Prenatal diagnosis of familial 2p15 microduplication associated with pulmonary artery stenosis, single umbilical artery and left foot postaxial polydactyly on fetal ultrasound.

OBJECTIVE: We present prenatal diagnosis of familial 2p15 microduplication associated with pulmonary artery stenosis, single umbilical artery and left foot postaxial polydactyly on fetal ultrasound. CASE REPORT: A 34-year-old woman underwent amniocentesis at 17 weeks of gestation because of advanced maternal age. Amniocentesis revealed the karyotype of 46,XX. Prenatal ultrasound examination at 21 weeks of gestation showed pulmonary artery stenosis, single umbilical artery and left foot postaxial polydactyly. Repeat amniocentesis was performed at 22 weeks of gestation and array comparative genomic hybridization (aCGH) analysis on the DNAs extracted from amniocytes revealed the result of arr 2p15 (61, 495, 220-62,885,679) × 3.0 [GRCh37 (hg19)] with a 1.391-Mb 2p15 duplication encompassing seven Online Mendelian Inheritance in Man (OMIM) genes of USP34, XPO1, FAM161A, CCT4, COMMD1, B3GNT2 and TMEM17. aCGH analysis on the DNAs extracted from parental bloods confirmed a familial transmission from a normal carrier mother who had no phenotypic abnormality. A 3270-g female baby was delivered at term with mild pulmonary artery stenosis and left foot postaxial polydactyly. The infant had normal physical and psychomotor development when follow-up at age of one year. CONCLUSION: Prenatal diagnosis of fetal structural abnormalities should include aCGH analysis in addition to conventional cytogenetic analysis.

Dysregulated BMP signaling through ACVR1 impairs digit joint development in fibrodysplasia ossificans progressiva (FOP).

The development of joints in the mammalian skeleton depends on the precise regulation of multiple interacting signaling pathways including the bone morphogenetic protein (BMP) pathway, a key regulator of joint development, digit patterning, skeletal growth, and chondrogenesis. Mutations in the BMP receptor ACVR1 cause the rare genetic disease fibrodysplasia ossificans progressiva (FOP) in which extensive and progressive extra-skeletal bone forms in soft connective tissues after birth. These mutations, which enhance BMP-pSmad1/5 pathway activity to induce ectopic bone, also affect skeletal development. FOP can be diagnosed at birth by symmetric, characteristic malformations of the great toes (first digits) that are associated with decreased joint mobility, shortened digit length, and absent, fused, and/or malformed phalanges. To elucidate the role of ACVR1-mediated BMP signaling in digit skeletal development, we used an Acvr1R206H/+;Prrx1-Cre knock-in mouse model that mimics the first digit phenotype of human FOP. We have determined that the effects of increased Acvr1-mediated signaling by the Acvr1R206H mutation are not limited to the first digit but alter BMP signaling, Gdf5+ joint progenitor cell localization, and joint development in a manner that differently affects individual digits during embryogenesis. The Acvr1R206H mutation leads to delayed and disrupted joint specification and cleavage in the digits and alters the development of cartilage and endochondral ossification at sites of joint morphogenesis. These findings demonstrate an important role for ACVR1-mediated BMP signaling in the regulation of joint and skeletal formation, show a direct link between failure to restrict BMP signaling in the digit joint interzone and failure of joint cleavage at the presumptive interzone, and implicate impaired, digit-specific joint development as the proximal cause of digit malformation in FOP.

WNT5A-Ca2+-CaN-NFAT signalling plays a permissive role during cartilage differentiation in embryonic chick digit development.

During digit development, the correct balance of chondrogenic signals ensures the recruitment of undifferentiated cells into the cartilage lineage or the maintenance of cells at the undifferentiated stage. WNT/ß catenin maintains the pool of progenitor cells, whereas TGFß signalling promotes cartilage differentiation by inducing Sox9 expression. Moreover, WNT5A promotes the degradation of ß catenin during mouse limb development. Although these mechanisms are well established, it is still unknown whether the signalling pathway downstream WNT5A is also involved in early chondrogenesis during digit formation. Thus, the aim of this study was to determine the role of WNT5A during the recruitment of progenitor cells during digit development. Our results showed that WNT5A activated calcium (Ca2+) release in the undifferentiated region during digit development. Further, the blockade of Ca2+ release or calcineurin (CaN) or nuclear factor of activated T-cells (NFAT) functions resulted in an inhibition of cartilage differentiation. Together, our results demonstrate that non canonical WNT5A-Ca2+-CaN-NFAT signalling plays a key role during embryonic digit development in vivo promoting the competence for chondrogenic signals and also acts as a permissive factor for chondrogenesis independently of cell death mechanisms.

Epithelial Migration and Non-adhesive Periderm Are Required for Digit Separation during Mammalian Development.

The fusion of digits or toes, syndactyly, can be part of complex syndromes, including van der Woude syndrome. A subset of van der Woude cases is caused by dominant-negative mutations in the epithelial transcription factor Grainyhead like-3 (GRHL3), and Grhl3-/-mice have soft-tissue syndactyly. Although impaired interdigital cell death of mesenchymal cells causes syndactyly in multiple genetic mutants, Grhl3-/- embryos had normal interdigital cell death, suggesting alternative mechanisms for syndactyly. We found that in digit separation, the overlying epidermis forms a migrating interdigital epithelial tongue (IET) when the epithelium invaginates to separate the digits. Normally, the non-adhesive surface periderm allows the IET to bifurcate as the digits separate. In contrast, in Grhl3-/- embryos, the IET moves normally between the digits but fails to bifurcate because of abnormal adhesion of the periderm. Our study identifies epidermal developmental processes required for digit separation.

Pacinian corpuscles in the human fetal foot: A study using 3D reconstruction and immunohistochemistry.

PURPOSE: Our group had recently described human hand Pacinian corpuscles (PCs): the hand PCs are not simply arranged along the digital palmar nerves but often exhibited specific morphologies known uncommonly. However, there is still no or few information about human foot PCs. MATERIALS AND METHODS: We observed transverse sections of all five toes including the interdigital area obtained from 12 feet of eight fetuses at 28-33 weeks (crown-rump length 230-290mm). Serial sections were prepared for 3D reconstructions and measurement. RESULTS: Foot PCs were characterized by (1) a dense distribution in the interdigital area in contrast to a few PCs in the distal tip of the all five toes; (2) abundant dorsal PCs including those in the nail bed and: (3) a long chain of PCs in the flexor tendon sheath of all five toes. Therefore, a distal dominance was not evident in the foot in contrast to the hand and, a tendon sheath contained much greater numbers of PCs than the hand. A tree-like or bouquet-like arrangement of PCs along a short perforating artery to the palmar digital skin was seen in the foot as we had described in the hand. The tree of foot PCs was sometimes seen laying transversely along the digital skin surface, not toward the skin. CONCLUSION: It is still unknown that, in utero, how the PCs distribution became different between the hand and foot: it might be determined genetically in a region-specific manner.

Cooperation of BMP and IHH signaling in interdigital cell fate determination.

The elaborate anatomy of hands and feet is shaped by coordinated formation of digits and regression of the interdigital mesenchyme (IM). A failure of this process causes persistence of interdigital webbing and consequently cutaneous syndactyly. Bone morphogenetic proteins (BMPs) are key inductive factors for interdigital cell death (ICD) in vivo. NOGGIN (NOG) is a major BMP antagonist that can interfere with BMP-induced ICD when applied exogenously, but its in vivo role in this process is unknown. We investigated the physiological role of NOG in ICD and found that Noggin null mice display cutaneous syndactyly and impaired interdigital mesenchyme specification. Failure of webbing regression was caused by lack of cell cycle exit and interdigital apoptosis. Unexpectedly, Noggin null mutants also exhibit increased Indian hedgehog (Ihh) expression within cartilage condensations that leads to aberrant extension of IHH downstream signaling into the interdigital mesenchyme. A converse phenotype with increased apoptosis and reduced cell proliferation was found in the interdigital mesenchyme of Ihh mutant embryos. Our data point towards a novel role for NOG in balancing Ihh expression in the digits impinging on digit-interdigit cross talk. This suggests a so far unrecognized physiological role for IHH in interdigital webbing biology.

Interdigital tissue remodelling in the embryonic limb involves dynamic regulation of the miRNA profiles.

Next-generation sequencing in combination with quantitative polymerase chain reaction analysis revealed a dynamic miRNA signature in the interdigital mesoderm of the chick embryonic hinlimb in the course of interdigit remodelling. During this period, 612 previously known chicken miRNAs (gga-miRNAs) and 401 non-identified sequences were expressed in the interdigital mesoderm. Thirty-six microRNAs, represented by more than 750 reads per million, displayed differential expression between stages HH29 (6 id) and HH32 (7.5 id), which correspond to the onset and the peak of interdigital cell death. Twenty miRNAs were upregulated by at least 1.5-fold, and sixteen were downregulated by at least 0.5-fold. Upregulated miRNAs included miRNAs with recognized proapoptotic functions in other systems (miR-181 family, miR-451 and miR-148a), miRNAs associated with inflammation and cell senescence (miR-21 and miR-146) and miRNAs able to induce changes in the extracellular matrix (miR-30c). In contrast, miRNAs with known antiapoptotic effects in other systems, such as miR-222 and miR-205, became downregulated. In addition, miR-92, an important positive regulator of cell proliferation, was also downregulated. Together, these findings indicate a role for miRNAs in the control of tissue regression and cell death in a characteristic morphogenetic embryonic process based on massive apoptosis.

The origins, scaling and loss of tetrapod digits.

Many of the great morphologists of the nineteenth century marvelled at similarities between the limbs of diverse species, and Charles Darwin noted these homologies as significant supporting evidence for descent with modification from a common ancestor. Sir Richard Owen also took great care to highlight each of the elements of the forelimb and hindlimb in a multitude of species with focused attention on the homology between the hoof of the horse and the middle digit of man. The ensuing decades brought about a convergence of palaeontology, experimental embryology and molecular biology to lend further support to the homologies of tetrapod limbs and their developmental origins. However, for all that we now understand about the conserved mechanisms of limb development and the development of gross morphological disturbances, little of what is presented in the experimental or medical literature reflects the remarkable diversity resulting from the 450 million year experiment of natural selection. An understanding of conserved and divergent limb morphologies in this new age of genomics and genome engineering promises to reveal more of the developmental potential residing in all limbs and to unravel the mechanisms of evolutionary variation in limb size and shape. In this review, we present the current state of our rapidly advancing understanding of the evolutionary origin of hands and feet and highlight what is known about the mechanisms that shape diverse limbs.This article is part of the themed issue 'Evo-devo in the genomics era, and the origins of morphological diversity'.

Side-specific effect of yolk testosterone elevation on second-to-fourth digit ratio in a wild passerine.

Second-to-fourth digit ratio is a widely investigated sexually dimorphic morphological trait in human studies and could reliably indicate the prenatal steroid environment. Conducting manipulative experiments to test this hypothesis comes up against ethical limits in humans. However, oviparous tetrapods may be excellent models to experimentally investigate the effects of prenatal steroids on offspring second-to-fourth digit ratio. In this field study, we injected collared flycatcher (Ficedula albicollis) eggs with physiological doses of testosterone. Fledglings from eggs with elevated yolk testosterone, regardless of their sex, had longer second digits on their left feet than controls, while the fourth digit did not differ between groups. Therefore, second-to-fourth digit ratio was higher in the testosterone-injected group, but only on the left foot. This is the first study which shows experimentally that early testosterone exposure can affect second-to-fourth digit ratio in a wild population of a passerine bird.

The tumor suppressor BTG1 is expressed in the developing digits and regulates skeletogenic differentiation of limb mesodermal progenitors in high density cultures.

In the developing limb, differentiation of skeletal progenitors towards distinct connective tissues of the digits is correlated with the establishment of well-defined domains of Btg1 gene expression. Zones of high expression of Btg1 include the earliest digit blastemas, the condensing mesoderm at the tip of the growing digits, the peritendinous mesenchyme, and the chondrocytes around the developing interphalangeal joints. Gain- and loss-of function experiments in micromass cultures of skeletal progenitors reveal a negative influence of Btg1 in cartilage differentiation accompanied by up-regulation of Ccn1, Scleraxis and PTHrP. Previous studies have assigned a role to these factors in the aggregation of progenitors in the digit tips (Ccn1), in the differentiation of tendon blastemas (Scleraxis) and repressing hypertrophic cartilage differentiation (PTHrP). Overexpression of Btg1 up-regulates the expression of retinoic acid and thyroid hormone receptors, but, different from other systems, the influence of BTG1 in connective tissue differentiation appears to be independent of retinoic acid and thyroid hormone signaling.

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.

Shared rules of development predict patterns of evolution in vertebrate segmentation.

Phenotypic diversity is not uniformly distributed, but how biased patterns of evolutionary variation are generated and whether common developmental mechanisms are responsible remains debatable. High-level 'rules' of self-organization and assembly are increasingly used to model organismal development, even when the underlying cellular or molecular players are unknown. One such rule, the inhibitory cascade, predicts that proportions of segmental series derive from the relative strengths of activating and inhibitory interactions acting on both local and global scales. Here we show that this developmental design rule explains population-level variation in segment proportions, their response to artificial selection and experimental blockade of putative signals and macroevolutionary diversity in limbs, digits and somites. Together with evidence from teeth, these results indicate that segmentation across independent developmental modules shares a common regulatory 'logic', which has a predictable impact on both their short and long-term evolvability.

Formation of proximal and anterior limb skeleton requires early function of Irx3 and Irx5 and is negatively regulated by Shh signaling.

Limb skeletal pattern relies heavily on graded Sonic hedgehog (Shh) signaling. As a morphogen and growth cue, Shh regulates identities of posterior limb elements, including the ulna/fibula and digits 2 through 5. In contrast, proximal and anterior structures, including the humerus/femur, radius/tibia, and digit 1, are regarded as Shh independent, and mechanisms governing their specification are unclear. Here, we show that patterning of the proximal and anterior limb skeleton involves two phases. Irx3 and Irx5 (Irx3/5) are essential in the initiating limb bud to specify progenitors of the femur, tibia, and digit 1. However, these skeletal elements can be restored in Irx3/5 null mice when Shh signaling is diminished, indicating that Shh negatively regulates their formation after initiation. Our data provide genetic evidence supporting the concept of early specification and progressive determination of anterior limb pattern.

Digit evolution in gymnophthalmid lizards.

The tetrapod limb is a highly diverse structure, and reduction or loss of this structure accounts for many of the limb phenotypes observed within species. Squamate reptiles are one of the many tetrapod lineages in which the limbs have been greatly modified from the pentadactyl generalized pattern, including different degrees of reduction in the number of limb elements to complete limblessness. Even though limb reduction is widespread, the evolutionary and developmental mechanisms involved in the formation of reduced limb morphologies remains unclear. In this study, we present an overview of limb morphology within the microteiid lizard group Gymnophthalmidae, focusing on digit arrangement. We show that there are two major groups of limb-reduced gymnophthalmids. The first group is formed by lizard-like (and frequently pentadactyl) species, in which minor reductions (such as the loss of 1-2 phalanges mainly in digits I and V) are the rule; these morphologies generally correspond to those seen in other squamates. The second group is formed by species showing more drastic losses, which can include the absence of an externally distinct limb in adults. We also present the expression patterns of Sonic Hedgehog (Shh) in the greatly reduced fore and hindlimb of a serpentiform gymnophthalmid. Our discussion focuses on identifying shared patterns of limb reduction among tetrapods, and explaining these patterns and the morphological variation within the gymnophthalmids based on current knowledge of the molecular signaling pathways that coordinate limb development.

Slow stretching that mimics embryonic growth rate stimulates structural and mechanical development of tendon-like tissue in vitro.

A distinctive feature of embryonic tendon development is the steady increase in collagen fibril diameter and associated improvement of tissue mechanical properties. A potential mechanical stimulus for these changes is slow stretching of the tendon during limb growth. Testing this hypothesis in vivo is complicated by the presence of other developmental processes including muscle development and innervation. Here we used a cell culture tendon-like construct to determine if slow stretch can explain the increases in fibril diameter and mechanical properties that are observed in vivo. Non-stretched constructs had an ultrastructural appearance and mechanical properties similar to those of early embryonic tendon. However, slowly stretching during 4 days in culture increased collagen fibril diameter, fibril packing volume, and mechanical stiffness, and thereby mimicked embryonic development. 3D EM showed cells with improved longitudinal alignment and elongated nuclei, which raises the hypothesis that nuclear deformation could be a novel mechanism during tendon development.

Embryological evidence identifies wing digits in birds as digits 1, 2, and 3.

The identities of the digits of the avian forelimb are disputed. Whereas paleontological findings support the position that the digits correspond to digits one, two, and three, embryological evidence points to digit two, three, and four identities. By using transplantation and cell-labeling experiments, we found that the posteriormost digit in the wing does not correspond to digit four in the hindlimb; its progenitor segregates early from the zone of polarizing activity, placing it in the domain of digit three specification. We suggest that an avian-specific shift uncouples the digit anlagen from the molecular mechanisms that pattern them, resulting in the imposition of digit one, two, and three identities on the second, third, and fourth anlagens.

The fused toes locus is essential for somatic-germ cell interactions that foster germ cell maturation in developing gonads in mice.

Ovarian development absolutely depends on communication between somatic and germ cell components. In contrast, it is not until after birth that interactions between somatic and germ cells play an important role in testicular maturation and spermatogenesis. Previously, we discovered that Irx3 expression was localized specifically to female gonads during embryonic development; therefore, we sought to determine the function of this genetic locus in developing gonads of both sexes. The fused toes (Ft) mutant mouse is missing 1.6 Mb of chromosome 8, which includes the entire IrxB cluster (Irx3, Irx5, Irx6), Ftm, Fts, and Fto genes. Homozygote Ft mutant embryos die around embryonic day 13.5 (E13.5); therefore, to assess later development, we harvested gonads at E11.5 and transplanted them into nude mouse hosts. Our results show defects in somatic and germ cell maturation in developing gonads of both sexes. Testis development was normal initially; however, by 3-wk posttransplantation, expression of Sertoli and peritubular myoid cell markers were decreased. In many cases, gonocytes failed to migrate to structurally impaired basement membranes of seminiferous cords. Developmental abnormalities of the ovary appeared earlier and were more severe. Over time, the Ft mutant ovary formed very few primordial or primary follicles, which contained oocytes that failed to grow and were surrounded by scarce granulosa cells that expressed low levels of FOXL2. By 3 wk after transplantation, it was difficult to identify ovarian tissue in Ft mutant ovary transplants. In summary, we conclude that the Ft locus contains genes essential for somatic-germ cell interactions, without which the germ cell niche fails to mature in both sexes.

Relationships of toe-length ratios to finger-length ratios, foot preference, and wearing of toe rings.

Sexually differentiated digit ratios of the hand (2D:4D and other) are currently widely studied, owing to their presumed role as a retrospective diagnostic window into prenatal androgen action. This study was only the second one (following McFadden & Shubel, 2002) to examine all 6 possible finger-length ratios (excluding the thumb) and all 10 possible toe-length ratios (including the big toe). Data from a sample of 59 male and 69 female Austrian adults (M age = 27 yr., SD = 7.9) were collected. Replicating the prior study, the majority of toe-length ratios exhibited significant sex differences, which was similar to finger-length ratios, but of weaker effect size. Ipsilateral correlations of toe-length and finger-length ratios were largely absent, except for those of corresponding or adjacent length ratios of the right body side among women. However, these associations were directionally opposite, such that among women, feminized finger-length ratios corresponded to masculinized toe-length ratios. Foot preference (among both sexes) and wearing of toe rings (among women) were not correlates of toe-length ratios. Discussed are implications of these findings for digit ratio research, along with ideas for further inquiry on this topic.

A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning.

Embryogenesis depends on self-regulatory interactions between spatially separated signaling centers, but few of these are well understood. Limb development is regulated by epithelial-mesenchymal (e-m) feedback loops between sonic hedgehog (SHH) and fibroblast growth factor (FGF) signaling involving the bone morphogenetic protein (BMP) antagonist Gremlin1 (GREM1). By combining mouse molecular genetics with mathematical modeling, we showed that BMP4 first initiates and SHH then propagates e-m feedback signaling through differential transcriptional regulation of Grem1 to control digit specification. This switch occurs by linking a fast BMP4/GREM1 module to the slower SHH/GREM1/FGF e-m feedback loop. This self-regulatory signaling network results in robust regulation of distal limb development that is able to compensate for variations by interconnectivity among the three signaling pathways.

Examination of the accessory tendons of extensor hallucis longus muscle in fetuses.

There are various data about the incidence of accessory tendons (AT) of extensor hallucis longus (EHL) muscle; however, their function is unknown. This study aimed to determine the incidence and morphometric features of the AT of EHL muscle in fetuses in order to provide more information to discuss its possible function. Forty-five fetuses (26 female and 19 male) were used in this study. Fetuses were grouped as Group A (16-21 weeks), Group B (22-27 weeks), and Group C (28-34 weeks) according to their age. In 23 (51%) out of 45 fetuses, there were AT. These were bilateral in 15 fetuses (65%) and unilateral in eight fetuses (35%). Fifty-two percent of the fetuses in group A, 43% in group B, and 67% in group C had AT. AT were observed in 14 female (54%) and 9 male (47%) fetuses. In all cases, the AT were always diverging to the medial side of the main EHL tendons and attached to the metatarsophalangeal joint capsule distal to the joint space. Significant correlations were observed in this study between EHL and AT widths as well as between EHL width and EHL-AT distance on both sides. The present study is the first to provide morphometric data about the AT of EHL muscle in fetuses which will be of use in understanding their function, particularly in biomechanics of the great toe.