3b functional experiments revealed an inverse regulation of cell death and cartilage differentiation, by transcriptional regulation of key genes including Sox9, Scleraxis, p21 and Bak1, via differential methylation of CpG islands across their promoters. Our findings point to a regulation of cell death versus chondrogenesis of limb skeletal precursors based on epigenetic mechanisms.

Missing Midline Metatarsals Conform to Plantar Arterial Arch Dysgenesis.

Midline metatarsal ray deficiencies, which occur in approximately half of congenital short limbs with fibular deficiency, provide the most distal and compelling manifestation of a fluid spectrum of human lower-extremity congenital long bone reductions; this spectrum syndromically affects the long bone triad of the proximal femur, fibula, and midline metatarsals. The bony deficiencies correspond to sites of rapid embryonic arterial transitioning. Long bones first begin to ossify because of vascular invasions of their respective mesenchymal/cartilage anlagen, proceeding in a proximal-to-distal sequence along the forming embryonic limb. A single-axis artery forms initially in the embryonic lower limb by means of vasculogenesis. Additional arteries evolve in overlapping transitional waves, in proximity to the various anlagen, during the sixth and seventh weeks after fertilization. An adult pattern of vessels presents by the eighth week. Arterial alterations, in the form of retained primitive embryonic vessels and/or reduced absent adult vessels, have been observed clinically at the aforementioned locations where skeletal reductions occur. Persistence of primitive vessels in association with the triad of long bone reductions allows a heuristic estimation of the time, place, and nature of such coupled vascular and bony dysgeneses. Arterial dysgenesis is postulated to have occurred when the developing arterial and skeletal structures were concurrently vulnerable to teratogenic insults because of embryonic arterial instability, a risk factor during arterial transition. It is herein hypothesized that flawed arterial transitions subject the prefigured long bone cartilage models of the rapidly growing limb to the risk of teratogenesis at one or more of the then most rapidly growing sites. Midline metatarsal deficiency forms the keystone of this developmental concept of an error of limb development, which occurs as a consequence of failed completion of the medial portion of the plantar arch. Therefore, the historical nomenclature of congenital long bone deficiencies will benefit from modification from a current reliance on empirical physical taxonomies to a developmental foundation.