The development of the medial longitudinal arch in the intrauterine period.

In this study, we aimed to reveal whether the medial longitudinal arch is formed in the intrauterine period and the structural features of the medial longitudinal arch. The study was conducted on 146 feet of 73 fetuses (38 male, 35 female) aged between 15 and 40 weeks of gestation. The fetuses were grouped by trimesters. The footprints taken were photographed with a millimeter ruler, and the development of the medial longitudinal arch was examined on footprints based on the Clarke index, Chipaux-Smirak index, and Staheli index. In Clarke index and Staheli index, it was observed that the arch height was normalized in the transition from the second trimester to the third trimester, the arch decreased in the transition to full-term, and the rate of pes planus increased. All indices detected pes planus by 81.81% in the full-term period. The rate of pes planus determined according to Clarke index and Staheli index, especially in the third trimester period, was 6.94% and 11.11%, respectively. We have provided a perspective on how the development of the medial longitudinal arch is shaped in the intrauterine period. Based on the results of study, we consider that the data on the medial longitudinal arch, especially in the third trimester period, may be more significant. In the evaluations made from the footprints of premature infants in the intrauterine third trimester period in the future, a study, in which infants detected with pes planus can be followed up and the development of their medial longitudinal arch is evaluated, can be conducted.

Updating fetal foot length to gestational age references: a chart review of abortion cases from 2012 to 2014.

OBJECTIVES: We sought to develop an updated fetal foot length-to-gestational week reference range from patient-reported last menstrual period (LMP), ultrasound, and best-estimate for gestational age based on American College of Obstetricians and Gynecologists' (ACOG) pregnancy dating guidelines. Subsequently, we aimed to determine the impact, if any, of race/ethnicity on our findings and compare our measurements to commonly referenced ranges. METHODS: We performed a chart review of 610 dilation and evacuation records with gestational ages 14 weeks 0 days to 21 weeks 6 days from October 2012 to December 2014 in Honolulu, Hawaii. We analyzed records containing pathology-measured fetal foot length and three gestational age estimation methods using ANOVA tests, determined if race/ethnicity affected fetal foot length, and compared our measurements to previously published studies. RESULTS: Linear regression analysis demonstrated that ultrasound-derived gestational age dating provided the best-fit regression formula with an R-squared and adjusted R-squared value of 0.92. Patient body mass index (p=.15), parity (p=.15), and race (p=.99) did not affect the equation. Mean fetal foot length per gestational age differed from historically referenced ranges by 0.36-3.92 mm in either direction. CONCLUSIONS: Our population's fetal foot length per gestational age differed from ranges typically referenced by pathologists following abortion at all gestational ages, using all three methods of fetal gestational age estimation. As gestational age increased, the variability of fetal foot length measurements per gestational week increased. If post-abortion fetal foot length measurements are obtained, it is important to use an updated reference range. IMPLICATIONS: Reference ranges frequently used by pathologists to assess fetal foot length following abortion may be outdated, limiting their utility. If facilities routinely obtain these measurements to estimate gestational age, it is important to use an updated reference range.

Comparing preoperative dating and postoperative dating for second-trimester surgical abortions.

OBJECTIVES: To assess relationships between preoperative and postoperative dating of second-trimester surgical abortion. STUDY DESIGN: We used a deidentified institutional database to extract demographic, dating and pathology data for surgical abortions performed at 14 to 23-6/7 weeks' gestational age (GA) from 9/2015 to 5/2017. We excluded women with multiple gestations, fetal anomalies and missing fetal biometric measurements. We assigned preoperative GA by ultrasonography for unknown last menstrual period (LMP) or when discrepancy between sonographic and LMP dating exceeded 7 days (<15-6/7 weeks), 10 days (16 to 21-6/7 weeks) or 14 days (22 to 23-6/7 weeks). We determined postoperative GA using fetal foot length pathology standards published by Streeter in 1920 and Drey et al. in 2005. We performed regression analysis to estimate the relationship between pre- and postoperative estimates of GA and to assess demographic effects on these estimates, and χ2 tests to assess whether fetal foot lengths were concordant with, larger than or smaller than the expected range for the preoperative GA. RESULTS: The 469 patients analyzed had a median preoperative GA of 19-4/7 weeks (range 14-0/7 to 23-6/7 weeks). Preoperative dating highly correlated with postoperative dating using both pathology standards (r2=0.95, p<.001), without any clinically relevant effect by body mass index (Streeter and Drey, p=.79), parity (Streeter p=.89; Drey p=.71), race (Streeter p=.06; Drey p=.07) or GA. Fetal foot lengths were larger than expected in 134 (28.6%) women using Streeter and 17 (3.6%) women using Drey standards (p<.001). CONCLUSIONS: Preoperative dating and postoperative dating for second-trimester surgical abortion highly correlate. Use of Streeter standards results in more women with a postoperative GA greater than expected compared to Drey standards. IMPLICATIONS: Increasing legal gestational age restrictions have placed additional burden on clinicians providing safe abortions, but guidelines on gestational age determination are lacking. Contemporary pathology standards consistent with modern practice and universally accepted by abortion providers and gynecologic pathologists are critical to our goal of safe and legal abortion provision.

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.

Clubfoot Versus Positional Foot Deformities on Prenatal Ultrasound Imaging.

Clubfoot and positional foot deformities (eg, pes spinatus) may have the same aspects on prenatal ultrasound (US) imaging. Nevertheless, differentiating these entities is essential because their prognoses are different. This pictorial review illustrates the US findings of clubfoot and positional foot deformities. On the basis of clinical postnatal images, we describe a prenatal US technique that could give an accurate diagnosis. In this essay, we demonstrate that when a foot malposition is suspected, a systematic analysis with 3 rigorous planes could help differentiate positional foot deformities from malformations and define their types.

Pigeon foot feathering reveals conserved limb identity networks.

The tetrapod limb is a stunning example of evolutionary diversity, with dramatic variation not only among distantly related species, but also between the serially homologous forelimbs (FLs) and hindlimbs (HLs) within species. Despite this variation, highly conserved genetic and developmental programs underlie limb development and identity in all tetrapods, raising the question of how limb diversification is generated from a conserved toolkit. In some breeds of domestic pigeon, shifts in the expression of two conserved limb identity transcription factors, PITX1 and TBX5, are associated with the formation of feathered HLs with partial FL identity. To determine how modulation of PITX1 and TBX5 expression affects downstream gene expression, we compared the transcriptomes of embryonic limb buds from pigeons with scaled and feathered HLs. We identified a set of differentially expressed genes enriched for genes encoding transcription factors, extracellular matrix proteins, and components of developmental signaling pathways with important roles in limb development. A subset of the genes that distinguish scaled and feathered HLs are also differentially expressed between FL and scaled HL buds in pigeons, pinpointing a set of gene expression changes downstream of PITX1 and TBX5 in the partial transformation from HL to FL identity. We extended our analyses by comparing pigeon limb bud transcriptomes to chicken, anole lizard, and mammalian datasets to identify deeply conserved PITX1- and TBX5-responsive components of the limb identity program. Our analyses reveal a suite of predominantly low-level gene expression changes that are conserved across amniotes to regulate the identity of morphologically distinct limbs.

Determination and classification of cutaneous innervation of the dorsum of the foot in foetal cadavers.

BACKGROUND: The aim of this study is to determine cutaneous innervation of the dorsum of the foot on foetal cadavers. MATERIALS AND METHODS: In this study. 200 limbs from 100 embalmed foetuses (54 males and 46 females) were studied in Anatomy Laboratory. Contributions of medial, lateral and intermediate dorsal cutaneous nerves (MDCN, LDCN and IDCN) of the foot were identified. RESULTS: Cutaneous innervation of the dorsum of the foot was classified into five types. Type I (75%) where MDCN innervated medial border of the foot and second interdigital cleft; IDCN innervated third, fourth and fifth interdigital clefts; and LDCN innervated the lateral border of the foot. Type II (21%) where MDCN innervated medial border of the foot, second and third interdigital clefts; IDCN innervated fourth and fifth interdigital clefts; and LDCN innervated the lateral border of the foot. Type III (1.5%) where saphenous nerve innervated medial border of the foot; MDCN innervated second and third interdigital clefts; IDCN innervated fourth and fifth interdigital clefts; and LDCN innervated the lateral border of the foot. Type IV (1.5%) was similar to type I, with an extra connection between the MDCN and IDCN on the dorsum of the foot. Type V (1%) where superficial fibular nerve innervated medial border of the foot, and second, third and fourth interdigital clefts; and sural nerve innervated fifth interdigital cleft and lateral border of the foot. CONCLUSIONS: The present study provides a new classification for the cutaneous innervation of the dorsum of the foot.

The plantar aponeurosis in fetuses and adults: an aponeurosis or fascia? / La aponeurosis plantar en fetos y adultos: ¿aponeurosis o fascia?

The plantar aponeurosis (PA), which is a thickened layer of deep fascia located on the plantar surface of the foot, is comprised of three parts. There are differing opinions on its nomenclature since various authors use the terms PA and plantar fascia (PF) interchangeably. In addition, the variable classifications of its parts has led to confusion. In order to assess the nature of the PA, this study documented its morphology. Furthermore, a pilot histological analysis was conducted to examine whether the structure is an aponeurosis or fascia. This study comprised of a morphological analysis of the three parts of the PA by micro- and macro-dissection of 50 fetal and 50 adult cadaveric feet, respectively (total n=100). Furthermore, a pilot histological analysis was conducted on five fetuses (n=10) and five adults (n=10) (total n=20). In each foot, the histological analysis was conducted on the three parts of the plantar aponeurosis, i.e. the central, lateral, and medial at their calcaneal origin (total n=60). Fetuses: i) Morphology: In 66 % (33/50) of the specimens, the standard anatomical pattern was observed, viz. three parts (i.e. central, lateral, medial) that originated from the medial and lateral processes of the calcaneal tuberosity and inserted onto the metatarsals. In 18 % (9/50) of the specimens, a two-part structure was observed. Variable origins of the medial part were noted in 16 % (8/50) of the specimens. In order to document these variations, the central part of the PA was divided into three segments (i.e. upper, middle, lower): a) In 63 % (5/8) of the specimens, the medial part arose from the middle segment; b) In 37 % (3/8) of the specimens, the medial part arose from the middle and upper segments. ii) Histological analysis: a) The central part contained longitudinally arranged semi-dense type I collagen fibres with fibroblasts; b) The lateral part displayed semi-dense type I collagen fibres with fibroblasts, hyaluronic acid, corpusculum sensorium fusiforme (Ruffini corpuscle) and corpusculum lamellosum (Pacinian corpuscle); c) The medial part comprised of loose connective tissue with elastic and reticular fibres. Adults: i) Morphology: In 100 % of the specimens, the standard anatomical pattern was observed. ii) Histological Analysis: a) In the central part, longitudinally arranged type I collagen fibres with fibroblasts were visible; b) The lateral part contained longitudinally arranged type I collagen fibres with fibroblasts; c) The medial part comprised of loose connective tissue, type I and type III collagen fibres, elastic and reticular fibres. In the current study, the morphology of the PA in fetuses and adults conformed to the standard anatomical description with variations in the origin of the medial part observed in fetuses. In addition, the fetal specimens displayed a two-part structure of the PA when the medial part was absent. Microscopically, the findings suggest that only the central and lateral parts may be considered as the PA, whilst the medial part may be termed the PF.

La aponeurosis plantar (AP), que es una capa engrosada de fascia profunda localizada en la superficie plantar del pie, está compuesta de tres partes. Hay diferentes opiniones sobre su nomenclatura, ya que varios autores utilizan los términos AP y fascia plantar (FP) de forma intercambiable. Además, las distintas clasificaciones de sus partes han dado lugar a confusión. Con el fin de evaluar la naturaleza de la AP, este estudio documentó su morfología. Además, se realizó un análisis histológico para examinar si la estructura es una aponeurosis o fascia. Este estudio consistió en un análisis morfológico de las tres partes de la AP de 50 pies de fetos y 50 pies de cadáveres adultos, por micro y macrodisección, respectivamente (total n = 100). Además, se realizó un análisis histológico en cinco fetos (n = 10) y cinco adultos (n = 10) (total n = 20). En cada pie, el análisis histológico se realizó sobre las tres partes de la aponeurosis plantar, es decir, la central, lateral y medial en su origen calcáneo (total n = 60). Fetos: i) Morfología: En el 66 % (33/50) de los especímenes, se observó el patrón anatómico estándar, es decir, tres partes (central, lateral y medial) que se originaron a partir de los procesos medial y lateral de la tuberosidad calcánea y se insertaban en los metatarsianos. En 18 % (9/50) de los especímenes, se observó una estructura de dos partes. Los orígenes variables de la parte mediana se visualizaron en el 16 % (8/50) de los especímenes. Para documentar estas variaciones, la parte central de la AP se dividió en tres segmentos (superior, medio, inferior): en el 63 % (5/8) de los casos, la parte mediana surgió del segmento medio; en el 37 % (3/8) de los casos, los casos la parte medial surgió de los segmentos medio y superior. ii) Análisis histológico: a) La parte central contenía fibras de colágeno tipo I semi-densas dispuestas longitudinalmente con fibroblastos; b) La parte lateral mostró fibras de colágeno tipo I semi-densas con fibroblastos, ácido hialurónico, corpúsculo sensorial fusiform (corpúsculo de Ruffini) y corpúsculo lamellosum (corpúsculo de Pacini); c) La parte medial comprende tejido conjuntivo suelto con fibras elásticas y reticulares. Adultos: i) Morfología: En el 100 % de los especímenes se observó el patrón anatómico estándar. ii) Análisis histológico: a) En la parte central, se observaron fibras de colágeno de tipo I con disposición longitudinal de fibroblastos; b) La parte lateral contenía fibras de colágeno de tipo I dispuestas longitudinalmente con fibroblastos; c) La parte medial estaba compuesta de tejido conectivo suelto, fibras de colágenos tipo I y tipo III, fibras elásticas y reticulares. En el presente estudio, la morfología de la AP en fetos y adultos se ajustó a la descripción anatómica estándar con variaciones en el origen de la parte medial observada en fetos. Además, los especímenes fetales mostraron una estructura de dos partes de la AP cuando la parte medial estaba ausente. Microscópicamente, los hallazgos sugieren que sólo las partes central y lateral pueden considerarse como AP, mientras que la parte medial puede denominarse FP.

Greater Growth of Proximal Metatarsals in Bird Embryos and the Evolution of Hallux Position in the Grasping Foot.

In early theropod dinosaurs-the ancestors of birds-the hallux (digit 1) had an elevated position within the foot and had lost the proximal portion of its metatarsal. It no longer articulated with the ankle, but was attached at about mid-length of metatarsal 2 (mt2). In adult birds, the hallux is articulated closer to the distal end of mt2 at ground level with the other digits. However, on chick embryonic day 7, its position is as in early theropods at half-length of mt2. The adult distal location is acquired during embryonic days 8-10. To assess how the adult phenotype is acquired, we produced fate maps of the metatarsals of day 6 chicken embryos injecting the lipophilic tracer DiI. The fates of these marks indicate a larger expansion of the metatarsals at their proximal end, which creates the illusory effect that d1 moves distally. This larger proximal expansion occurs concomitantly with growth and early differentiation of cartilage. Histological analysis of metatarsals shows that the domains of flattened and prehypertrophic chondrocytes are larger toward the proximal end. The results suggest that the distal position of the hallux in the avian foot evolved as a consequence of an embryological period of expansion of the metatarsus toward the proximal end. It also brings attention to the developmental mechanisms leading to differential growth between epiphyses and their evolutionary consequences.

Early embryonic development of long tendons in the human foot.

To examine a common plantar tendinous plate for long flexors of the toe and fingers in human embryos, we observed sections of 10 embryos at 5-6 weeks (crown-rump length or CRL 15-21 mm). The heel or tuber of the calcaneus was underdeveloped in 3 embryos with CRL 15 mm and the talus appeared not to be piled up on the calcaneus but these two bones were arranged along the lateromedial axis. As reported in the hand, we demonstrated, in the deep side of tarsal bones, a common tendinous plate formed by a joining of the flexor halluces longus and flexor digitorum longus tendons. In the tendinous plate, much or less, some connections between tendons seemed to remain even after birth to provide much greater types of tendon anomalies than in the hand. In addition, we postulated a hypothetical change in course of the peroneus longus tendon. In the initial phase, because of the underdeveloped calcaneus, the peroneus tendon might take an almost straight course similar to long flexor tendons. However, at 6 weeks and later, the inferomedially expanding calcaneus beneath the talus was likely to push the tendon to the cuboid bone.

Skeletal plasticity in response to embryonic muscular activity underlies the development and evolution of the perching digit of birds.

Most birds have an opposable digit 1 (hallux) allowing the foot to grasp, which evolved from the non-opposable hallux of early theropod dinosaurs. An important morphological difference with early theropods is the twisting of the long axis of its metatarsal. Here, we show how embryonic musculature and the onset of its activity are required for twisting of metatarsal 1 (Mt1) and retroversion of the hallux. Pharmacologically paralyzed embryos do not fully retrovert the hallux and have a straight Mt1 shaft, phenocopying the morphology of early tetanuran dinosaurs. Molecular markers of cartilage maturation and ossification show that differentiation of Mt1 is significantly delayed compared to Mt2-4. We hypothesize on how delayed maturation may have increased plasticity, facilitating muscular twisting. Our experimental results emphasize the importance of embryonic muscular activity in the evolutionary origin of a crucial adaptation.

The developmental origin of zygodactyl feet and its possible loss in the evolution of Passeriformes.

The zygodactyl orientation of toes (digits II and III pointing forwards, digits I and IV pointing backwards) evolved independently in different extant bird taxa. To understand the origin of this trait in modern birds, we investigated the development of the zygodactyl foot of the budgerigar (Psittaciformes). We compared its muscular development with that of the anisodactyl quail (Galliformes) and show that while the musculus abductor digiti IV (ABDIV) becomes strongly developed at HH36 in both species, the musculus extensor brevis digiti IV (EBDIV) degenerates and almost disappears only in the budgerigar. The asymmetric action of those muscles early in the development of the budgerigar foot causes retroversion of digit IV (dIV). Paralysed budgerigar embryos do not revert dIV and are anisodactyl. Both molecular phylogenetic analysis and palaeontological information suggest that the ancestor of passerines could have been zygodactyl. We followed the development of the zebra finch (Passeriformes) foot muscles and found that in this species, both the primordia of the ABDIV and of the EBDIV fail to develop. These data suggest that loss of asymmetric forces of muscular activity exerted on dIV, caused by the absence of the ABDIV, could have resulted in secondary anisodactyly in Passeriformes.

Cephalometric investigation of craniomaxillofacial structures during the prenatal period: a cadaver study.

INTRODUCTION: We aimed to investigate the morphometric development of the cranial base and its related structures, and their growth rate changes from the ninth gestational week to full term in a large group of human fetuses. METHODS: We selected 203 (109 male, 94 female) fetuses between 9 and 40 weeks of gestation and without any external anomalies. From each fetus, standard lateral and posteroanterior cephalometric images were taken using a dental digital panoramic and cephalometric x-ray machine. Fourteen linear and 9 angular parameters were measured. RESULTS: The cranial base angle showed a statistically significant increase between the groups from only the second to the third trimester periods. The sagittal translation of the maxilla increased during the prenatal period, whereas the mandibular sagittal relation grew at a steady rate. The vertical plane angles of the maxilla and the mandible did not show any significant changes. The maxillary length to mandibular length ratio remained stable. CONCLUSIONS: The cranial base angle increased, especially in the second through the third trimesters. The maxilla and the mandible demonstrated different growth patterns in the sagittal direction. The findings of this study could be a guide for interpreting the relationships among the craniofacial structures.

Fetal development of ligaments around the tarsal bones with special reference to contribution of muscles.

Through a histological examination of eight mid-term human fetuses (10-15 weeks) and seven late-stage fetuses (30-34 weeks), we attempted to determine how and when fetal ligaments around the tarsal bones form the regular arrangement seen in adults. Ligaments along the dorsal aspect of the tarsal bones developed early as an elongation of the perichondrium, in contrast to the late development of the plantar-sided ligaments. In contrast, a distal elongation of the tibialis posterior tendon was a limited plantar ligament in the early stage; finally, it extended from the navicular, ran obliquely to cross the dorsal side of the fibularis longus tendon, and inserted to the lateral cuneiform and fourth metatarsal. In the late stage, the adductor hallucis muscle origin provided multiple ligamentous structures along the cuneiforms and metatarsals. The tarsal sinus contained multiple fibrous bundles (possibly, the putative interosseous talocalcanean ligaments) that were derived from (1) insertion tendons of the extensor digitorus brevis muscle and (2) the fibrous sheath of the extensor digitorus longus tendon. The aponeurotic origin of the quadratus plantae muscle seemed to contribute to formation of the long plantar ligament. Therefore, tarsal ligaments appeared likely to develop from the long tendons, their fibrous sheaths and aponeuroses and intramuscular tendons of the proper foot muscles. Under in utero conditions with little or no stress from the plantar side of the foot, the muscle-associated connective tissue seems to play a crucial role in providing a regular arrangement of the ligaments in accordance with tensile stress from muscle contraction.

Prenatal diagnosis of type 1 fibular hemimelia.

Fibular hemimelia (FH) is a congenital longitudinal limb deficiency characterized by complete or partial absence of the fibula. Typically, it has been diagnosed at birth, when the neonate is seen to have lower limb shortening and a foot with missing toes. Although it is the most frequent lower limb deficiency anomaly, there are few published reports of prenatally diagnosed cases. Most of these published cases have involved the complete absence of the fibula, which is relatively easy to diagnose with antenatal ultrasound. In our opinion, our case is the first case of unilateral partial absence of the fibula detected using prenatal ultrasound imaging. Herein, we report a FH case associated with foot equinovalgus, and absence of the fourth and fifth foot rays diagnosed at 24 weeks' gestation. The anomaly was confirmed after birth by X-ray, and conservative orthopedic management was chosen. Our case shows that partial limb defects can also be detected by prenatal ultrasound imaging.

Fetal dorsalis pedis artery velocimetry in the second and third trimesters.

OBJECTIVES: This study aimed to establish reference ranges for Doppler parameters of the fetal dorsalis pedis artery and to compare them with those of the anterior tibial artery. METHODS: Dorsalis pedis artery velocimetry was performed in 138 singleton fetuses. Intraobserver repeatability coefficients and differences between measurements of bilateral legs were also evaluated. Comparisons were made between the pulsatility index in the dorsalis pedis and anterior tibial arteries. RESULTS: The average maximum velocity of the dorsalis pedis artery increased from approximately 12.2 cm/s at 18 weeks' gestation to 33.6 cm/s at 39 weeks' gestation, whereas the minimum velocity did not show any significant variation during the observed gestational weeks. The average pulsatility index increased from about 2.0 at 18 weeks' gestation to 3.1 at 39 weeks' gestation. The pulsatility index was lower in the dorsalis pedis artery than in the anterior tibial artery. CONCLUSIONS: Doppler parameters of the dorsalis pedis artery can be easily and accurately acquired by trained examiners and therefore are potential means for evaluating related fetal vascular development. However, it is still unclear whether changes exist in fetuses with limb diseases, and further investigation is needed.

Copy number analysis of 413 isolated talipes equinovarus patients suggests role for transcriptional regulators of early limb development.

Talipes equinovarus is one of the most common congenital musculoskeletal anomalies and has a worldwide incidence of 1 in 1000 births. A genetic predisposition to talipes equinovarus is evidenced by the high concordance rate in twin studies and the increased risk to first-degree relatives. Despite the frequency of isolated talipes equinovarus and the strong evidence of a genetic basis for the disorder, few causative genes have been identified. To identify rare and/or recurrent copy number variants, we performed a genome-wide screen for deletions and duplications in 413 isolated talipes equinovarus patients using the Affymetrix 6.0 array. Segregation analysis within families and gene expression in mouse E12.5 limb buds were used to determine the significance of copy number variants. We identified 74 rare, gene-containing copy number variants that were present in talipes equinovarus probands and not present in 759 controls or in the Database of Genomic Variants. The overall frequency of copy number variants was similar between talipes equinovarus patients compared with controls. Twelve rare copy number variants segregate with talipes equinovarus in multiplex pedigrees, and contain the developmentally expressed transcription factors and transcriptional regulators PITX1, TBX4, HOXC13, UTX, CHD (chromodomain protein)1, and RIPPLY2. Although our results do not support a major role for recurrent copy number variations in the etiology of isolated talipes equinovarus, they do suggest a role for genes involved in early embryonic patterning in some families that can now be tested with large-scale sequencing methods.

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development.

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 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/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 Cdc42(fl/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.