Quantitative anatomy of the ulna's shaft primary ossification center in the human fetus.

PURPOSE: There has been little information in the medical literature regarding the growing ulna in the human fetus, though such knowledge appears to be potentially useful in diagnosing skeletal dysplasias, characterized by a disrupted or completely halted growth of the fetus. Therefore, longitudinal measurements of long bones are extremely conducive in assessing both pregnancy and fetal anatomy. MATERIALS AND METHODS: Using methods of CT, digital-image analysis and statistics, the size of the ulna's shaft primary ossification center in 48 (26 males and 22 females) spontaneously aborted human fetuses aged 17-30 weeks was studied. RESULTS: With no sex differences, the best fit growth dynamics for the ulna's shaft primary ossification center was modeled by the following functions: y = - 8.476 + 1.561 × age ± 0.019 for its length, y = - 2.961 + 0.278 × age ± 0.016 for its proximal transverse diameter, y = - 0.587 + 0.107 × age ± 0.027 for its middle transverse diameter, y = - 2.865 + 0.226 × age ± 0.295 for its distal transverse diameter, y = - 50.758 + 0.251 × (age)2 ± 0.016 for its projection surface area, and y = - 821.707 + 52.578 × age ± 0.018 ± 102.944 for its volume. CONCLUSIONS: The morphometric characteristics of the ulna's shaft primary ossification center show neither sex nor bilateral differences. The ulna's shaft primary ossification center grows linearly with respect to its length, transverse dimensions and volume, and follows a quadratic function with respect to its projection surface area. The obtained morphometric data of the ulna's shaft primary ossification center is considered normative for respective prenatal weeks and may be of relevance in both the estimation of fetal ages and the diagnostic process of congenital defects.

Cross-sectional study of fetal long-bone length in an Iranian population at 17-25 weeks of gestation.

OBJECTIVE: To construct improved reference charts for fetal long bones in an Iranian setting and to compare them with previous studies. METHODS: The present prospective cross-sectional study included singleton fetuses assessed by ultrasonography at 17-25 weeks of gestation at the Comprehensive Medical Genetics Center, Shahid Soltani, Shiraz, Iran between May 1, 2012, and February 28, 2014. Exclusion criteria included conditions that could affect fetal growth. Fetal long bones (femur, humerus, tibia, fibula, ulna, and radius) were measured with ultrasonography and biometric charts were produced. Regression models were fitted to estimate bone lengths. The models produced were compared with those from previous studies in other populations. RESULTS: There were 660 singleton fetuses included and 660 femur, 633 humerus, 512 tibia, 498 fibula, 505 ulna, and 498 radius biometric measurements were recorded. The models generated to predict the length of the tibia, fibula, ulna, and radius from the length of the femur and humerus demonstrated a high goodness of fit when the predicted lengths were plotted against the actual lengths. Comparisons of mean lengths with previous studies suggested that long-bone length was affected by maternal ethnicity. CONCLUSION: The equations generated could be used to predict long-bone length in an Iranian population and ethnicity should be considered when using fetal long-bone length as a prenatal diagnostic tool.

Mouse TBX3 mutants suggest novel molecular mechanisms for Ulnar-mammary syndrome.

The transcription factor TBX3 plays critical roles in development and TBX3 mutations in humans cause Ulnar-mammary syndrome. Efforts to understand how altered TBX3 dosage and function disrupt the development of numerous structures have been hampered by embryonic lethality of mice bearing presumed null alleles. We generated a novel conditional null allele of Tbx3: after Cre-mediated recombination, no mRNA or protein is detectable. In contrast, a putative null allele in which exons 1-3 are deleted produces a truncated protein that is abnormally located in the cytoplasm. Heterozygotes and homozygotes for this allele have different phenotypes than their counterparts bearing a true null allele. Our observations with these alleles in mice, and the different types of TBX3 mutations observed in human ulnar-mammary syndrome, suggest that not all mutations observed in humans generate functionally null alleles. The possibility that mechanisms in addition to TBX3 haploinsufficiency may cause UMS or other malformations merits investigation in the human UMS population.

Orofaciodigital syndrome type IV (Mohr-Majewski syndrome): report of a family with two affected siblings.

We report on sibling fetuses with orofaciodigital syndrome (OFDS) type IV (Mohr-Majewski syndrome). The 1st was a 13-week-old fetus with hypertelorism; a median cleft defect of the upper lip, soft palate, and uvula; a polypoid lower lip and multiple frenula of the tongue adherent to the mandible; a congenital heart defect; pre- and postaxial polydactyly of the upper and preaxial polydactyly of the lower limbs; and an intersex genitalia. However, the shortening of both arms and forearms was particularly striking, with shortening of the ulna and ulnar deviation of both hands. The 2nd fetus was of the same parents, was 11 weeks old, and presented with a similar spectrum of malformations. The features of both fetuses showed a transitional phenotype between the OFDS type II (Mohr syndrome) and the short rib-polydactyly syndrome type II (Majewski syndrome), thus extending the known spectrum of the OFDS type IV.

Possible roles of Runx1 and Sox9 in incipient intramembranous ossification.

UNLABELLED: We evaluated the detailed expression patterns of Runx1 and Sox9 in various types of bone formation, and determined whether Runx1 expression was affected by Runx2 deficiency and Runx2 expression by Runx1 deficiency. Our results indicate that both Runx1 and Sox9 are intensely expressed in the future osteogenic cell compartment and in cartilage. The pattern of Runx1 and Sox9 expression suggests that both genes could potentially be involved in incipient intramembranous bone formation during craniofacial development. INTRODUCTION: Runx1, a gene essential for hematopoiesis, contains RUNX binding sites in its promoter region, suggesting possible cross-regulation with Runx2 and potential regulatory roles in bone development. On the other hand, Sox9 is essential for chondrogenesis, and haploinsufficiency of Sox9 leads to premature ossification of the skeletal system. In this study, we studied the possible roles of Runx1 and Sox9 in bone development. MATERIALS AND METHODS: Runx1, Runx2/Osf2, and Sox9 expression was evaluated by in situ hybridization in the growing craniofacial bones of embryonic day (E)12-16 mice and in the endochondral bone-forming regions of embryonic and postnatal long bones. In addition, we evaluated Runx2/Osf2 expression in the growing face of Runx1 knockout mice at E12.5 and Runx1 expression in Runx2 knockout mice at E14.5. RESULTS: Runx1 and Sox9 were expressed in cartilage, and the regions of expression expanded to the neighboring Runx2-expressing osteogenic regions. Expression of both Runx1 and Sox9 was markedly downregulated on ossification. Runx1 and Sox9 expression was absent in the regions of endochondral bone formation and in actively modeling or remodeling bone tissues in the long bones as well as in ossified craniofacial bones. Runx2 expression was not affected by gene disruption of Runx1, whereas the expression domains of Runx1 were extended in Runx2(-/-) mice compared with wildtype mice. CONCLUSIONS: Runx1 and Sox9 are specifically expressed in the osteogenic cell compartments in the craniofacial bones and the bone collar of long bones, and this expression is downregulated on terminal differentiation of osteoblasts. Our results suggest that Runx1 may play a role in incipient intramembranous bone formation.

Protection from ethanol-induced limb malformations by the superoxide dismutase/catalase mimetic, EUK-134.

Based on previous in vitro studies that have illustrated prevention of ethanol-induced cell death by antioxidants, using an in vivo model, we have tested the anti-teratogenic potential of a potent synthetic superoxide dismutase plus catalase mimetic, EUK-134. The developing limb of C57BL/6J mice, which is sensitive to ethanol-induced reduction defects, served as the model system. On their ninth day of pregnancy, C57BL/6J mice were administered ethanol (two intraperitoneal doses of 2.9 g/kg given 4 h apart) alone or in combination with EUK-134 (two doses of 10 mg/kg). Pregnant control mice were similarly treated with either vehicle or EUK-134, alone. Within 15 h of the initial ethanol exposure, excessive apoptotic cell death was observed in the apical ectodermal ridge (AER) of the newly forming forelimb buds. Forelimb defects, including postaxial ectrodactyly, metacarpal, and ulnar deficiencies, occurred in 67.3% of the ethanol-exposed fetuses that were examined at 18 days of gestation. The right forelimbs were preferentially affected. No limb malformations were observed in control fetuses. Cell death in the AER of embryos concurrently exposed to ethanol and EUK-134 was notably reduced compared with that in embryos from ethanol-treated dams. Additionally, the antioxidant treatment reduced the incidence of forelimb malformations to 35.9%. This work illustrates that antioxidants can significantly improve the adverse developmental outcome that results from ethanol exposure in utero, diminishing the incidence and severity of major malformations that result from exposure to this important human teratogen.

Teratogenic mechanisms of longitudinal deficiency and cleft hand.

In order to better understand the teratogenic mechanisms of congenital defects of the digits, we analyzed clinical cases and induced similar types of congenital hand anomalies in rat fetuses by oral administration of busulfan. In clinical cases, radial and ulnar deficiencies had common characteristic features. We induced radial and ulnar deficiencies in rat fetuses with the same drug. Radial and ulnar deficiencies induced in rats have similar clinical manifestations and these anomalies might be caused by the same teratogenic mechanism. Then, the formation of the digital rays was examined histologically. The results of histological examination suggested that these deficiencies were not caused by localized damage of the limb bud. They also suggested that the cause of missing digits in longitudinal deficiency is closely related to a deficit of mesenchymal cells in the limb bud. Cleft hand is considered to be one of the types of longitudinal deficiency. However, several investigators have suggested that the abnormal induction of finger rays in the process of formation of fingers induced central polydactyly, osseous syndactyly and also cleft hand. X-rays of the clinical cases and skeletal changes of the anomalies induced in rats appear to demonstrate that cleft hand formation proceeds from osseous syndactyly and central polydactyly. The results of our experimental study show that the critical periods of central polydactyly, osseous syndactyly and cleft hand are the same. They also suggest that central polydactyly, syndactyly and cleft hand might be induced when the same teratogenic factor acts on embryos at the same developmental stage in the human being. Because they have a similar causation, cleft hand, syndactyly and central polydactyly should be classified into the same entity, that is, abnormal induction of digital rays. Based on these clinical and experimental studies, we modified the Swanson classification. In our modified classification, typical cleft hand, syndactyly and polydactyly are included in the same category of abnormal induction of digital rays as the fourth new category.

The cyclin-dependent kinase inhibitor p57(Kip2) mediates proliferative actions of PTHrP in chondrocytes.

Parathyroid hormone-related peptide (PTHrP) is a positive regulator of chondrocyte proliferation during bone development. In embryonic mice lacking PTHrP, chondrocytes stop proliferating prematurely, with accelerated differentiation. Because the bone phenotype of mice lacking the cyclin-dependent kinase inhibitor p57(Kip2) is the opposite of the PTHrP-null phenotype, we hypothesized that PTHrP's proliferative actions in chondrocytes might be mediated by opposing p57. We generated p57/PTHrP-null embryos, which showed partial rescue of the PTHrP-null phenotype. There was reversal of the loss of proliferative chondrocytes in most bones, with reversal of the accelerated differentiation that occurs in the PTHrP-null phenotype. p57 mRNA and protein were upregulated in proliferative chondrocytes in the absence of PTHrP. Metatarsal culture studies confirmed the action of PTHrP to decrease p57 mRNA and protein levels in a model in which parathyroid hormone (PTH), used as an analog of PTHrP, increased chondrocyte proliferation rate and the length of the proliferative domain. PTH treatment of p57-null metatarsals had no effect on proliferation rate in round proliferative chondrocytes but still stimulated proliferation in columnar chondrocytes. These studies suggest that the effects of PTHrP on both the rate and extent of chondrocyte proliferation are mediated, at least in part, through suppression of p57 expression.

Multiple roles of Hoxa11 and Hoxd11 in the formation of the mammalian forelimb zeugopod.

Mutations in the 5' or posterior murine Hox genes (paralogous groups 9-13) markedly affect the formation of the stylopod, zeugopod and autopod of both forelimbs and hindlimbs. Targeted disruption of Hoxa11 and Hoxd11 or Hoxa10, Hoxc10 and Hoxd10 result in gross mispatterning of the radius and ulna or the femur, respectively. Similarly, in mice with disruptions of both Hoxa13 and Hoxd13, development of the forelimb and hindlimb autopod is severely curtailed. Although these examples clearly illustrate the major roles played by the posterior Hox genes, little is known regarding the stage or stages at which Hox transcription factors intersect with the limb development program to ensure proper patterning of the principle elements of the limb. Moreover, the cellular and/or molecular bases for the developmental defects observed in these mutant mice have not been described. In this study, we show that malformation of the forelimb zeugopod in Hoxa11/Hoxd11 double mutants is a consequence of interruption at multiple steps during the formation of the radius and ulna. In particular, reductions in the levels of Fgf8 and Fgf10 expression may be related to the observed delay in forelimb bud outgrowth that, in turn, leads to the formation of smaller mesenchymal condensations. However, the most significant defect appears to be the failure to form normal growth plates at the proximal and distal ends of the zeugopod bones. As a consequence, growth and maturation of these bones is highly disorganized, resulting in the creation of amorphous bony elements, rather than a normal radius and ulna.

Which morphologies of synovial folds result from degeneration and/or aging of the radiohumeral joint: an anatomic study with cadavers and embryos.

The synovial folds of the radiohumeral joints in cadaveric elbows from 179 elderly subjects and 40 embryos were investigated macroscopically and histologically to determine any morphologic changes caused by aging or degeneration. The anterior and posterior folds found in the elderly population shared characteristics of folds seen in embryos, with some modifications, and were thought to originate from the primitive septum. Proportionally, the length, width, and thickness of these folds were consistent between adults and embryos. However, the embryonic folds showed a homogenous morphology. In contrast, in the adult the anterior fold was characterized by a shorter and narrower villous pattern, and the posterior fold tended to be wider. Lateral extension of the anterior or posterior folds was also observed. Moreover, the lateral fold, never seen in embryos, was present and characterized by a hard plicate pattern in the adult. These derived or specific morphologies in adults probably result from alterations in the movement of the radial head caused by aging.

IL-1- and TNF-induced bone resorption is mediated by p38 mitogen activated protein kinase.

We have previously shown that p38 mitogen-activated protein kinase (MAPK) inhibitors, which block the production and action of inflammatory cytokines such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), are effective in models of bone and cartilage degradation. To further investigate the role of p38 MAPK, we have studied its activation in osteoblasts and chondrocytes, following treatment with a panel of proinflammatory and osteotropic agents. In osteoblasts, significant activation of p38 MAPK was observed following treatment with IL-1 and TNF, but not parathyroid hormone, transforming growth factor-beta (TGF-beta), 1,25(OH)(2)D(3), insulin-like growth factor-1 (IGF-1), or IGF-II. Similar results were obtained using primary bovine chondrocytes and an SV40-immortalized human chondrocyte cell line, T/C28A4. SB 203580, a selective inhibitor of p38 MAPK, inhibited IL-1 and TNF-induced p38 MAPK activity and IL-6 production (IC(50)s 0.3--0.5 microM) in osteoblasts and chondrocytes. In addition, IL-1 and TNF also activated p38 MAPK in fetal rat long bones and p38 MAPK inhibitors inhibited IL-1- and TNF-stimulated bone resorption in vitro in a dose-dependent manner (IC(50)s 0.3--1 microM). These data support the contention that p38 MAPK plays a central role in regulating the production of, and responsiveness to, proinflammatory cytokines in bone and cartilage. Furthermore, the strong correlation between inhibition of kinase activity and IL-1 and TNF-stimulated biological responses indicates that selective inhibition of the p38 MAPK pathway may have therapeutic utility in joint diseases such as rheumatoid arthritis (RA).

Developmental morphological and histological studies on structures of the human fetal elbow joint.

In the present work, morphological changes in the developing human elbow joint were studied at different prenatal ages (8, 12, 16, 20, 29 and 40 weeks) and were compared with the same structures in the adult joint. The elbow joint had gone through its most important developmental changes during the 20th week of prenatal life, probably due to the direct dynamic effect of the newly developed fetal movement. During later prenatal development, the articular surfaces of the lower end of humerus and the upper ends of radius and ulna developed their characteristic congruencies, so that the highly curved convexities always articulate with the highly curved concavities. That process progressed postnatally and even till adult age. In full-term infants it was found that the lower end of humerus had acquired its adult shape, while the shape of the upper ends of radius and ulna were still not fully developed. They continued development in postnatal life even till adult age. In the present work, histological prenatal studies were done on longitudinal sections from the back of the capsule and synovial tissue, early (8 weeks) and late in full term, and the results were also compared with the same structures in adults. It was found that at all ages, the capsules were formed of cellular and fibrous elements, but at early prenatal age (8 weeks), this cellular condensation was more massive and prominent while in full-term infants, it became generally more fibrous, but was still different compared to adults. Basic cellular structures of the synovial tissue changed very little during the late prenatal developmental stage, as it did not become more fibrous than cellular during these periods, but differences in vascularity became more obvious. The cartilaginous content of the articular surface at 8 weeks was highly cellular with very little intercellular matrix. In contrast to that of full term, this cartilage became fully chondrogenous with a notable decrease in cellular density and massive increase in matrix content.

Development of the human elbow joint.

Many studies have been published on the development of the human elbow joint, but authors disagree on its morphogenetic timetable. Most discrepancies center on the cavitation of the elbow joint (including the humeroradial, humeroulnar, and superior radioulnar joints), and the organization of the tunnel of the ulnar nerve. We summarize our observations on the development of the elbow joint in 49 serially sectioned human embryonic (n = 28) and fetal (n = 21) upper limbs. During week 12, ossification begins in the epiphyses of the elements comprising the elbow joint. At the end of the embryonic period, the shallow groove between the posterior aspect of the medial epicondyle and the olecranon process, begins to be visible. The elbow joint cavity appears in O'Rahilly stage 21 (51 days) at the level of the humeroulnar and humeroradial interzones. Formation of the cavity begins at the medialmost portion of the humeroradial interzone and the lateralmost portion of the humeroulnar interzone. The annular ligament begins to develop in O'Rahilly stage 21 (51 days), and the superior radioulnar joint cavity appears between this ligament and the lateral aspect of the head of the radius during O'Rahilly stage 23 (56 days). We established the morphogenetic timetable of the human elbow joint.

Prenatal ultrasound diagnosis of a femur-fibula-ulna complex during the first half of pregnancy.

A case of fetal femur-fibula-ulna (FFU) complex diagnosed by ultrasound is presented. Ultrasonographic features of a fetus displaying bilateral femoral hypoplasia, aplasia of the right forearm and the right hand, ray defects of the left hand are described. The importance of an early diagnosis of this malformation is emphasized with respect to parental counselling concerning prognosis and further prenatal management.

1,25-dihydroxyvitamin D3 inhibits parathyroid hormone-related peptide mRNA expression in fetal rat long bones in culture.

When fetal rat long bones are incubated in the presence of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], steady-state parathyroid hormone-related peptide (PTHrP) mRNA levels are decreased. This decrease is temporary: it is observed as soon as after 3 h of exposure and reaches a nadir after 6 h. At that time, PTHrP mRNA levels are significantly lower in the experimental than in the control bones. However the inhibitory effect vanishes after 24 h, despite continuous exposure to 1,25(OH)2D3 for even 48 h. This is the first report showing that PTHrP mRNA expression can be regulated in rat fetal long bones in vitro by 1,25(OH)2D3.

Retinoid signaling is required for chondrocyte maturation and endochondral bone formation during limb skeletogenesis.

Retinoids have long been known to influence skeletogenesis but the specific roles played by these effectors and their nuclear receptors remain unclear. Thus, it is not known whether endogenous retinoids are present in developing skeletal elements, whether expression of the retinoic acid receptor (RAR) genes alpha, beta, and gamma changes during chondrocyte maturation, or how interference with retinoid signaling affects skeletogenesis. We found that immature chondrocytes present in stage 27 (Day 5.5) chick embryo humerus exhibited low and diffuse expression of RARalpha and gamma, while RARbeta expression was strong in perichondrium. Emergence of hypertrophic chondrocytes in Day 8-10 embryo limbs was accompanied by a marked and selective up-regulation of RARgamma gene expression. The RARgamma-rich type X collagen-expressing hypertrophic chondrocytes lay below metaphyseal prehypertrophic chondrocytes expressing Indian hedgehog (Ihh) and were followed by mineralizing chondrocytes undergoing endochondral ossification. Bioassays revealed that cartilaginous elements in Day 5.5, 8.5, and 10 chick embryo limbs all contained endogenous retinoids; strikingly, the perichondrial tissues surrounding the cartilages contained very large amounts of retinoids. Implantation of beads filled with retinoid antagonist Ro 41-5253 or AGN 193109 near the humeral anlagens in stage 21 (Day 3.5) or stage 27 chick embryos severely affected humerus development. In comparison to their normal counterparts, antagonist-treated humeri in Day 8.5-10 chick embryos were significantly shorter and abnormally bent; their diaphyseal chondrocytes had remained prehypertrophic Ihh-expressing cells, did not express RARgamma, and were not undergoing endochondral ossification. Interestingly, formation of an intramembranous bony collar around the diaphysis was not affected by antagonist treatment. Using chondrocyte cultures, we found that the antagonists effectively interfered with the ability of all-trans-retinoic acid to induce terminal cell maturation. The results provide clear evidence that retinoid-dependent and RAR-mediated mechanisms are required for completion of the chondrocyte maturation process and endochondral ossification in the developing limb. These mechanisms may be positively influenced by cooperative interactions between the chondrocytes and their retinoid-rich perichondrial tissues.

Regulation of chondrocyte differentiation by Cbfa1.

Cbfa1, a developmentally expressed transcription factor of the runt family, was recently shown to be essential for osteoblast differentiation. We have investigated the role of Cbfa1 in endochondral bone formation using Cbfa1-deficient mice. Histology and in situ hybridization with probes for indian hedgehog (Ihh), collagen type X and osteopontin performed at E13.5, E14.5 and E17.5 demonstrated a lack of hypertrophic chondrocytes in the anlagen of the humerus and the phalanges and a delayed onset of hypertrophy in radius/ulna in Cbfa1-/- mice. Detailed analysis of Cbfa1 expression using whole mount in situ hybridization and a lacZ reporter gene reveled strong expression not only in osteoblasts but also in pre-hypertrophic and hypertrophic chondrocytes. Our studies identify Cbfa1 as a major positive regulator of chondrocyte differentiation.

The evolution of the distal radio-ulnar joint.

The pectoral girdle and the distal radio-ulnar joint have evolved over a period of 400 million years. Clinical circumstances exist that may represent atavistic development in modern man. Current understanding of this complex area does not explain all clinical situations.

Ossification and growth rates of the limb long bones during the prehatching period in the quail (Coturnix coturnix japonica).

The timing of ossification and the growth of six long bones of the prehatching period in the quail was studied. Ninety-nine quail eggs were incubated and in total nine fetuses were selected daily from the sixth to the sixteenth day of incubation. The fetuses were stained with alizarine and alcian blue double colouration. The fetuses were studied under the stereoscopic microscope and linear measurements were obtained from the humerus, ulna, radius, femur, tibia and fibula. The first appearance of the primary ossification centres in the diaphysis of the studied bones was found to occur between the sixth and the seventh day of incubation. Different growth patterns between the bones of the leg and of the wing were observed. Humerus and tibia showed the greatest growth rate while the radius and fibula showed the lowest.

The mouse Ulnaless mutation deregulates posterior HoxD gene expression and alters appendicular patterning.

The semi-dominant mouse mutation Ulnaless alters patterning of the appendicular but not the axial skeleton. Ulnaless forelimbs and hindlimbs have severe reductions of the proximal limb and less severe reductions of the distal limb. Genetic and physical mapping has failed to separate the Ulnaless locus from the HoxD gene cluster (Peichel, C. L., Abbott, C. M. and Vogt, T. F. (1996) Genetics 144, 1757-1767). The Ulnaless limb phenotypes are not recapitulated by targeted mutations in any single HoxD gene, suggesting that Ulnaless may be a gain-of-function mutation in a coding sequence or a regulatory mutation. Deregulation of 5' HoxD gene expression is observed in Ulnaless limb buds. There is ectopic expression of Hoxd-13 and Hoxd-12 in the proximal limb and reduction of Hoxd-13, Hoxd-12 and Hoxd-11 expression in the distal limb. Skeletal reductions in the proximal limb may be a consequence of posterior prevalence, whereby proximal misexpression of Hoxd-13 and Hoxd-12 results in the transcriptional and/or functional inactivation of Hox group 11 genes. The Ulnaless digit phenotypes are attributed to a reduction in the distal expression of Hoxd-13, Hoxd-12, Hoxd-11 and Hoxa-13. In addition, Hoxd-13 expression is reduced in the genital bud, consistent with the observed alterations of the Ulnaless penian bone. No alterations of HoxD expression or skeletal phenotypes were observed in the Ulnaless primary axis. We propose that the Ulnaless mutation alters a cis-acting element that regulates HoxD expression specifically in the appendicular axes of the embryo.