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Background: Limb length discrepancy (LLD) is one of the most common complications of primary total hip arthroplasty (THA) causing patients dissatisfaction. In this study we determined the mean of preoperative limb length discrepancy (LLD), postoperative LLD after THA and their difference. Previously very little literature is available on this topic in Pakistan, so the results of my study will be a useful addition in the existing as well as local literature. Also, on the basis of these results, need of some preoperative planning and executing the plan in the operating room to eliminate LLD can be recommended. Methods: It is a descriptive case series study (Clinical Practice Article) conducted on 44 patients undergoing primary Total Hip arthroplasty (THA) at orthopaedics department Madinah Teaching Hospital Faisalabad from Sep 2018 to Sept 2019. Patients having age between 18--70 years including both genders were studied with non-probability, consecutive sampling. Results: In our study mean of pre-operative LLD is -19.02 with standard deviation of 21.31 and mean of post-operative LLD is 7.25 mm with standard deviation of 9.46. This study tells that 91% of patients undergoing primary THA without using any method to eliminate LLD have postoperative LLD but 88.6% of patients (39 patients out of 44) have postoperative LLD in the range of 0 to ±10 mm which is not much significant. But 11.4% of patients (5 patients out of 44) have postoperative LLD >10 mm which causes functional impairment. Conclusion: We can say that in most of the patients postoperative LLD after primary THA is very minimal and within acceptable range in experienced hands but a few patients can have a significant LLD in order to gain better joint stability which can be compensated by shoe wears. [ABSTRACT FROM AUTHOR]

More than sixty years ago, while studying feather tracks on the shoulder of the chick embryo, Dr. John Saunders used Nile Blue dye to stain the tissue. There, he noticed a darkly stained line of cells that neatly rims the tip of the growing limb bud. Rather than ignoring this observation, he followed it up by removing this tissue and found that it led to a striking truncation of the limb skeletons. This landmark experiment marks the serendipitous discovery of the apical ectodermal ridge (AER), the quintessential embryonic structure that drives the outgrowth of the limb. Dr. Saunders continued to lead the limb field for the next fifty years, not just through his own work, but also by inspiring the next generation of researchers through his infectious love of science. Together, he and those who followed ushered in the discovery of fibroblast growth factor (FGF) as the AER molecule. The seamless marriage of embryology and molecular biology that led to the decoding of the AER serves as a shining example of how discoveries are made for the rest of the developmental biology field. [ABSTRACT FROM AUTHOR]

John Saunders was a highly skilled embryologist who pioneered the study of limb development. His studies on chick embryos provided the fundamental framework for understanding how vertebrate limbs develop. This framework inspired generations of scientists and formed the bridge from experimental embryology to molecular mechanisms. Saunders investigated how feathers become organized into tracts in the skin of the chick wing and also identified regions of programmed cell death. He discovered that a region of thickened ectoderm that rims the chick wing bud - the apical ectodermal ridge - is required for outgrowth and the laying down of structures along the proximo-distal axis (long axis) of the wing, identified the zone of polarizing activity (ZPA; polarizing region) that controls development across the anteroposterior axis (“thumb to little finger “axis) and contributed to uncovering the importance of the ectoderm in development of structures along the dorso-ventral axis ( “back of hand to palm” axis). This review looks in depth at some of his original papers and traces how he made the crucial findings about how limbs develop, considering these findings both in the context of contemporary knowledge at the time and also in terms of their immediate impact on the field. [ABSTRACT FROM AUTHOR]

Among John Saunders' many seminal contributions to developmental biology, his discovery of the limb ‘zone of polarizing activity’ (ZPA) is arguably one of the most memorable and ground-breaking. This discovery introduced the limb as a premier model for understanding developmental patterning and promoted the concept of patterning by a morphogen gradient. In the 50 years since the discovery of the ZPA, Sonic hedgehog (Shh) has been identified as the ZPA factor and the basic components of the signaling pathway and many aspects of its regulation have been elucidated. Although much has also been learned about how it regulates growth, the mechanism by which Shh patterns the limb, how it acts to instruct digit ‘identity’, nevertheless remains an enigma. This review focuses on what has been learned about Shh function in the limb and the outstanding puzzles that remain to be solved. [ABSTRACT FROM AUTHOR]

John W. Saunders, Jr. made seminal discoveries unveiling how chick embryos develop their limbs. He discovered the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the domains of interdigital cell death within the developing limb and determined their function through experimental analysis. These discoveries provided the basis for subsequent molecular understanding of how vertebrate limbs are induced, patterned, and differentiated. These mechanisms are strongly conserved among the vast diversity of tetrapod limbs suggesting that relatively minor changes and tweaks to the molecular cascades are responsible for the diversity observed in nature. Analysis of the pathway systems first identified by Saunders in the context of animals displaying limb reduction show how alterations in these pathways have resulted in multiple mechanisms of limb and digit loss. Other classes of modification to these same patterning systems are seen at the root of other, novel limb morphological alterations and elaborations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: From bat wings to whale flippers, limb diversification has been crucial to the evolutionary success of mammals. We performed the first transcriptome-wide study of limb development in multiple species to explore the hypothesis that mammalian limb diversification has proceeded through the differential expression of conserved shared genes, rather than by major changes to limb patterning. Specifically, we investigated the manner in which the expression of shared genes has evolved within and among mammalian species. Results: We assembled and compared transcriptomes of bat, mouse, opossum, and pig fore- and hind limbs at the ridge, bud, and paddle stages of development. Results suggest that gene expression patterns exhibit larger variation among species during later than earlier stages of limb development, while within species results are more mixed. Consistent with the former, results also suggest that genes expressed at later developmental stages tend to have a younger evolutionary age than genes expressed at earlier stages. A suite of key limb-patterning genes was identified as being differentially expressed among the homologous limbs of all species. However, only a small subset of shared genes is differentially expressed in the fore- and hind limbs of all examined species. Similarly, a small subset of shared genes is differentially expressed within the fore- and hind limb of a single species and among the forelimbs of different species. Conclusions: Taken together, results of this study do not support the existence of a phylotypic period of limb development ending at chondrogenesis, but do support the hypothesis that the hierarchical nature of development translates into increasing variation among species as development progresses. [ABSTRACT FROM AUTHOR]

mRNA variance has been proposed to play key roles in normal development, population fitness, adaptability, and disease. While variance in gene expression levels may be beneficial for certain cellular processes, for example in a cell's ability to respond to external stimuli, variance may be detrimental for the development of some organs. In the bilaterally symmetric vertebrate limb buds, the amount of Sonic Hedgehog (SHH) protein present at specific stages of development is essential to ensure proper patterning of this structure. To our surprise, we found that SHH protein variance is present during the first 10 hr of limb development. The variance is virtually eliminated after the first 10 hr of limb development. By examining mutant animals, we determined that the ability of the limb bud apical ectodermal ridge (AER) to respond to SHH protein was required for reducing SHH variance during limb formation. One consequence of the failure to eliminate variance in SHH protein was the presence of polydactyly and an increase in digit length. These data suggest a potential novel mechanism in which alterations in SHH variance during evolution may have driven changes in limb patterning and digit length. [ABSTRACT FROM AUTHOR]

Genetic aberrations of several unlinked loci cause human congenital split hand/foot malformation (SHFM) development. Mutations of the DLX5 (distalless) transcription factor-encoding gene in chromosome 7 cause SHFM through haploinsufficiency, but the vast majority of cases result from heterozygous chromosomal aberrations of the region without mutating the DLX5 gene. To resolve this paradox, we invoke a chromosomal epigenetic mechanism for limb development. It is composed of amonochromatid gene expression phenomenon that we discovered in two fission yeasts with the selective chromosome copy segregation phenomenon that we discovered in mouse cells. Accordingly, one daughter cell inherits both expressed DLX5 copies while the other daughter inherits both epigenetically silenced ones from a single deterministic cell of the developing limb. Thus, differentiated daughter cells after further proliferation will correspondingly produce proximal/distal-limb tissues. Published results of a Chr. 7 translocation with a centromere-proximal breakpoint situated over 41 million bases away from the DLX locus, centromeric and DLX5-region inversions have satisfied key genetic and developmental biology predictions of the mechanism. Further genetic tests of the mechanism are proposed. We propose that the DNA double helical structure itself causes the development of sister cells' gene regulation asymmetry. We also argue against the conventionally invoked morphogen model of development. [ABSTRACT FROM AUTHOR]

Summary Limb reduction and loss are hallmarks of snake evolution. Although advanced snakes are completely limbless, basal and intermediate snakes retain pelvic girdles and small rudiments of the femur. Moreover, legs may have re-emerged in extinct snake lineages [ 1–5 ], suggesting that the mechanisms of limb development were not completely lost in snakes. Here we report that hindlimb development arrests in python embryos as a result of mutations that abolish essential transcription factor binding sites in the limb-specific enhancer of Sonic hedgehog ( SHH ). Consequently, SHH transcription is weak and transient in python hindlimb buds, leading to early termination of a genetic circuit that drives limb outgrowth. Our results suggest that degenerate evolution of the SHH limb enhancer played a role in reduction of hindlimbs during snake evolution. By contrast, HOXD digit enhancers are conserved in pythons, and HOXD gene expression in the hindlimb buds progresses to the distal phase, forming an autopodial (digit) domain. Python hindlimb buds then develop transitory pre-chondrogenic condensations of the tibia, fibula, and footplate, raising the possibility that re-emergence of hindlimbs during snake evolution did not require de novo re-evolution of lost structures but instead could have resulted from persistence of embryonic legs. Video Abstract [ABSTRACT FROM AUTHOR]

ABSTRACT The mesopodium forms at the boundary between the zeugopod and autopod and is composed of short nodular bones that typically lack growth plates. Hoxa11 and Hoxa13 are expressed in mutually exclusive proximal-distal domains that demarcate the zeugopod/autopod boundary. Similarly, Hoxd genes are deployed in two distinct phases during limb development. The early phase corresponds to proximal segments including the zeugopod, and a late phase occurs in the digits. This arrangement produces a gap of low Hoxd expression that is traditionally viewed to correspond to the mesopodium. In contrast to the other mesopodials, the mammalian pisiform and calcaneus form true growth plates. We show that these bones, along with other proximal mesopodials, develop within the Hoxa11 and Hoxd11 expression domains. We also observe that the pisiform growth plate becomes disorganized with Hoxa11 or Hoxd11 loss of function, indicating a direct role for Hox11 in its development. Hoxa13 loss of function has minimal effect on the pisiform and proximal calcaneus as these bones still form secondary centers and undergo longitudinal growth. Consideration of the phenotypes resulting from hypodactyly ( Hd) and synpolydactyly homolog ( spdh) mutations, which result from altered HOXA13 and HOXD13 proteins, respectively, confirms that Hox13 plays a limited role in the development of the pisiform and calcaneus and suggests that they lie within the early phase of Hox expression. Therefore, with respect to patterns of ossification and gene expression, these bones share much more in common with the zeugopod than the autopod. Such an interpretation fits with the timing of autopod origins during tetrapod evolution. [ABSTRACT FROM AUTHOR]

Involvement of proliferation and apoptosis in the human limb development was analyzed electronmicroscopically and immunohistochemically in histological sections of 8 human embryos, 4 th −10 th week old, using apoptotic (caspase-3, AIF, BAX), anti-apoptotic (Bcl-2) and proliferation (Ki-67) markers, and TUNEL method. The data were analyzed by Mann–Whitney test, Kruskal–Wallis and Dunn’s post hoc test. Initially, developing human limbs consisted of mesenchymal core and surface ectoderm with apical ectodermal ridge (AER). During progression of development, strong proliferation activity gradually decreased in the mesenchyme (from 78% to 68%) and in the epithelium (from 62% to 42%), while in the differentiating finger cartilages proliferation was constantly low (26–7%). Apoptotic caspase-3 and AIF—positive cells characterized mesenchyme and AER at earliest stages, while during digit separation they appeared in interdigital mesenchyme as well. Strong Bcl-2 expression was observed in AER, subridge mesenchyme and phalanges, while BAX expression charaterized limb areas undergoing apoptosis. Ultrastructurally, proliferating cells showed mitotic figures, while apoptotic cells were characterized by nuclear fragmentation. Macrophages were observed around the apoptotic cells. We suggest that intense proliferation enables growth and elongation of human limb primordia, and differential growth of digits. Both caspase-3 and AIF–dependant pathways of cell death control the extent of AER and numer of cells in the subridge mesenchyme at earliest developmental stages, as well as process of digit separation at later stages of limb development. Spatio-temporal co-expresson of Bcl-2 and BAX indicates their role in suppression of apoptosis and selective stimulation of growth during human limb morphogenesis. [ABSTRACT FROM AUTHOR]

The molecular analysis of limb bud development in vertebrates continues to fuel our understanding of the gene regulatory networks that orchestrate the patterning, proliferation and differentiation of embryonic progenitor cells. In recent years, systems biology approaches have moved our understanding of the molecular control of limb organogenesis to the next level by incorporating next generation 'omics' approaches, analyses of chromatin architecture, enhancer-promoter interactions and gene network simulations based on quantitative datasets into experimental analyses. This Review focuses on the insights these studies have given into the gene regulatory networks that govern limb development and into the fin-to-limb transition and digit reductions that occurred during the evolutionary diversification of tetrapod limbs. [ABSTRACT FROM AUTHOR]

Parameter optimization coupled with model selection is a convenient approach to infer gene regulatory networks from experimental gene expression data, but so far it has been limited to single cells or static tissues where growth is not significant. Here, we present a computational study in which we determine an optimal gene regulatory network from the spatiotemporal dynamics of gene expression patterns in a complex 2D growing tissue (nonisotropic and heterogeneous growth rates). We use this method to predict the regulatory mechanisms that underlie proximodistal (PD) patterning of the developing limb bud. First, we map the expression patterns of the PD markers Meis1, Hoxa11 and Hoxa13 into a dynamic description of the tissue movements that drive limb morphogenesis. Secondly, we use reverse-engineering to test how different gene regulatory networks can interpret the opposing gradients of fibroblast growth factors (FGF) and retinoic acid (RA) to pattern the PD markers. Finally, we validate and extend the best model against various previously published manipulative experiments, including exogenous application of RA, surgical removal of the FGF source and genetic ectopic expression of Meis1. Our approach identifies the most parsimonious gene regulatory network that can correctly pattern the PD markers downstream of FGF and RA. This network reveals a new model of PD regulation which we call the "crossover model", because the proximal morphogen (RA) controls the distal boundary of Hoxa11, while conversely the distal morphogens (FGFs) control the proximal boundary. [ABSTRACT FROM AUTHOR]

A record of an adult Large Striped Skink Ctenotus robustus, from the northern outskirts of metropolitan Melbourne, with four regenerating tails is described. The rarity of this record is confirmed with reference to other field surveys and published studies. [ABSTRACT FROM AUTHOR]

During limb development Pax3 positive myoblasts delaminate from the hypaxial dermomyotome of limb level somites and migrate into the limb bud where they form the dorsal and ventral muscle masses. Only then do they begin to differentiate and express markers of myogenic commitment and determination such as Myf5 and MyoD . However the signals regulating this process remain poorly characterised. We show that FGF18, which is expressed in the distal mesenchyme of the limb bud, induces premature expression of both Myf5 and MyoD and that blocking FGF signalling also inhibits endogenous MyoD expression. This expression is mediated by ERK MAP kinase but not PI3K signalling. We also show that retinoic acid (RA) can inhibit the myogenic activity of FGF18 and that blocking RA signalling allows premature induction of MyoD by FGF18 at HH19. We propose a model where interactions between FGF18 in the distal limb and retinoic acid in the proximal limb regulate the timing of myogenic gene expression during limb bud development. [ABSTRACT FROM AUTHOR]

Urodele amphibians have remarkable organ regeneration capability, and their limb regeneration capability has been investigated as a representative phenomenon. In the early 19th century, nerves were reported to be an essential tissue for the successful induction of limb regeneration. Nerve substances that function in the induction of limb regeneration responses have long been sought. A new experimental system called the accessory limb model (ALM) has been established to identify the nerve factors. Skin wounding in urodele amphibians results in skin wound healing but never in limb induction. However, nerve deviation to the wounded skin induces limb formation in ALM. Thus, nerves can be considered to have the ability to transform skin wound healing to limb formation. In the present study, co-operative Bmp and Fgf application, instead of nerve deviation, to wounded skin transformed skin wound healing to limb formation in two urodele amphibians, axolotl ( Ambystoma mexicanum ) and newt ( Pleurodeles waltl ). Our findings demonstrate that defined factors can induce homeotic transformation in postembryonic bodies of urodele amphibians. The combination of Bmp and Fgf(s) may contribute to the development of novel treatments for organ regeneration. [ABSTRACT FROM AUTHOR]

Fibroblast growth factor ( FGF) signalling appears essential for the regulation of limb development, but a full complexity of this regulation remains unclear. Here, we addressed the effect of three different chemical inhibitors of FGF receptor tyrosine kinases ( FGFR) on growth and patterning of the chicken wings. The inhibitor PD173074 caused shorter and thinner wing when using lower concentration. Microinjection of higher PD173074 concentrations (25 and 50 mmol/L) into the wing bud at stage 20 resulted in the development of small wing rudiment or the total absence of the wing. Skeletal analysis revealed the absence of the radius but not ulna, deformation of metacarpal bones and/or a reduction of digits. Treatment with PD161570 resembled the effects of PD173074. NF449 induced shortening and deformation of the developing wing with reduced autopodium. These malformed embryos mostly died at the stage HH25-29. PD173074 reduced chondrogenesis also in the limb micromass cultures together with early inhibition of cartilaginous nodule formation, evidenced by lack of sulphated proteoglycan and peanut agglutinin expression. The effect of FGFR inhibition on limb development observed here was unlikely mediated by excessive cell death as none of the inhibitors caused massive apoptosis at low concentrations. More probably, FGFR inhibition decreased both the proliferation and adhesion of mesenchymal chondroprogenitors. We conclude that FGFR signalling contributes to the regulation of the anterior-posterior patterning of zeugopod during chicken limb development. [ABSTRACT FROM AUTHOR]

Fibroblast growth factors (FGFs) expressed in the apical ectodermal ridge (AER) and FGF10 expressed in the underlying mesoderm are essential for limb bud outgrowth. Their expression is maintained through a positive feedback loop. We identified the cis-regulatory element and trans-acting factors involved in the AER-FGF-dependent transactivation of Fgf 10. Etv1 and Ewsr1 stimulated transcription from the Fgf 10 promoter in the sub-AER mesenchyme of mouse and chick limb buds in a conserved AGAAAR cluster-dependent manner. We found that both Etv 1 and Ewsr 1 were necessary for Fgf 10 expression and elongation of the limb bud. In addition, Etv1 and AER-FGF synergistically stimulated Fgf 10 promoter activity in an Ewsr1-dependent manner. We also found that Etv1 and Ewsr1 bound to the segment of DNA containing the AGAAAR cluster in vivo and in vitro . Moreover, Etv1 directly bound to the AGAAAR sequence in vitro. Our results suggest that Etv1 and Ewsr1 transactivate Fgf 10 directly and cooperatively in response to AER-FGFs. [ABSTRACT FROM AUTHOR]

Mutations in the Bone Morphogenetic Protein (BMP) pathway are associated with a range of defects in skeletal formation. Genetic analysis of BMP signaling requirements is complicated by the presence of three partially redundant BMPs that are required for multiple stages of limb development. We generated an inducible allele of a BMP inhibitor, Gremlin , which reduces BMP signaling. We show that BMPs act in a dose and time dependent manner in which early reduction of BMPs result in digit loss, while inhibiting overall BMP signaling between E10.5 and E11.5 allows polydactylous digit formation. During this period, inhibiting BMPs extends the duration of FGF signaling. Sox9 is initially expressed in normal digit ray domains but at reduced levels that correlate with the reduction in BMP signaling. The persistence of elevated FGF signaling likely promotes cell proliferation and survival, inhibiting the activation of Sox9 and secondarily, inhibiting the differentiation of Sox9 -expressing chondrocytes. Our results provide new insights into the timing and clarify the mechanisms underlying BMP signaling during digit morphogenesis. [ABSTRACT FROM AUTHOR]

Gestational diabetes has an adverse impact on fetal musculoskeletal development, but the mechanism involved is still not completely understood. In this study, we investigated the effects of high glucose on the developing somites and their derivate using the chick embryo as a model. We demonstrated that under high glucose, the number of generated somites was reduced and their morphology altered in 2-day old chick embryos. In addition, high glucose repressed the development of the limb buds in 5.5-day old chick embryos. We also demonstrated that high glucose abridged the development of the sclerotome and the cartilage in the developing limb bud. The sonic hedgehog (Shh) gene has been reported to play a crucial role in the development and differentiation of sclerotome. Hence, we examined how Shh expression in the sclerotome was affected under high glucose. We found that high glucose treatment significantly inhibited Shh expression. The high glucose also impaired myotome formation at trunk level – as revealed by immunofluorescent staining with MF20 antibodies. In the neural tube, we established that Wnt3a expression was also significantly repressed. In summary, our study demonstrates that high glucose concentrations impair somite and limb bud development in chick embryos, and suggests that Shh and Wnt genes may play a role in the underlying mechanism. [ABSTRACT FROM AUTHOR]

During limb development, digits emerge from the undifferentiated mesenchymal tissue that constitutes the limb bud. It has been proposed that this process is controlled by a self-organizing Turing mechanism, whereby diffusible molecules interact to produce a periodic pattern of digital and interdigital fates. However, the identities of the molecules remain unknown. By combining experiments and modeling, we reveal evidence that a Turing network implemented by Bmp, Sox9, and Wnt drives digit specification. We develop a realistic two-dimensional simulation of digit patterning and show that this network, when modulated by morphogen gradients, recapitulates the expression patterns of Sox9 in the wild type and in perturbation experiments. Our systems biology approach reveals how a combination of growth, morphogen gradients, and a self-organizing Turing network can achieve robust and reproducible pattern formation. [ABSTRACT FROM AUTHOR]

The regulatory architecture that controls developmental genes is often a collection of enhancers that, in combination, generate a complex spatial and temporal pattern of expression. These enhancers populate domains operating at long distances and, in the case of the sonic hedgehog ( Shh ) locus, this regulatory domain covers ∼900–1000 kb. Within this context each embryonic tissue that expresses Shh has acquired its own regulatory apparatus which may require the activity from several distinct enhancers. Expression of Shh in the developing limb bud is driven by a single enhancer that interprets a myriad of genetic information to initiate expression in the posterior margin of the limb bud, inhibits expression along the anterior margin, defines the level of expression, and sets the tissue boundary of expression. [ABSTRACT FROM AUTHOR]

The large spectrum of limb morphologies reflects the wide evolutionary diversification of the basic pentadactyl pattern in tetrapods. In even-toed ungulates (artiodactyls, including cattle), limbs are adapted for running as a consequence of progressive reduction of their distal skeleton to symmetrical and elongated middle digits with hoofed phalanges. Here we analyse bovine embryos to establish that polarized gene expression is progressively lost during limb development in comparison to the mouse. Notably, the transcriptional upregulation of the Ptch1 gene, which encodes a Sonic hedgehog (SHH) receptor, is disrupted specifically in the bovine limb bud mesenchyme. This is due to evolutionary alteration of a Ptch1 cis-regulatory module, which no longer responds to graded SHH signalling during bovine handplate development. Our study provides a molecular explanation for the loss of digit asymmetry in bovine limb buds and suggests that modifications affecting the Ptch1 cis-regulatory landscape have contributed to evolutionary diversification of artiodactyl limbs. [ABSTRACT FROM AUTHOR]

While growth has been studied extensively in invertebrates, the mechanisms by which it is controlled in vertebrates, particularly in mammals, remain poorly understood. In this study, we investigate the cellular basis of differential limb growth in postnatal Monodelphis domestica, the gray short-tailed opossum, to gain insights into the mechanisms regulating mammalian growth. Opossums are an ideal model for the study of growth because they are born with relatively large, well-developed forelimbs and small hind limbs that must 'catch up' to the forelimb before the animal reaches adulthood. Postnatal Days 1-17 were identified as a key period of growth for the hind limbs, during which they undergo accelerated development and nearly quadruple in length. Histology performed on fore- and hind limbs from this period indicates a higher rate of cellular differentiation in the long bones of the hind limbs. Immunohistochemical assays indicate that cellular proliferation is also occurring at a significantly greater rate in the long bones of the hind limb at 6 days after birth. Taken together, these results suggest that a faster rate of cellular proliferation and differentiation in the long bones of the hind limb relative to those of the forelimb generates a period of accelerated growth through which the adult limb phenotype of M. domestica is achieved. Assays for gene expression suggest that the molecular basis of this differential growth differs from that previously identified for differential pre-natal growth in opossum fore- and hind limbs. J. Exp. Zool. (Mol. Dev. Evol.) 322B: 221-229, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: The role of the T-box transcription factor Tbx5 in heart and limb development has been well documented; however, how posttranslational modification is involved in mediating its activity is unknown. Here we report that Tbx5 is a novel target by SUMO conjugation, a posttranslational modification that is involved in a variety of cellular events. Sumoylation potentiated the transcriptional activity of Tbx5, and PIAS family members, a group of SUMO E3 ligase, differentially mediated sumoylation and function of Tbx5. PIAS1 potently stimulated SUMO conjugation to Tbx5, and the physical association of Tbx5 with PIAS1 was required for its full sumoylation. PIAS1 also enhanced the functional cooperation between Tbx5 and its interaction partners. Overlapping expression pattern and colocalization of PIAS1 and Tbx5 in the mouse embryonic hearts and on the native target gene promoter were observed, pointing to a potential functional interaction of these two factors in vivo. These findings provide novel insights into how the transcriptional activity of a cardiac-specific factor, Tbx5, is regulated both directly and indirectly via posttranslational modification by a non-tissue-specific factor, PIAS1. [Copyright &y& Elsevier]

Summary: The patterning and growth of the embryonic vertebrate limb is dependent on Sonic hedgehog (Shh), a morphogen that regulates the activity of Gli transcription factors. However, Shh expression is not observed during the first 12 hr of limb development. During this phase, the limb bud is prepatterned into anterior and posterior regions through the antagonistic actions of transcription factors Gli3 and Hand2. We demonstrate that precocious activation of Shh signaling during this early phase interferes with the Gli3-dependent specification of anterior progenitors, disturbing establishment of signaling centers and normal outgrowth of the limb. Our findings illustrate that limb development requires a sweet spot in the level and timing of pathway activation that allows for the Shh-dependent expansion of posterior progenitors without interfering with early prepatterning functions of Gli3/Gli3R or specification of anterior progenitors. [Copyright &y& Elsevier]

The tail organizer has been assessed by such transplantation methods as the Einsteck procedure. However, we found that simple wounding of blastocoel roof ( BCR) made it possible to form secondary tails without any transplantation in Xenopus laevis. We revealed that the ectopic expression of Xbra was blocked by inhibiting the contact between BCR and blastocoel floor ( BCF), and wounding per se seemed to be not directly related to the secondary tail formation. Therefore, the secondary tail might be induced by the contact between BCR and BCF due to the leak of blastocoel fluid from the wound. This secondary tail was similar to the original tail in the expression pattern of tail genes, and in the fact that the inhibition of fibroblast growth factor signaling prevented the secondary tail induction. Our results imply that the secondary tail formation reflects the developmental processes of the original tail, indicating that simple wounding of BCR is useful for the analysis of tail formation in normal development. [ABSTRACT FROM AUTHOR]

Vertebrate limbs first emerge as small buds at specific locations along the trunk. Although a fair amount is known about the molecular regulation of limb initiation and outgrowth, the cellular events underlying these processes have remained less clear. We show that the mesenchymal limb progenitors arise through localized epithelial-to-mesenchymal transition (EMT) of the coelomic epithelium specifically within the presumptive limb fields. This EMT is regulated at least in part by and, two genes known to control limb initiation. This work shows that limb buds Tbx5 Fgf10 initiate earlier than previously thought, as a result of localized EMT rather than differential proliferation rates. [ABSTRACT FROM AUTHOR]

A current hypothesis states that the ancestral limb of arthropods is composed of only two segments. The proximal segment represents the main part of the modern leg, and the distal segment represents the tarsus and claw of the modern leg. If the distal part of the limb is an ancestral feature, one would expect conserved regulatory gene networks acting in distal limb development in all arthropods and possibly even their sister group, the onychophorans. We investigated the expression patterns of six genes known to function during insect distal limb development in the onychophoran Euperipatoides kanangrensis, i.e., clawless ( cll), aristaless ( al), spineless ( ss), zinc finger homeodomain 2 ( zfh2), rotund ( rn), and Lim1. We find that all investigated genes are expressed in at least some of the onychophoran limbs. The expression patterns of most of these genes, however, display crucial differences to the known insect patterns. The results of this study question the hypothesis of conserved distal limb evolution in arthropods and highlight the need for further studies on arthropod limb development. [ABSTRACT FROM AUTHOR]

ABSTRACT Objectives The purpose of this study is to document the appearance of adult patterns in intralimb indices during ontogeny in a skeletal sample from the Kellis 2 cemetery, Dakhleh Oasis, Egypt. In addition, this study explores evolvability in intralimb indices to understand relative differences in sensitivity to ecogeographic variables. Methods Brachial and crural indices were compared across age cohorts with Welch's ANOVA tests and post-hoc Dunnett-Tukey-Kramer (DTK) pairwise multiple comparison tests. Spearman's rank correlation coefficients were used to examine developmental conservation and evolvability in intralimb proportions. Results Brachial and crural indices are greatest in the fetus/perinate cohort as compared to all other cohorts, decrease during infancy and early childhood, and increase during middle/late childhood. The adult pattern in the brachial index is first evident in infancy, but is not maintained throughout development. Conversely, the adult pattern in the crural index appears during early childhood and is maintained throughout development. The brachial index shows a higher degree of evolvability than the crural index in utero. Conclusions The shifting pattern in intralimb proportions during development in the Kellis 2 sample is similar to that previously reported from globally diverse samples, which likely reflects the differential growth acceleration of proximal and distal intralimb skeletal elements during ontogeny. The brachial index may be more responsive to climatic conditions while the crural index may be more conserved due to functional demands. The data indicate that Kellis 2 juveniles were under strong selective pressures from climatic factors. Am. J. Hum. Biol. 26:221-228, 2014. © 2014 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Tetrapod limb development has been studied extensively for decades, yet the strength and role of developmental constraints in this process remains unresolved. Mammals exhibit a particularly wide array of limb morphologies associated with various locomotion modes and behaviors, providing a useful system for identifying periods of developmental constraint and conserved developmental mechanisms or morphologies. In this study, landmark-based geometric morphometrics are used to investigate levels and patterns of morphological diversity (disparity) among the developing forelimbs of four mammals with diverse limb morphologies: mice, opossums, horses, and pigs. Results indicate that disparity among the forelimbs of these species slightly decreases or stays the same from the appearance of the limb ridge to the bud stage, and increases dramatically from the paddle through tissue regression stages. Heterochrony exhibited by the precocial opossum limb was not found to drive these patterns of morphological disparity, suggesting that the low disparity of the middle stages of limb development (e.g., paddle stage) is driven by processes operating within the limb and is likely not a result of embryo-wide constraint. [ABSTRACT FROM AUTHOR]

The vertebrate limbs develop through coordinated series of inductive, growth and patterning events. Fibroblast Growth Factor receptor 2b (FGFR2b) signaling controls the induction of the Apical Ectodermal Ridge (AER) but its putative roles in limb outgrowth and patterning, as well as in AER morphology and cell behavior have remained unclear. We have investigated these roles through graded and reversible expression of soluble dominant-negative FGFR2b molecules at various times during mouse limb development, using a doxycycline/transactivator/tet(O)-responsive system. Transient attenuation (≤24 hours) of FGFR2b-ligands signaling at E8.5, prior to limb bud induction, leads mostly to the loss or truncation of proximal skeletal elements with less severe impact on distal elements. Attenuation from E9.5 onwards, however, has an irreversible effect on the stability of the AER, resulting in a progressive loss of distal limb skeletal elements. The primary consequences of FGFR2b-ligands attenuation is a transient loss of cell adhesion and down-regulation of P63, β1-integrin and E-cadherin, and a permanent loss of cellular β-catenin organization and WNT signaling within the AER. Combined, these effects lead to the progressive transformation of the AER cells from pluristratified to squamous epithelial-like cells within 24 hours of doxycycline administration. These findings show that FGFR2b-ligands signaling has critical stage-specific roles in maintaining the AER during limb development. [ABSTRACT FROM AUTHOR]

Studies of the vertebrate limb development have contributed significantly to understanding the fundamental mechanisms underlying growth, patterning, and morphogenesis of a complex multicellular organism. In the limb, well-defined signaling centers interact to coordinate limb growth and patterning along the three axes. Recent analyses of live imaging and mathematical modeling have provided evidence that polarized cell behaviors governed by morphogen gradients play an important role in shaping the limb bud. Furthermore, the Wnt/planar cell polarity (PCP) pathway that controls uniformly polarized cell behaviors in a field of cells has emerged to be critical for directional morphogenesis in the developing limb. Directional information coded in the morphogen gradient may be interpreted by responding cells through regulating the activities of PCP components in a Wnt morphogen dose-dependent manner. [Copyright &y& Elsevier]

Summary: The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution. [ABSTRACT FROM AUTHOR]

Limb development relies on an exquisite coordination between growth and patterning, but the underlying mechanisms remain elusive. Anterior-posterior and proximal-distal specification initiates in early limb bud concomitantly with the proliferative expansion of limb cells. Previous studies have shown that limb bud growth initially relies on fibroblast growth factors (FGFs) produced in the apical ectodermal ridge (AER-FGFs), the maintenance of which relies on a positive-feedback loop involving sonic hedgehog (Shh) and the BMP antagonist gremlin 1 (Grem1). The positive cross-regulation between Shh and the HoxA and HoxD clustered genes identified an indirect effect of Hox genes on the maintenance of AER-FGFs but the respective function of Shh and Hox genes in this process remains unknown. Here, by uncoupling Hox and Shh function, we show that HoxA and HoxD genes are required for proper AER-FGFs expression, independently of their function in controlling Shh expression. In addition, we provide evidence that the Hoxdependent control of AER-FGF expression is achieved through the regulation of key mesenchymal signals, namely Grem1 and Fgf10, ensuring proper epithelial-mesenchymal interactions. Notably, HoxA and HoxD genes contribute to both the initial activation of Grem1 and the subsequent anterior expansion of its expression domain. We propose that the intricate interactions between Hox genes and the FGF and Shh signaling pathways act as a molecular network that ensures proper limb bud growth and patterning, probably contributing to the coordination of these two processes. [ABSTRACT FROM AUTHOR]

Direct developing frogs, like Eleutherodactylus coqui, provide opportunities to investigate limb early development in anuran amphibians that are less available in species with tadpoles. We have found that myosin heavy chain 6 ( myh6), a myosin gene usually considered heart-specific in Xenopus and other animals, is expressed in limbs of E. coqui embryos. The gene for microRNA(miR)-208 is contained in an intron of the E. coqui myh6 gene as in mammals, and miR -208 was detected as a microRNA, more highly expressed in a microarray of E. coqui limb buds, compared to Xenopus laevis limb buds. Myh6 is also expressed in several muscles of tadpoles and froglets of Xenopus tropicalis. These connections raise the possibility of an involvement of myh6 and miR-208 in the thyroid dependent metamorphosis of anurans. [ABSTRACT FROM AUTHOR]

The formation of repetitive structures (such as stripes) in nature is often consistent with a reaction-diffusion mechanism, or Turing model, of self-organizing systems. We used mouse genetics to analyze how digit patterning (an iterative digit/nondigit pattern) is generated. We showed that the progressive reduction in Hoxa13 and Hoxd11-Hoxd13 genes (hereafter referred to as distal Hox genes) from the G//5-null background results in progressively more severe polydactyly, displaying thinner and densely packed digits. Combined with computer modeling, our results argue for a Turing-type mechanism underlying digit patterning, in which the dose of distal Hox genes modulates the digit period or wavelength. The phenotypic similarity with fish-fin endoskeleton patterns suggests that the pentadactyl state has been achieved through modification of an ancestral Turing-type mechanism. [ABSTRACT FROM AUTHOR]

Members of vertebrates are complex structures with three lines to consider, proximal-distal, anterior-posterior and dorsal-ventral. The battery of genes involved in the formation of these structures is well conserved in evolution. The outline of the member is composed of undifferentiated mesenchymal cells derived from somatic lateral mesoderm covered by ectoderm. The apical ectodermal ridge is a signal producing center for development and is located in the distal margin of the outline of a member. The area of progress is below and allows the growth of the member. The type of structures formed along the proximal distal axis is specified by Hox genes. Sonic Hedgehog protein is involved in regulating the activity of a second signaling center known as the zone of polarizing activity. Members also have a dorsal ventral polarity. The Wnt protein secreted from the dorsal ectoderm, instructs the surrounding mesenchymal cells to differentiate into dorsal structures whereas Engrafted 1 expressed in the ventral ectoderm, inhibit the expression of WNT7A outline in this membership area promoting the formation of ventral structures. [ABSTRACT FROM AUTHOR]

Tbx4 and Tbx5 are two closely related T-box genes that encode transcription factors expressed in the prospective hindlimb and forelimb territories, respectively, of all jawed vertebrates. Despite their striking limb type-restricted expression pattern, we have shown that these genes do not participate in the acquisition of limb type-specific morphologies. Instead, Tbx4 and Tbx5 play similar roles in the initiation of hindlimb and forelimb outgrowth, respectively. We hypothesized that different combinations of Hox proteins expressed in different rostral and caudal domains of the lateral plate mesoderm, where limb induction occurs, might be involved in regulating the limb type-restricted expression of Tbx4 and Tbx5 and in the later determination of limb type-specific morphologies. Here, we identify the minimal regulatory element sufficient for the earliest forelimb-restricted expression of the mouse Tbx5 gene and show that this sequence is Hox responsive. Our results support a mechanism in which Hox genes act upstream of Tbx5 to control the axial position of forelimb formation. [ABSTRACT FROM AUTHOR]

A late phase of HoxD activation is crucial for the patterning and growth of distal structures across the anterior-posterior (A-P) limb axis of mammals. Polycomb complexes and chromatin compaction have been shown to regulate Hox loci along the main body axis in embryonic development, but the extent to which they have a role in limb-specific HoxD expression, an evolutionary adaptation defined by the activity of distal enhancer elements that drive expression of 5' Hoxd genes, has yet to be fully elucidated. We reveal two levels of chromatin topology that differentiate distal limb A-P HoxD activity. Using both immortalised cell lines derived from posterior and anterior regions of distal E10.5 mouse limb buds, and analysis in E10.5 dissected limb buds themselves, we show that there is a loss of polycomb-catalysed H3K27me3 histone modification and a chromatin decompaction over HoxD in the distal posterior limb compared with anterior. Moreover, we show that the global control region (GCR) long-range enhancer spatially colocalises with the 5' HoxD genomic region specifically in the distal posterior limb. This is consistent with the formation of a chromatin loop between 5' HoxD and the GCR regulatory module at the time and place of distal limb bud development when the GCR participates in initiating Hoxd gene quantitative collinearity and Hoxd13 expression. This is the first example of A-P differences in chromatin compaction and chromatin looping in the development of the mammalian secondary body axis (limb). [ABSTRACT FROM AUTHOR]

Objective: To assess the frequency and types of limb abnormalities observed among patients with trisomy 18, or Edwards syndrome (ES). Method: The sample consisted of consecutive patients evaluated by a clinical genetics service in the period from 1975 to 2008. The results of the cytogenetic analysis, as well as the clinical data were retrieved from the medical records, with special attention to limb abnormalities findings. All the karyotype analysis was performed at the same laboratory. Results: During the study period, 50 patients were identified, 33 (66%) of them females, with ages at the first evaluation ranging from 1 day to 16 years (median 14 days). The single lineage with free trisomy 18 was the most frequent chromosomal disorder (90%). Mosaicism was observed in 10% of the cases. Clenched fist with overlapping fingers was the predominant anomaly of the upper limbs (70%). Other common disorders included the single palmar crease (42%) and hypoplastic nails (36%). Radial abnormalities were found in 11 patients (22%). As for the lower limbs, hypoplastic nails were the most common abnormality (58%), followed by the rocker bottom foot with prominent calcaneus (50%). One patient had unilateral ectrodactyly as well. Conclusions: Despite the classical description, limb anomalies can be much variable in ES. Some patients may show unusual abnormalities, such as radial defects and ectrodactyly. These findings are extremely important for the clinical suspicion and early identification of patients with ES. [ABSTRACT FROM AUTHOR]

Abstract: The developing limb is one of the best described vertebrate systems for understanding how coordinated gene expression during embryogenesis leads to the structures present in the mature organism. This knowledge, derived from decades of research, is largely based upon gain- and loss-of-function experiments. These studies have provided limited information about how the key signaling pathways interact with each other and the downstream effectors of these pathways. We summarize our current understanding of known genetic interactions in the context of three temporally defined gene regulatory networks. These networks crystallize our current knowledge, depicting a dynamic process involving multiple feedback loops between the ectoderm and mesoderm. At the same time, they highlight the fact that many essential processes are still largely undescribed. Much of the dynamic transcriptional activity occurring during development is regulated by distal cis-regulatory elements. Modern genomic tools have provided new approaches for studying the function of cis-regulatory elements and we discuss the results of these studies in regard to understanding limb development. Ultimately, these genomic techniques will allow scientists to understand how multiple signaling pathways are integrated in space and time to drive gene expression and regulate the formation of the limb. [Copyright &y& Elsevier]

Abstract: Retinoic acid (RA) plays a necessary role in limb development and regeneration, but the precise mechanism by which it acts during these processes is unclear. The role of RA in limb regeneration was first highlighted by the remarkable effect that it has on respecifying the proximodistal axis of the regenerating limb so that serially repeated limbs are produced. To facilitate the study of RA signaling during development and then during regeneration of the same structure we have turned to the axolotl, the master of vertebrate regeneration, and generated transgenic animals that fluorescently report RA signaling in vivo. Characterization of these animals identified an anterior segment of the developing embryo where RA signaling occurs revealing conserved features of the early vertebrate embryo. During limb development RA signaling was present in the developing forelimb bud mesenchyme, but was not detected during hindlimb development. During limb regeneration, RA signaling was surprisingly almost exclusively observed in the apical epithelium suggesting a different role of RA during limb regeneration. After the addition of supplemental RA to regenerating limbs that leads to pattern duplications, the fibroblast stem cells of the blastema responded showing that they are capable of transcriptionally responding to RA. These findings are significant because it means that RA signaling may play a multifunctional role during forelimb development and regeneration and that the fibroblast stem cells that regulate proximodistal limb patterning during regeneration are targets of RA signaling. [Copyright &y& Elsevier]

The developing limb is one of the first systems where it was proposed that a signalling gradient is involved in pattern formation. This gradient for specifying positional information across the antero-posterior axis is based on Sonic hedgehog signalling from the polarizing region. Recent evidence suggests that Sonic hedgehog signalling also specifies positional information across the antero-posterior axis by a timing mechanism acting in parallel with graded signalling. The progress zone model for specifying proximo-distal pattern, involving timing to provide cells with positional information, continues to be challenged, and there is further evidence that graded signalling by retinoic acid specifies the proximal part of the limb. Other recent papers present the first evidence that gradients of signalling by Wnt5a and FGFs govern cell behaviour involved in outgrowth and morphogenesis of the developing limb. [Copyright &y& Elsevier]

Abstract: Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42fl/fl ; Prx1-Cre) mice. Cdc42fl/fl ; Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42fl/fl ; Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development. [Copyright &y& Elsevier]

Although estrogen action is indispensable for normal bone growth in both genders, the roles of estrogen receptors (ERs) in mediating bone growth are not fully understood. The effects of ER inactivation on bone growth are sex and age dependent, and may differ between the axial and appendicular regions. In this study, the spatial and temporal expression of ERα and β in the tibial and spinal growth plates of the female and male rats during postnatal development was examined to explore the possible mechanisms. The level of mRNA was examined and compared with quantitative real-time PCR. The spatial location was determined by immunohistochemical analysis. The 1-, 4-, 7-, 12- and 16-week age stages correspond to early life, puberty and early adulthood after puberty, respectively. Gender- and region-specific differences in ERα and β expression were shown in the growth plates. Mainly nuclear staining of ERα and β immunoreactivity was demonstrated in the spinal and tibial growth plate chondrocytes for both genders. Moreover, our study indicated significant effect of gender on temporal ERα and β expression and of region on temporal ERα/ERβ expression ratio. However, spatial differences of region-related ERα and β expression were not observed. Gender-related spatial changes were detected only at 16 weeks of both spine and limb growth plates. ERα and β immunoreactivity was detected in the resting, proliferative and prehypertrophic chondrocytes in the early life stage and during puberty. After puberty, ERα expression was mainly located in the late proliferative and hypertrophic chondrocytes in female, whereas the expression still extended from the resting to hypertrophic chondrocytes in males. Gender- and region-specific expression patterns of ERα and β gene might be one possible reason for differences in sex- and region-related body growth phenotypes. Gender, age and region differences should be taken into consideration when the roles of ERs in the growth plate are investigated. [ABSTRACT FROM AUTHOR]

The molecular mechanisms underpinning vertebrate body plan evolution are beginning to be unravelled. In this issue of Cell, Kvon et al. spectacularly demonstrate how transplanting snake-specific genetic changes found uniquely in serpent enhancers leads to limb loss in mice. [ABSTRACT FROM AUTHOR]

Canalization and developmental stability refer to the tendency of developmental processes to follow particular trajectories, despite external or internal perturbation. Canalization is the tendency for development of a specific genotype to follow the same trajectory under different conditions (different environments or different genetic backgrounds), while developmental stability is the tendency for the development of a specific genotype to follow the same trajectory under the same conditions. Morphological integration refers to the tendency for structures to show correlated variation because they develop in response to shared developmental processes or function in concert with other structures. All three phenomena are emergent properties of developmental systems that can affect the interaction of development and evolution. In this paper, we review the topics of canalization, developmental stability, and morphological integration and their relevance to primate and human evolution. We then test three developmentally motivated hypotheses about the patterning of variability components in the mammalian limb. We find that environmental variances and fluctuating asymmetries (FA) increase distally along the limb in adult macaques but not in fetal mice. We infer that the greater variability of more distal segments in macaques is due to postnatal mechanical effects. We also find that heritability and FA are significantly correlated when different limb measurements are compared in fetal mice. This supports the idea that the mechanisms underlying canalization and developmental stability are related. Finally, we report that the covariation structure of fore- and hindlimb skeletal elements shows evidence for morphological integration between serially homologous structures between the limbs. This is evidence for the existence of developmental modules that link structures between the limbs. Such modules would produce covariation that would need to be overcome by selection for divergence in hind- and forelimb morphology. Yrbk Phys Anthropol 45:131-158, 2002. © 2002 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]