The spatiotemporal expression patterns of MSC-associated markers contribute to the identification of progenitor subpopulations in developing limbs.

During limb development, skeletal tissues differentiate from their progenitor cells in an orchestrated manner. Mesenchymal stromal cells (MSCs), which are considered to be adult undifferentiated/progenitor cells, have traditionally been identified by the expression of MSC-associated markers (MSC-am) and their differentiation capacities. However, although MSCs have been isolated from bone marrow and a variety of adult tissues, their developmental origin is poorly understood. Remarkably, adult MSCs share similar differentiation characteristics with limb progenitors. Here, we determined the expression patterns of common MSC-am throughout mouse hindlimb development. Our results demonstrate that MSC-am expression is not restricted to undifferentiated cells in vivo. Results from the analysis of MSC-am spatiotemporal expression in the embryonic hindlimb allowed us to propose five subpopulations which represent all limb tissues that potentially correspond to progenitor cells for each lineage. This work contributes to the understanding of MSC-am expression dynamics throughout development and underlines the importance of considering their expression patterns in future MSC studies of the limb.

Feasibility of a fetal anatomy 3D atlas by computer-assisted anatomic dissection.

OBJECTIVE: To assess the feasibility of 3D modelisation of fetal anatomy by using the Computer-assisted anatomic dissection (CAAD) based on immunolabeled histologic slices and MRI slices with a specific 3D software. STUDY DESIGN: For pelvis and lower limbs, subjects came from legal abortion, medical pregnancy termination, or late miscarriage. Specimens were fixed in 10 % formalin, then embedded in paraffin wax and serially sectioned. The histological slices were stained using HES and Masson Trichrome. Protein S-100 and D2-40 markers were used for immuno-labelling. Serial transverse sections were digitalized and manually aligned. Fetal brain slices were obtained from in utero or post-mortem MRI. RESULTS: CAAD was performed on 10 fetuses: pelvis was modelised with 3 fetuses of 13, 15 and 24 W G, lower limbs with 2 fetuses of 14 and 15 W G and brain with 5 fetuses aged between 19 and 37 W G. Fetal pelvis innervation was analysed after immunolabelling and nerves appeared proportionally bigger than in adults with the same topography. Lower limbs analysis revealed that nerve development was guided by vascular development: the sciatic nerve along the big axial vein, the saphen nerve along the big saphen vein and the sural nerve along the small saphen vein. Fetal brain study allowed to describe the gyration process and the lateral ventricle development. CONCLUSION: CAAD technique provides an accurate 3D reconstruction of fetal anatomy for lower limbs and pelvis but has to be improved for brain model since midline structures were not amendable for analysis. These results need to be confirmed with larger series of specimens at different stages of development.

Development of human limb muscles based on whole-mount immunostaining and the links between ontogeny and evolution.

We provide the first detailed ontogenetic analysis of human limb muscles using whole-mount immunostaining. We compare our observations with the few earlier studies that have focused on the development of these muscles, and with data available on limb evolution, variations and pathologies. Our study confirms the transient presence of several atavistic muscles - present in our ancestors but normally absent from the adult human - during normal embryonic human development, and reveals the existence of others not previously described in human embryos. These atavistic muscles are found both as rare variations in the adult population and as anomalies in human congenital malformations, reinforcing the idea that such variations/anomalies can be related to delayed or arrested development. We further show that there is a striking difference in the developmental order of muscle appearance in the upper versus lower limbs, reinforcing the idea that the similarity between various distal upper versus lower limb muscles of tetrapod adults may be derived.

Osteomyelitis in limb amputated by amniotic band sequence.

A preterm (30+2 week) neonate with below-knee amputation (right lower limb), constriction rings and syndactyly, subsequent to amniotic band sequence, developed pus discharge from the right tibial stump. The neonate did not have clinical features of systemic sepsis. Blood culture was sterile. The pus culture, however, grew methicillin-resistant coagulase-negative Staphylococcus and bone scan was suggestive of osteomyelitis of right proximal tibial stump. Osteomyelitis was likely caused by the contiguous spread of infection from the exposed stump. Neonate was treated with intravenous antibiotics for 4 weeks and discharged on oral feeds.

Apical and Basal Matrix Remodeling Control Epithelial Morphogenesis.

Epithelial tissues can elongate in two dimensions by polarized cell intercalation, oriented cell division, or cell shape change, owing to local or global actomyosin contractile forces acting in the plane of the tissue. In addition, epithelia can undergo morphogenetic change in three dimensions. We show that elongation of the wings and legs of Drosophila involves a columnar-to-cuboidal cell shape change that reduces cell height and expands cell width. Remodeling of the apical extracellular matrix by the Stubble protease and basal matrix by MMP1/2 proteases induces wing and leg elongation. Matrix remodeling does not occur in the haltere, a limb that fails to elongate. Limb elongation is made anisotropic by planar polarized Myosin-II, which drives convergent extension along the proximal-distal axis. Subsequently, Myosin-II relocalizes to lateral membranes to accelerate columnar-to-cuboidal transition and isotropic tissue expansion. Thus, matrix remodeling induces dynamic changes in actomyosin contractility to drive epithelial morphogenesis in three dimensions.

Prenatal ultrasound evaluation of segmental level of neurological lesion in fetuses with myelomeningocele: development of a new technique.

OBJECTIVES: To report our preliminary experience in the use of prenatal ultrasound examination to assess lower-limb movements in fetuses with myelomeningocele. We aimed to determine the accuracy of this method to establish the segmental level of neurological lesion, as this is the best known predictor of the future ability to walk. METHODS: This was a preliminary, observational study including fetuses with myelomeningocele operated on prenatally. The patterns of movements present and absent in the affected fetuses' lower limbs were evaluated systematically by ultrasound examination. According to the known nerve function associated with each muscle group, the segmental level of neurological lesion was established before birth. The agreement for the segmental levels assigned, between the prenatal ultrasound technique and the classical neurological clinical examination after birth (gold standard), was tested using the weighed kappa (wκ) index. RESULTS: Seventy-one fetuses with myelomeningocele were evaluated at the Hospital Universitari Vall d'Hebron. After counseling, the parents opted for prenatal surgery (26 cases), termination of pregnancy (43 cases) or postnatal repair (two cases). Five patients did not fulfil the inclusion criteria for prenatal surgery and three were excluded after birth. In the 18 fetuses that underwent surgery and were analyzed, the agreement between prenatal and postnatal segmental levels assigned was 91.7% for the right limb (wκ = 0.80) and 88.9% for the left limb (wκ = 0.73). CONCLUSIONS: The agreement found between prenatal and postnatal assignment of level of lesion in this preliminary study suggests that neurological sonographic evaluation is feasible before birth. This may provide accurate individualized information about the motor function and future ambulation prognosis of fetuses with myelomeningocele.

Developmental stages of the climbing gecko Tarentola annularis with special reference to the claws, pad lamellae, and subdigital setae.

Studying the in ovo mode of development of squamates has the advantage of allowing easy access to embryos without surgically compromising gravid females. Despite the non-ophidian squamates being a very diverse lineage of reptiles, embryonic tables for individuals of this group are very few. Here, I present the first in ovo embryonic table for a basal multi-scansored, pad-bearing gecko, Tarentola annularis. In this gecko, only the III and IV digits bear claws. Eleven embryonic stages are described based on chronological development of morphological characteristics. In contrast to other previously studied geckos, this species exhibits a longer incubation period. Comparison with other squamates, embryonic development of T. annularis is an indicative of a conserved developmental strategy. Interestingly, the clawless digits of this gecko do exhibit claws during the first half of embryonic development. Thus, regression of claws in these digits could be an advantage of studying this particular taxon, as it raises the question, to be answered in future study, of which mechanisms could be responsible for such claw regression. Before hatching, the outer periderm layer sloughs revealing the functional setae. The present study provides not only a model for pentadactyl limbs and digit development, but also an example of a unique developmental phenomenon, as represented by claw regression.

Knee joint morphogenesis of the quail (Coturnix japonica) embryo.

Knee joint development and its morphogenetic events have been studied in human, chicken and other animal models and differences have been found in the pattern of the knee joint morphogenesis among the studied species. According to the small number of studies which have focused on the chronology of knee morphogenesis, a "morphogenetic timely pattern" is hard to suggest. Quail is an animal model for which there is no information about knee joint morphogenesis. This study was planned to define the time table of the knee joint structures formation in this bird. For this purpose embryonated Japanese quail eggs were incubated for 3 to 12 days. Embryos were removed from their eggs every twelve hours and staged according to Ainsworth et al. The hind limbs of the embryos at the stages 17 to 41 were dissected and 6 µm thick slides were prepared from their knee region. The time of appearance of menisci, ligaments, articular cavity and other knee joint components were identified in the quail embryo. During quail knee morphogenesis we observed the appearance of a three layered interzone, femorotibial cavitation and long bone ossification earlier than in chicken. A hypothesis is presented on the differential role of the flexor and extensor muscles of the knee joint on embryonic knee development in birds as compared with humans.

When right differs from left: human limb directional asymmetry emerges during very early development.

The often observed directional asymmetry (DA) in human limb bones may have a genetic/developmental basis and/or could emerge from different mechanical loadings across sides due to handedness. Because behavioural lateralization in itself has a genetic basis, it has been suggested that DA in limbs could develop prenatally as a pre-adaptation to adult life. However, the presence of consistent differences in the size of left and right limb bones in early development is understudied. We study asymmetry in limb bones during early development (10-20 weeks of gestation) in a sample of 178 aborted foetuses. Statistically significant DA was found in several upper and lower limb bones, where the right-hand side was consistently larger than the left. We argue that this pattern is probably the consequence of developmental processes related to internal asymmetric positioning of organs.

Sirenomelia phenotype in bmp7;shh compound mutants: a novel experimental model for studies of caudal body malformations.

Sirenomelia is a severe congenital malformation of the lower body characterized by the fusion of the legs into a single lower limb. This striking external phenotype consistently associates severe visceral abnormalities, most commonly of the kidneys, intestine, and genitalia that generally make the condition lethal. Although the causes of sirenomelia remain unknown, clinical studies have yielded two major hypotheses: i) a primary defect in the generation of caudal mesoderm, ii) a primary vascular defect that leaves the caudal part of the embryo hypoperfused. Interestingly, Sirenomelia has been shown to have a genetic basis in mice, and although it has been considered a sporadic condition in humans, recently some possible familial cases have been reported. Here, we report that the removal of one or both functional alleles of Shh from the Bmp7-null background leads to a sirenomelia phenotype that faithfully replicates the constellation of external and internal malformations, typical of the human condition. These mutants represent an invaluable model in which we have analyzed the pathogenesis of sirenomelia. We show that the signaling defect predominantly impacts the morphogenesis of the hindgut and the development of the caudal end of the dorsal aortas. The deficient formation of ventral midline structures, including the interlimb mesoderm caudal to the umbilicus, leads to the approximation and merging of the hindlimb fields. Our study provides new insights for the understanding of the mechanisms resulting in caudal body malformations, including sirenomelia.

Wls-mediated Wnts differentially regulate distal limb patterning and tissue morphogenesis.

Wnt proteins are diffusible morphogens that play multiple roles during vertebrate limb development. However, the complexity of Wnt signaling cascades and their overlapping expression prevent us from dissecting their function in limb patterning and tissue morphogenesis. Depletion of the Wntless (Wls) gene, which is required for the secretion of various Wnts, makes it possible to genetically dissect the overall effect of Wnts in limb development. In this study, the Wls gene was conditionally depleted in limb mesenchyme and ectoderm. The loss of mesenchymal Wls prevented the differentiation of distal mesenchyme and arrested limb outgrowth, most likely by affecting Wnt5a function. Meanwhile, the deletion of ectodermal Wls resulted in agenesis of distal limb tissue and premature regression of the distal mesenchyme. These observations suggested that Wnts from the two germ layers differentially regulate the pool of undifferentiated distal limb mesenchyme cells. Cellular behavior analysis revealed that ectodermal Wnts sustain mesenchymal cell proliferation and survival in a manner distinct from Fgf. Ectodermal Wnts were also shown for the first time to be essential for distal tendon/ligament induction, myoblast migration and dermis formation in the limb. These findings provide a comprehensive view of the role of Wnts in limb patterning and tissue morphogenesis.

Deletions in PITX1 cause a spectrum of lower-limb malformations including mirror-image polydactyly.

PITX1 is a bicoid-related homeodomain transcription factor implicated in vertebrate hindlimb development. Recently, mutations in PITX1 have been associated with autosomal-dominant clubfoot. In addition, one affected individual showed a polydactyly and right-sided tibial hemimelia. We now report on PITX1 deletions in two fetuses with a high-degree polydactyly, that is, mirror-image polydactyly. Analysis of DNA from additional individuals with isolated lower-limb malformations and higher-degree polydactyly identified a third individual with long-bone deficiency and preaxial polydactyly harboring a heterozygous 35 bp deletion in PITX1. The findings demonstrate that mutations in PITX1 can cause a broad spectrum of isolated lower-limb malformations including clubfoot, deficiency of long bones, and mirror-image polydactyly.

[Assessment and management of rotation and angulation of lower limbs in children].

Most rotational and angulational variations in young children are benign and resolved spontaneously. However, they may cause great concerns to parents. In order to provide a deliberate assessment and management of lower limb rotational and angulational problems, it is necessary for pediatricians to understand the normal variations of lower limb development in healthy children combined with the features of child growth and development and detail physical examinations.

MicroRNA-142-3p regulates TGF-ß3-mediated region-dependent chondrogenesis by regulating ADAM9.

Position-dependent chondrogenesis is regulated by processes that are both common to and differ among all limb types and limb skeletal elements. Despite intrinsic differences between wing and leg bud mesenchyme, the exact regulatory molecules and mechanisms involved in these processes have not been elucidated. Here, we show the limb type-specific role of TGF-ß3 during chondrogenic differentiation of chick limb mesenchymal cells. Exposure of wing cells to TGF-ß3 stimulated chondrogenic differentiation, whereas in leg bud mesenchymal cells, TGF-ß3 induced apoptotic cell death via G2M arrest. Consistent with a limb type-specific effect of TGF-ß3 on chondrogenic differentiation, we found different levels of miR-142-3p induction. Inhibition of miR-142-3p via PNA-based antisense oligonucleotides (ASOs) markedly promoted cell migration and precartilage condensation, while exogenous induction of miR-142-3p reduced cell survival and increased cell death. Overexpression of ADAM9 significantly reduced chondrogenic differentiation via downregulation of cell migration and cell survival and upregulation of apoptotic cell death. Limb type-specific expression levels of ADAM9 induced by TGF-ß3 were observed. Collectively, this study demonstrates that differential induction of miR-142-3p is involved in the limb type-specific effect of TGF-ß3 on wing vs. leg mesenchymal cells through direct modulation of ADAM9 transcription.

A context-dependent combination of Wnt receptors controls axis elongation and leg development in a short germ insect.

Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. We analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). We show that simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. We identify Tc-Arr as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation. We discuss how Wnt signalling is regulated at the receptor and co-receptor levels in a dose-dependent fashion.

Cross- and triple-ratios of human body parts during development.

Recently developed landmark-based geometric morphometry has been used to depict the morphological development of organisms. In geometry, four landmarks can be mapped to any other four by Möbius transformations, if the cross-ratio of the landmarks is invariant and vice versa. To geometrically analyze the morphological development of the human body, we examined the cross-ratio of three consecutive body parts that are segmented by four landmarks in their configuration. Moreover, we introduced the triple-ratio of five landmarks that segments four consecutive parts (e.g., the shoulder, upper arm, forearm, and hand) and examined their growth patterns. The cross- and triple-ratios of the upper limb and shoulder girdle in fetuses were constant when biomechanical landmarks were used, although the cross-ratio of the upper limb varied when anatomical landmarks were used. The cross-ratios of the lower limbs, trunk, and pelvic girdles in fetuses differed from their corresponding cross-ratios in adults. These results suggest Möbius growth in the fetal upper limb and shoulder girdle but not in the other body parts examined. However, the growth balance of the three contiguous body parts was represented by the developmental change in the cross-ratio. Therefore, the cross- and triple-ratios may be applicable for simple but significant assessments of growth balance or proportion of the body parts.

Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development.

The radically distinct morphologies of arthropod and tetrapod legs argue that these appendages do not share a common evolutionary origin. Yet, despite dramatic differences in morphology, it has been known for some time that transcription factors encoded by the Distalless (Dll)/Dlx gene family play a critical role in the development of both structures. Here we show that a second transcription factor family encoded by the Sp8 gene family, previously implicated in vertebrate limb development, also plays an early and fundamental role in arthropod leg development. By simultaneously removing the function of two Sp8 orthologs, buttonhead (btd) and Sp1, during Drosophila embryogenesis, we find that adult leg development is completely abolished. Remarkably, in the absence of these factors, transformations from ventral to dorsal appendage identities are observed, suggesting that adult dorsal fates become derepressed when ventral fates are eliminated. Further, we show that Sp1 plays a much more important role in ventral appendage specification than btd and that Sp1 lies genetically upstream of Dll. In addition to these selector-like gene functions, Sp1 and btd are also required during larval stages for the growth of the leg. Vertebrate Sp8 can rescue many of the functions of the Drosophila genes, arguing that these activities have been conserved, despite more than 500 million years of independent evolution. These observations suggest that an ancient Sp8/Dlx gene cassette was used in an early metazoan for primitive limb-like outgrowths and that this cassette was co-opted multiple times for appendage formation in multiple animal phyla.

Sonographic identification of lower limb venous hypoplasia in the prenatal diagnosis of Klippel-Trénaunay syndrome.

We report the prenatal identification of lower-limb venous hypoplasia to support a provisional prenatal diagnosis of Klippel-Trénaunay syndrome (KTS). Ultrasound assessment of a fetus with marked lower-limb edema, cystic areas in the abdomen/pelvis/lower limbs and abnormal development of the feet demonstrated bilateral hypoplasia of the femoral and popliteal veins. The external iliac veins and the great saphenous veins were seen to be normal. The lower limb arterial system was present. These findings supported KTS as the most likely provisional diagnosis, and postnatal clinical evaluation confirmed that the infant is best classified in the spectrum of KTS. Venous hypoplasia was confirmed with a postnatal ultrasound examination of the lower limbs. This case suggests that careful examination of the lower-limb venous system may be helpful in making the prenatal diagnosis of KTS.

Dendritic targeting in the leg neuropil of Drosophila: the role of midline signalling molecules in generating a myotopic map.

Neural maps are emergent, highly ordered structures that are essential for organizing and presenting synaptic information. Within the embryonic nervous system of Drosophila motoneuron dendrites are organized topographically as a myotopic map that reflects their pattern of innervation in the muscle field. Here we reveal that this fundamental organizational principle exists in adult Drosophila, where the dendrites of leg motoneurons also generate a myotopic map. A single postembryonic neuroblast sequentially generates different leg motoneuron subtypes, starting with those innervating proximal targets and medial neuropil regions and producing progeny that innervate distal muscle targets and lateral neuropil later in the lineage. Thus the cellular distinctions in peripheral targets and central dendritic domains, which make up the myotopic map, are linked to the birth-order of these motoneurons. Our developmental analysis of dendrite growth reveals that this myotopic map is generated by targeting. We demonstrate that the medio-lateral positioning of motoneuron dendrites in the leg neuropil is controlled by the midline signalling systems Slit-Robo and Netrin-Fra. These results reveal that dendritic targeting plays a major role in the formation of myotopic maps and suggests that the coordinate spatial control of both pre- and postsynaptic elements by global neuropilar signals may be an important mechanism for establishing the specificity of synaptic connections.

Essential roles for lines in mediating leg and antennal proximodistal patterning and generating a stable Notch signaling interface at segment borders.

The Drosophila leg imaginal disc provides a paradigm with which to understand the fundamental developmental mechanisms that generate an intricate appendage structure. Leg formation depends on the subdivision of the leg proximodistal (PD) axis into broad domains by the leg gap genes. The leg gap genes act combinatorially to initiate the expression of the Notch ligands Delta (Dl) and Serrate (Ser) in a segmental pattern. Dl and Ser induce the expression of a set of transcriptional regulators along the segment border, which mediate leg segment growth and joint morphogenesis. Here we show that Lines accumulates in nuclei in the presumptive tarsus and the inter-joints of proximal leg segments and governs the formation of these structures by destabilizing the nuclear protein Bowl. Across the presumptive tarsus, lines modulates the opposing expression landscapes of the leg gap gene dachshund (dac) and the tarsal PD genes, bric-a-brac 2 (bab), apterous (ap) and BarH1 (Bar). In this manner, lines inhibits proximal tarsal fates and promotes medial and distal tarsal fates. Across proximal leg segments, lines antagonizes bowl to promote Dl expression by relief-of-repression. In turn, Dl signals asymmetrically to stabilize Bowl in adjacent distal cells. Bowl, then, acts cell-autonomously, together with one or more redundant factors, to repress Dl expression. Together, lines and bowl act as a binary switch to generate a stable Notch signaling interface between Dl-expressing cells and adjacent distal cell. lines plays analogous roles in developing antennae, which are serially homologous to legs, suggesting evolutionarily conserved roles for lines in ventral appendage formation.