Forelimb Development Research Articles

Extracellular matrix protein composition dynamically changes during murine forelimb development

The extracellular matrix (ECM) is an integral part of multicellular organisms, connecting different cell layers and tissue types. During morphogenesis and growth, tissues undergo substantial reorganization. While it is intuitive that the ECM remodels in concert, little is known regarding how matrix composition and organization change during development. Here, we quantified ECM protein dynamics in the murine forelimb during appendicular musculoskeletal morphogenesis (embryonic days 11.5-14.5) using tissue fractionation, bioorthogonal non-canonical amino acid tagging, and mass spectrometry. Our analyses indicated that ECM protein (matrisome) composition in the embryonic forelimb changed as a function of development and growth, was distinct from other developing organs (brain), and was altered in a model of disease (osteogenesis imperfecta murine). Additionally, the tissue distribution for select matrisome was assessed via immunohistochemistry in the wild-type embryonic and postnatal musculoskeletal system. This resource will guide future research investigating the role of the matrisome during complex tissue development.

Ecomorphology and sensory biology of bats.

This special issue of The Anatomical Record is inspired by and dedicated to Professor Kunwar P. Bhatnagar, whose lifelong interests in biology, and long career studying bats, inspired many and advanced our knowledge of the world's only flying mammals. The 15 articles included here represent a broad range of investigators, treading topics familiar to Prof. Bhatnagar, who was interested in seemingly every aspect of bat biology. Key topics include broad themes of bat development, sensory systems, and specializations related to flight and diet. These articles paint a complex picture of the fascinating adaptations of bats, such as rapid fore limb development, ear morphologies relating to echolocation, and other enhanced senses that allow bats to exploit niches in virtually every part of the world. In this introduction, we integrate and contextualize these articles within the broader story of bat ecomorphology, providing an overview of each of the key themes noted above. This special issue will serve as a springboard for future studies both in bat biology and in the broader world of mammalian comparative anatomy and ecomorphology.

Direct development or endotrophic tadpole? Morphological aspects of the early ontogeny of the plump toad Osornophryne occidentalis (Anura: Bufonidae).

Andean Toads of the genus Osornophryne are suspected to have direct development on the basis of clutch and egg features. In this work, we describe the morphology of Osornophryne occidentalis embryos from a nest collected in the field. Several traits are similar to those reported in brachycephaloid Eleutherodactylus coqui and other direct-developing lineages. These include the pattern of formation of the body wall and the absence of oral and buccopharyngeal structures. Other features indicate the retention of ancestral larval characters. The development of forelimbs occurs in part within the operculum as in species with biphasic ontogeny; this has been reported in other direct-developing species, and is similar to what is described in African viviparous bufonids. This salient feature, along with the long, low-finned tail and the long and folded intestine early differentiated, gives these embryos a rather tadpole-like appearance. Our data confirm that development in O. occidentalis occurs within terrestrial eggs until advanced stages, and this would satisfy current definitions of direct development. At the same time, morphological differences regarding other species with comparable breeding reinforce interpretations about the wide anatomical and ontogenetic variations associated with endotrophic nutrition.

Exceptional features of the embryonic ontogeny of a direct‐developing Robber frog

AbstractIn frogs, the loss of the free‐living tadpole stage implies deep transformations in morphology and development, and the result is a combination of traits exclusive to direct development with the reduction or absence of typically larval features. Two traits stand out in terraranans, a mostly Neotropical clade with ca. 1200 species known or suspected to have direct development: (1) the development of forelimbs, which unlike in tadpoles and in several unrelated direct‐developing lineages, in this clade is described to occur completely exposed during ontogeny, and (2) the reduction of gills and their physiological replacement by highly vascularized tails for gas exchange. In this contribution, we report unusual aspects of the embryonic ontogeny in the Confused Robber frog Oreobates berdemenos, which complicate the interpretation of morphological diversity in Terrarana and in direct‐developing frogs in general. First, forelimbs develop initially entirely concealed by a larval operculum. Second, tail fins develop dorsal and ventral to the tail muscular core but their arrangement changes to lateral during ontogeny. Third, the expansion of the tail fins appears to be a plastic character whose variation likely responds to individual conditions of humidity and/or oxygenation of each egg within the clutch. Finally, we report cells with the morphology of hatching gland cells in the rostral region of embryos at stages about to hatch. If not vestigial, these cells could assist the egg tooth, which is characteristic of terraranans, with chemical softening of the egg capsules during hatching.

Wnt Signaling Coordinates the Expression of Limb Patterning Genes During Axolotl Forelimb Development and Regeneration.

After amputation, axolotl salamanders can regenerate their limbs, but the degree to which limb regeneration recapitulates limb development remains unclear. One limitation in answering this question is our lack of knowledge about salamander limb development. Here, we address this question by studying expression patterns of genes important for limb patterning during axolotl salamander limb development and regeneration. We focus on the Wnt signaling pathway because it regulates multiple functions during tetrapod limb development, including limb bud initiation, outgrowth, patterning, and skeletal differentiation. We use fluorescence in situ hybridization to show the expression of Wnt ligands, Wnt receptors, and limb patterning genes in developing and regenerating limbs. Inhibition of Wnt ligand secretion permanently blocks limb bud outgrowth when treated early in limb development. Inhibiting Wnt signaling during limb outgrowth decreases the expression of critical signaling genes, including Fgf10, Fgf8, and Shh, leading to the reduced outgrowth of the limb. Patterns of gene expression are similar between developing and regenerating limbs. Inhibition of Wnt signaling during regeneration impacted patterning gene expression similarly. Overall, our findings suggest that limb development and regeneration utilize Wnt signaling similarly. It also provides new insights into the interaction of Wnt signaling with other signaling pathways during salamander limb development and regeneration.

The functional diversity of marsupial limbs is influenced by both ecology and developmental constraint

Abstract Extant marsupials are less ecologically diverse than placentals, and this is reflected by placentals exhibiting a greater diversity of locomotor modes, including powered flight and fully aquatic swimming. One proposed explanation for this discrepancy is that the development of more disparate marsupial forelimbs is prevented by the neonate’s crawl to the pouch, which requires precocious forelimb development for climbing adaptations. To test predictions of this Developmental Constraint Hypothesis (DCH), we pursue a comparative morphometric study on osteological traits of mammalian limbs, with an emphasis on functional differentiation of marsupial limbs among locomotor modes. We apply multivariate analyses to a large dataset of limb metrics and a diverse sample of mammals, with the placental sample limited to taxa whose locomotor modes are exhibited in marsupials. Overall, we do not find consistent evidence in support of the DCH. Diprotodontia serves as an exception, with comparisons of their forelimbs to hind limbs supporting the DCH. Our results suggest that developmental constraints on marsupial forelimbs may have limited marsupial diversity to some degree. Despite this, the marsupial locomotor groups show unexpectedly high levels of morphological differentiation relative to placentals of the same locomotor modes, indicating that ecological functions may overcome developmental constraints on a macroevolutionary scale.

Generation of a transducible version of a bioactive recombinant human TBX5 transcription factor from E. Coli

• Protein purification of a recombinant TBX5 fusion protein was performed. • Recombinant TBX5 protein maintains its secondary structure after purification. • Purified TBX5 protein has cell penetration and nuclear translocation potential. • Purified TBX5 protein is bioactive. The protein T-box transcription factor 5 (TBX5) regulates heart and forelimb development in vertebrates, and its deficiency results in Holt-Oram syndrome. Notably, TBX5 functions as a tumor suppressor protein and is a key transcription factor in the reprogramming cocktail used to induce the formation of cardiomyocytes from fibroblasts. Here, we report the expression and purification of a transducible version of recombinant human TBX5 protein from the prokaryotic host Escherichia coli ( E. coli ) under native conditions. First, the protein-coding, codon-optimized TBX5 nucleotide sequence was fused in-frame with three tags (octahistidine, transactivator of transcription, and nuclear localization sequence) at either end of the sequence for affinity purification, cellular uptake, and nuclear delivery. Subsequently, this codon-optimized gene was cloned in a protein expression vector and expressed in E. coli . Various expression parameters were screened to obtain the highest expression of this protein with maximum solubility, to enable purification from the supernatant fraction. Furthermore, the TBX5 fusion protein was purified as a pure (>90%) recombinant protein under native conditions. The purity and identity were confirmed by Coomassie staining and immunoblotting, respectively. Subsequently, the retention of the secondary structure after purification was analyzed and established through far UV circular dichroism spectroscopy. The cellular uptake and nuclear translocation ability of purified TBX5 fusion protein and its biological activity in human cells were demonstrated. The cell-permeant recombinant protein tool established in this study should help elucidate the biological role of human TBX5 in various cellular processes and pave the way for more detailed understanding of cardiac reprogramming and disease-specific studies.

Embryonic Development of the Avian Sternum and Its Morphological Adaptations for Optimizing Locomotion

The sternum is part of the forelimb appendicular skeleton found in most terrestrial vertebrates and has become adapted across tetrapods for distinctive modes of locomotion. We review the regulatory mechanisms underlying sternum and forelimb development and discuss the possible gene expression modulation that could be responsible for the sternal adaptations and associated reduction in the forelimb programme found in flightless birds. In three phylogenetically divergent vertebrate lineages that all undertake powered flight, a ventral extension of the sternum, named the keel, has evolved independently, most strikingly in volant birds. In flightless birds, however, the sternal keel is absent, and the sternum is flattened. We review studies in a variety of species that have analysed adaptations in sterna morphology that are related to the animal’s mode of locomotion on land, in the sky and in water.

Radiographic and Biochemical Evaluations of Postnatal Development of Bones of the Forelimb in Nigerian Indigenous Dogs.

Bone is a hard and resilient tissue structure that provides mechanical support for the body of an organism. This study was aimed to evaluate the radiographical and biochemical changes during postnatal bone development of forelimbs in Nigerian indigenous puppies from 2-week-old to 24-week-old. Six (6) Nigerian indigenous puppies were chemically restrained using chlorpromazine at 2 mg/kg I.M. and positioned using ropes and sandbags for mediolateral and craniocaudal views of bones of the forelimbs. At 2-week-old, radiographic evaluation of bones of the forelimbs were undertaken and continued bi-weekly up to 24-week-old. Serum samples were analysed from 4-week-old to 24-week-old at four weeks’ interval for mineral (electrolytes) using colometric method for calcium and phosphorus and flame photometry method for sodium and potassium. At 2-week-old, there was an ill-defined radiopaque secondary ossification centre at the proximal epiphysis and a small ovoid radiopaque structure at the distal epiphysis of the humerus. There was not any sign of secondary ossification centre at proximal and distal epiphyses of both radius and ulna. There was the presence of a small radiopaque (ill-defined) accessory carpal. 1st to 5th metacarpals were present together with the proximal, middle and distal phalanges of all digits except the 1st digit that has proximal and distal phalanges. From 20-week-old, fully developed carpals and bony structures were observed through 24-week-old where the commencement of fusion of apophyses of olecranon (proximal epiphysis of ulna) at the centre and acrophyses of metacarpals and phalanges. The mean values (mean ± SEM) from serum biochemistry of calcium, phosphate, sodium and potassium in mmol/L from this study were within the normal limits throughout the study. Conclusively, the complementary information from calcium, phosphate, sodium and potassium in this study showed that these elements are within the normal limit that suggests normal bone development during the study period, which stressed the importance of the knowledge on serial changes of bone development which could be useful in clinical practice.

Ontogenetic changes in activity, locomotion and behavioural complexity in tadpoles

Abstract Metamorphosis is a widespread developmental process that involves considerable changes in morphology, habitat use, ecology and behaviour between early developmental (larval) stages and adult forms. Among amphibians, anuran larvae (tadpoles) undergo massive morphological and ecological changes during their development, with early stages characterized by somatic growth, whereas more conspicuous changes (i.e. metamorphosis) occur later during development. In this study, we examined how locomotor and behavioural traits covary with morphology (body size) and metamorphosis (hindlimb and forelimb development) across developmental stages in spined toad (Bufo spinosus) tadpoles. As expected, we found that locomotion and behaviour undergo significant changes during tadpole development. These changes are curvilinear across developmental stages, with a phase of increasing activity and locomotion followed by a phase of stasis and/or reduction in locomotion and behavioural complexity. All the metrics we investigated indicate that the peak of activity and associated behaviour is situated at a pivotal stage when somatic growth decreases and significant morphological changes occur (i.e. hindlimb growth). Future studies that aim to investigate determinants of locomotion should include developmental stages as covariates in order to assess whether the sensitivity of locomotion to environmental variables changes across developmental stages.

Regulation of vertebrate forelimb development and wing reduction in the flightless emu.

The vertebrate limb is a dynamic structure which has evolved into many diverse forms to facilitate complex behavioral adaptations. The principle molecular and cellular processes that underlie development of the vertebrate limb are well characterized. However, how these processes are altered to drive differential limb development between vertebrates is less well understood. Several vertebrate models are being utilized to determine the developmental basis of differential limb morphogenesis, though these typically focus on later patterning of the established limb bud and may not represent the complete developmental trajectory. Particularly, heterochronic limb development can occur prior to limb outgrowth and patterning but receives little attention. This review summarizes the genetic regulation of vertebrate forelimb diversity, with particular focus on wing reduction in the flightless emu as a model for examining limb heterochrony. These studies highlight that wing reduction is complex, with heterochronic cellular and genetic events influencing the major stages of limb development. Together, these studies provide a broader picture of how different limb morphologies may be established during development.

Placental Expression of the Forelimb Patterning Transcription Factor MEIS2 in Trisomy 15

Background Observations of first trimester human trisomy 15 (T15) embryos have identified meromelic changes in the upper limbs. These changes are similar to those observed in animal studies investigating the effects of overexpression of Meis2, a signaling transcription factor expressed during forelimb development. Although it would be exceedingly difficult to assess MEIS2 expression in the human embryonic arm, MEIS2 has been reported as consistently expressed in first trimester placental villus stroma. Methods: This study addresses whether gene dosage effect might underlie meromelia in T15 by comparing MEIS2 expression in placentas from T15 and euploid spontaneous abortions employing manual and automated assessment of MEIS2 immunohistochemical scoring. Results: Average MEIS2 expression is increased in T15 first trimester placental tissue compared to euploid controls but that the difference is marginal. Manual and automated scoring showed moderately strong correlation. Conclusion: Extrapolation of these results suggests MEIS2 overexpression may not be required for meromelia in T15.

Extracellular Matrix Protein Composition Dynamically Changes During Murine Forelimb Development

Extracellular Matrix Protein Composition Dynamically Changes During Murine Forelimb Development

Comparative Analyses of 35 Marine Mammal Genomes Provide Insights into the Evolution of Aquatic Life

Marine mammals, whose ancestors once roamed the land, have intrigued scientists for centuries. Although they share many similar morphological characteristics, the innovations for mammalian aquatic life were independently acquired at least seven times. Extant marine mammals are found in three orders: Artiodactyla, Sirenia, and Carnivora. To better understand the evolution of marine mammals, we generated the genomes of 17 marine mammals (11 cetaceans and six pinnipeds). Comparing these with publicly available marine mammal and terrestrial taxon genomes provides new insights into the evolutionary history of these iconic species. Focusing on the oldest groups of living marine mammals, the cetaceans, pinnipeds, and sirenians, we uncover genomic variations in marine mammals, including brown fat cell differentiation regulatory gene, NFIA, and forelimb development related genes, Xylt1 and Fam20b, which could underlie marine adaptations across these diverse taxonomic lineages.

HDAC4 and 5 repression of TBX5 is relieved by protein kinase D1

TBX5 is a T-box family transcription factor that regulates heart and forelimb development in vertebrates and functional deficiencies in this protein result in Holt-Oram syndrome. Recently, we have shown that acetylation of TBX5 potentiates its activity and is important for heart and limb development. Here we report that class II histone deacetylases HDAC4 and HDAC5 associate with TBX5 and repress its role in cardiac gene transcription. Both HDAC4 and HDAC5 deacetylate TBX5, which promotes its relocation to the cytoplasm and HDAC4 antagonizes the physical association and functional cooperation between TBX5 and MEF2C. We also show that protein kinase D1 (PRKD1) relieves the HDAC4/5-mediated repression of TBX5. Thus, this study reveals a novel interaction of HDAC4/5 and PRKD1 in the regulation of TBX5 transcriptional activity.

The Effect of Prenatal Exposure to 2.4 GHz Radio Frequency on the Histology and Expression of the osteocalcin and RUNX2 Gene of the Forelimb in an NMRI Mouse.

Introduction: Today the use of electromagnetic waves has dramatically increased in modern industrial societies. This study aimed to investigate the effect of prenatal exposure to 2.4 GHz wireless frequency on forelimb development in an NMRI mouse in vivo. Methods: A total of 21 female mice weighing 25-30 g were included in the present study. They were randomly assigned to 3 groups, namely control (n=7), sham (n=7), and experimental (n=7). After mating, the experimental group was exposed to 2.4 GHz radio frequency at a distance of 20-30 cm from the device, 4 h/d until the delivery. The sham group was placed at a distance of 20-30 cm from the device every day without exposure to electromagnetic waves, and the control group had a pregnancy period without any stress and electromagnetic wave exposure. After giving birth, the forelimbs were isolated from the infants and examined by stereological studies and RT-PCR for the evaluation of osteocalcin and RUNX2 gene expression. Results: Although, at first glance, there was no macroscopic teratogen effect in forelimbs in all groups, via a stereological method, we showed that bone and cartilage volume decreased in the experimental group compared to the other groups. We also found that the experimental group had lower expression of the osteocalcin and RUNX2 gene than the control and sham groups did. However, there were no significant differences between the control and sham groups in terms of bone and cartilage volume and gene expression. Conclusion: Although teratogen effect of prenatal exposure to 2.4 GHz radio frequency on forelimbs was not demonstrated macroscopically, further studies showed negative effects on the forelimb bone, cartilage volume, and gene expression.

Flap-Footed Lizards (Gekkota: Pygopodidae) Have Forelimbs, Albeit During Embryonic Development

Limb reduction among squamate clades is common; limbless (forelimbs, hindlimbs, or both) forms have had at least 25 independent origins among snakes and lizards. The general evolutionary pattern is the gradual loss of limb elements proceeding from the toes to the shoulder or hip. In contrast, forelimbs are not known to have ever existed in snakes. Forelimbs are not known for either stem or crown snakes; snake fossils lack forelimbs and the embryos of modern snakes lack the signaling pathways that initiate forelimb development. Both fossil and developmental data thus imply an abrupt loss of forelimbs. In contrast, the well-developed hindlimbs of the oldest fossil snakes and vestigial hindlimbs of some basal clades of modern snakes imply the historical presence of hindlimbs, and their gradual reduction during snake evolution. In contrast, observations on limb-reduced lizards are in accord with gradual reduction of both fore- and hindlimbs; embryos of even limbless species initiate limb development. The question then arises, have any clades of limbless lizards become limbless abruptly, as have snakes? To address this question, I examined limb development in the family Pygopodidae. Like basal snakes, pygopodids are characterized by the absence of forelimbs and by vestigial hindlimbs. My observations on the development of forelimb buds in Delma molleri support gradual limb reduction, as do observations on other limbless lizards. Nonetheless, many clades of limbless lizards are yet to be studied and hence provide a wealth of opportunities to address modes of limb reduction and loss in squamate reptiles.

Genomic Knockout of Two Presumed Forelimb Tbx5 Enhancers Reveals They Are Nonessential for Limb Development

SUMMARYA standard approach in the identification of transcriptional enhancers is the use of transgenic animals carrying DNA elements joined to reporter genes inserted randomly in the genome. We examined elements near Tbx5, a gene required for forelimb development in humans and other vertebrates. Previous transgenic studies reported a mammalian Tbx5 fore-limb enhancer located in intron 2 containing a putative retinoic acid response element and a zebrafish tbx5a forelimb (pectoral fin) enhancer located downstream that is conserved from fish to mammals. We used CRISPR/Cas9 gene editing to knockout the endogenous elements and unexpectedly found that deletion of the intron 2 and downstream elements, either singly or together in double knockouts, resulted in no effect on forelimb development. Our findings show that reporter transgenes may not identify endogenous enhancers and that in vivo genetic loss-of-function studies are required, such as CRISPR/Cas9, which is similar in effort to production of animals carrying reporter transgenes.

Three-Dimensional Growth of Flexural Slip Fault-Bend and Fault-Propagation Folds and Their Geomorphic Expression

The three-dimensional growth of fault-related folds is known to be an important process during the development of compressive mountain belts. However, comparatively little is known concerning the manner in which fold growth is expressed in topographic relief and local drainage networks. Here we report results from a coupled kinematic and surface process model of fault-related folding. We consider flexural slip fault-bend and fault-propagation folds that grow in both the transport and strike directions, linked to a surface process model that includes bedrock channel development and hillslope diffusion. We investigate various modes of fold growth under identical surface process conditions and critically analyse their geomorphic expression. Fold growth results in the development of steep forelimbs and gentler, wider backlimbs resulting in asymmetric drainage basin development (smaller basins on forelimbs, larger basins on backlimbs). However, topographies developed above fault-propagation folds are more symmetric than those developed above fault-bend folds as a result of their different forelimb kinematics. In addition, the surface expression of fault-bend and fault-propagation folds depends both on the slip distribution along the fault and on the style of fold growth. When along-strike plunge is a result of slip events with gently decreasing slip towards the fault tips (with or without lateral propagation), large plunge-panel drainage networks are developed at the expense of backpanel (transport-opposing) and forepanel (transport-facing) drainage basins. In contrast, if the fold grows as a result of slip events with similar displacements along strike, plunge-panel drainage networks are poorly developed (or are transient features of early fold growth) and restricted to lateral fold terminations, particularly when the number of propagation events is small. The absence of large-scale plunge-panel drainage networks in natural examples suggests that the latter mode of fold growth may be more common. The advective component of deformation (implicit in kink-band migration models of fault-bend and fault-propagation folding) exerts a strong control on drainage basin development. In particular, as drainage lengthens with fold growth, more linear, parallel drainage networks are developed as compared to the dendritic patterns developed above simple uplifting structures. Over the 1 Ma of their development the folds modelled here only attain partial topographic equilibrium, as new material is continually being advected through active axial surfaces on both fold limbs and faults are propagating in both the transport and strike directions. We also find that the position of drainage divides at the Earth’s surface has a complex relationship to the underlying fold axial surface locations.

Acetylation of TBX5 by KAT2B and KAT2A regulates heart and limb development

TBX5 plays a critical role in heart and forelimb development. Mutations in TBX5 cause Holt-Oram syndrome, an autosomal dominant condition that affects the formation of the heart and upper-limb. Several studies have provided significant insight into the role of TBX5 in cardiogenesis; however, how TBX5 activity is regulated by other factors is still unknown. Here we report that histone acetyltransferases KAT2A and KAT2B associate with TBX5 and acetylate it at Lys339. Acetylation potentiates its transcriptional activity and is required for nuclear retention. Morpholino-mediated knockdown of kat2a and kat2b transcripts in zebrafish severely perturb heart and limb development, mirroring the tbx5a knockdown phenotype. The phenotypes found in MO-injected embryos were also observed when we introduced mutations in the kat2a or kat2b genes using the CRISPR-Cas system. These studies highlight the importance of KAT2A and KAT2B modulation of TBX5 and their impact on heart and limb development.