Limb development genes underlie variation in human fingerprint patterns.

Fingerprints are of long-standing practical and cultural interest, but little is known about the mechanisms that underlie their variation. Using genome-wide scans in Han Chinese cohorts, we identified 18 loci associated with fingerprint type across the digits, including a genetic basis for the long-recognized "pattern-block" correlations among the middle three digits. In particular, we identified a variant near EVI1 that alters regulatory activity and established a role for EVI1 in dermatoglyph patterning in mice. Dynamic EVI1 expression during human development supports its role in shaping the limbs and digits, rather than influencing skin patterning directly. Trans-ethnic meta-analysis identified 43 fingerprint-associated loci, with nearby genes being strongly enriched for general limb development pathways. We also found that fingerprint patterns were genetically correlated with hand proportions. Taken together, these findings support the key role of limb development genes in influencing the outcome of fingerprint patterning.

Morphospace analysis leads to an evo-devo model of digit patterning.

Biological forms occupy a constrained portion of theoretical morphospaces. Developmental models accounting for empirical morphospaces are necessary to achieve a better understanding of this phenomenon. We analyzed the phalangeal formulas (PFs) in lizards and relatives' hands by comparing them with a set of simulated PFs that compose a theoretical morphospace. We detected that: (1) the empirical morphospace is severely limited in size, (2) the PFs comply with two properties of phalangeal count per digit, namely the ordering rule (DI ≤ DII ≤ DIII ≤ DIV ≥ DV), and the contiguity relationship (neighbor digits differ on average in one phalanx), (3) the totality of the PFs can be categorized into four categories of hands aligned along a feasibility gradient. We also reconstructed the evolution of PFs and found a stepwise trajectory from the plesiomorphic PF towards reduced conditions. Finally, we propose a developmental model as the generative mechanism behind the PFs. It is consistent with the bulk of evidence managed and involves an ordered digit primordia initialization timed with periodic signals of joint formation coming from digit tips. Our approach is also useful to address the study of other meristic sequences in nature such as dental, floral, and branchial formulas.

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs.

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding-a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

The prevalence and ossification pattern of the biphalangeal and triphalangeal lateral toes.

PURPOSE: Biphalangealism of the toes is an exclusively human phenomenon. The aim of this study was to evaluate the development of the lateral toes in childhood by following the ossification pattern of the phalanges. METHODS: Foot radiographs of 913 adults have been evaluated for biphalangealism of 3rd to 5th toe. The pediatric group, aged 6-15 years of age, was assessed for the number of ossification centers in the foot. RESULTS: In adults, the mean prevalence of biphalangealism in the 5th toe was 41.39%, in the 4th toe was 2.15%, and in the 3rd toe was 0.48%. In children, 45% feet had four ossification centers in the 5th toe. The epiphysis center of the middle and distal phalanx was missing. In the 4th toe, four centers were present in of 2.47% of cases. Those values are similar to the prevalence of the biphalangeal toes in adult population. The remaining toes had 5 or 6 ossification centers. In the 5-center toe, the epiphysis of the middle phalanx was missing. CONCLUSION: A missing distal phalanx epiphyseal ossification center is considered indicative of a biphalangeal toe, and the toes with 5 or 6 ossification centers are indicative of triphalangeal toes. The reason for such evolution of the lateral toes is still debated, but the differences in anatomy most likely have no impact on foot function.

Non-linear growth trends of toe flexor muscle strength among children, adolescents, and young adults: a cross-sectional study.

PURPOSE: There are only a few studies on the muscular strength of the foot in children and adolescents; thus, the developmental pattern and normative data of these populations during growth are unclear. We sought to elucidate the developmental pattern of the foot muscle strength among children, adolescents, and young adults compared with that of the hand. METHODS: A total of 747 children, adolescents, and young adults participated in this study, and their maximum isometric toe flexor strength (TFS), hand grip strength (HGS), and foot length were measured. RESULTS: TFS was correlated with HGS (r = 0.785), age (r = 0.659), height (r = 0.757), body mass (r = 0.737), and foot length (r = 0.594). Multiple regression analyses revealed that TFS was correlated with age (ß = 0.243 in boys; ß = 0.461 in girls), squared value of age (age2; ß = - 0.296 in boys; ß = - 0.260 in girls), and body mass (ß = 0.256 in boys; ß = 0.311 in girls) in both sexes, indicating a non-linear relationship between age and TFS development. In a regression model for HGS, age was a significant variable, but not age2. HGS increased linearly from childhood until young adulthood, whereas TFS increased from childhood until adolescence and then levelled off. CONCLUSION: Our results demonstrate that TFS has a different developmental pattern compared with HGS.

Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton.

Synovial joints are crucial for support and locomotion in vertebrates, and are the frequent site of serious skeletal defects and degenerative diseases in humans. Growth and differentiation factor 5 (Gdf5) is one of the earliest markers of joint formation, is required for normal joint development in both mice and humans, and has been genetically linked to risk of common osteoarthritis in Eurasian populations. Here, we systematically survey the mouse Gdf5 gene for regulatory elements controlling expression in synovial joints. We identify separate regions of the locus that control expression in axial tissues, in proximal versus distal joints in the limbs, and in remarkably specific sub-sets of composite joints like the elbow. Predicted transcription factor binding sites within Gdf5 regulatory enhancers are required for expression in particular joints. The multiple enhancers that control Gdf5 expression in different joints are distributed over a hundred kilobases of DNA, including regions both upstream and downstream of Gdf5 coding exons. Functional rescue tests in mice confirm that the large flanking regions are required to restore normal joint formation and patterning. Orthologs of these enhancers are located throughout the large genomic region previously associated with common osteoarthritis risk in humans. The large array of modular enhancers for Gdf5 provide a new foundation for studying the spatial specificity of joint patterning in vertebrates, as well as new candidates for regulatory regions that may also influence osteoarthritis risk in human populations.

Ihh and PTH1R signaling in limb mesenchyme is required for proper segmentation and subsequent formation and growth of digit bones.

Digit formation is a process, which requires the proper segmentation, formation and growth of phalangeal bones and is precisely regulated by several important factors. One such factor is Ihh, a gene linked to BDA1 and distal symphalangism in humans. In existing mouse models, mutations in Ihh have been shown to cause multiple synostosis in the digits but lead to perinatal lethality. To better study the exact biological and pathological events which occur in these fused digits, we used a more viable Prx1-Cre;Ihh(fl/fl) model in which Cre recombinase is expressed during mesenchymal condensation in the earliest limb buds at E9.5 dpc and found that mutant digits continuously fuse postnatally until phalanges are finally replaced by an unsegmented "one-stick bone". Mutant mice displayed osteocalcin-positive mature osteoblasts, but had reduced proliferation and abnormal osteogenesis. Because of the close interaction between Ihh and PTHrP during endochondral ossification, we also examined the digits of Prx1-Cre;PTH1R(fl/fl) mice, where the receptor for PTHrP was conditionally deleted. Surprisingly, we found PTH1R deletion caused symphalangism, demonstrating another novel function of PTH1R signaling in digit formation. We characterized the symphalangism process whereby initial cartilaginous fusion prevented epiphyseal growth plate formation, resulting in resorption and replacement of the remaining cartilage by bony tissue. Chondrocyte differentiation displayed abnormal directionality in both mutants. Lastly, Prx1-Cre;Ihh(fl/fl);Jansen Tg mice, in which a constitutively active PTH1R allele was introduced into Ihh mutants, were established to address the possible involvement of PTH1R signaling in Ihh mutant digits. These rescue mice failed to show significantly improved phenotype, suggesting that PTH1R signaling in chondrocytes is not sufficient to restore digit formation. Our results demonstrate that Ihh and PTH1R signaling in limb mesenchyme are both essential to regulate proper development of digit structures, although they appear to use different mechanisms.

Anatomical Network Comparison of Human Upper and Lower, Newborn and Adult, and Normal and Abnormal Limbs, with Notes on Development, Pathology and Limb Serial Homology vs. Homoplasy.

How do the various anatomical parts (modules) of the animal body evolve into very different integrated forms (integration) yet still function properly without decreasing the individual's survival? This long-standing question remains unanswered for multiple reasons, including lack of consensus about conceptual definitions and approaches, as well as a reasonable bias toward the study of hard tissues over soft tissues. A major difficulty concerns the non-trivial technical hurdles of addressing this problem, specifically the lack of quantitative tools to quantify and compare variation across multiple disparate anatomical parts and tissue types. In this paper we apply for the first time a powerful new quantitative tool, Anatomical Network Analysis (AnNA), to examine and compare in detail the musculoskeletal modularity and integration of normal and abnormal human upper and lower limbs. In contrast to other morphological methods, the strength of AnNA is that it allows efficient and direct empirical comparisons among body parts with even vastly different architectures (e.g. upper and lower limbs) and diverse or complex tissue composition (e.g. bones, cartilages and muscles), by quantifying the spatial organization of these parts-their topological patterns relative to each other-using tools borrowed from network theory. Our results reveal similarities between the skeletal networks of the normal newborn/adult upper limb vs. lower limb, with exception to the shoulder vs. pelvis. However, when muscles are included, the overall musculoskeletal network organization of the upper limb is strikingly different from that of the lower limb, particularly that of the more proximal structures of each limb. Importantly, the obtained data provide further evidence to be added to the vast amount of paleontological, gross anatomical, developmental, molecular and embryological data recently obtained that contradicts the long-standing dogma that the upper and lower limbs are serial homologues. In addition, the AnNA of the limbs of a trisomy 18 human fetus strongly supports Pere Alberch's ill-named "logic of monsters" hypothesis, and contradicts the commonly accepted idea that birth defects often lead to lower integration (i.e. more parcellation) of anatomical structures.

Suppressor of Fused Is Required for Determining Digit Number and Identity via Gli3/Fgfs/Gremlin.

The anterior-posterior patterning of the vertebrate limb bud requires closely coordinated signaling interactions, including Sonic Hedgehog (Shh)-mediated counteraction of the Gli3 transcription factor in the distal and posterior mesenchyme of the limb bud. Suppressor of Fused (Sufu), an intracellular negative regulator of Shh signaling via Gli2 and Gli3, is implicated in early development of the mouse limb bud. However, how Sufu is involved in the genetic regulation of limb bud patterning still remains elusive. In this study, we show that the conditional deletion of Sufu in the mesenchyme of the early limb bud results in polydactyly with loss of digit identity and supernumerary bones in the wrist and the ankle. These pattern alterations are associated with anterior expansion of HoxD genes located at the 5' end of the cluster. By focusing on gene expression analysis of Shh/Gremlin1/Fgf signaling critical for the establishment and maintenance of anterior-posterior patterning, we show that early response to loss of Sufu involves anterior prolongation of Fgf4 and Fgf8 expression in the apical ectodermal ridge at E10.5. We also reveal the anterior activation of Shh-dependent posterior markers Ptc1, Gli1 and Gremlin in limb buds lacking Sufu. Furthermore, we find that loss of Sufu leads to attenuated levels of repressor Gli2 and repressor Gli3 in the early limb bud. Moreover, expression of Hand2 is activated in the entire limb bud at the early outgrowth stage in the mutant lacking Sufu. Thus, we provide evidence that Sufu is involved in the genetic network that restricts the posterior expression of Gli2/3/Hand2 and Gremlin/Fgf in limb bud patterning.

From flat foot to fat foot: structure, ontogeny, function, and evolution of elephant "sixth toes".

Several groups of tetrapods have expanded sesamoid (small, tendon-anchoring) bones into digit-like structures ("predigits"), such as pandas' "thumbs." Elephants similarly have expanded structures in the fat pads of their fore- and hindfeet, but for three centuries these have been overlooked as mere cartilaginous curiosities. We show that these are indeed massive sesamoids that employ a patchy mode of ossification of a massive cartilaginous precursor and that the predigits act functionally like digits. Further, we reveal clear osteological correlates of predigit joint articulation with the carpals/tarsals that are visible in fossils. Our survey shows that basal proboscideans were relatively "flat-footed" (plantigrade), whereas early elephantiforms evolved the more derived "tip-toed" (subunguligrade) morphology, including the predigits and fat pad, of extant elephants. Thus, elephants co-opted sesamoid bones into a role as false digits and used them for support as they changed their foot posture.

Digit ratio in birds.

The Homeobox (Hox) genes direct the development of tetrapod digits. The expression of Hox genes may be influenced by endogenous sex steroids during development. Manning (Digit ratio. New Brunswick, NJ: Rutgers University Press, 2002) predicted that the ratio between the lengths of digits 2 (2D) and 4 (4D) should be sexually dimorphic because prenatal exposure to estrogens and androgens positively influence the lengths of 2D and 4D, respectively. We measured digits and other morphological traits of birds from three orders (Passeriformes, house sparrow, Passer domesticus; tree swallow, Tachycineta bicolor; Pscittaciformes, budgerigar, Melopsittacus undulates; Galliformes, chicken, Gallus domesticus) to test this prediction. None were sexually dimorphic for 2D:4D and there were no associations between 2D:4D and other sexually dimorphic traits. When we pooled data from all four species after we averaged right and left side digits from each individual and z-transformed the resulting digit ratios, we found that males had significantly larger 2D:4D than did females. Tetrapods appear to be sexually dimorphic for 2D:4D with 2D:4D larger in males as in some birds and reptiles and 2D:4D smaller in males as in some mammals. The differences between the reptile and mammal lineages in the directionality of 2D:4D may be related to the differences between them in chromosomal sex determination. We suggest that (a) natural selection for a perching foot in the first birds may have overridden the effects of hormones on the development of digit ratio in this group of vertebrates and (b) caution be used in making inferences about prenatal exposure to hormones and digit ratio in birds.

Secondary centers of ossification of the human toes: exceptional polymorphism and evolutionary perspectives.

As great morphological variability characterizes the phalanges of the human toes in adults, we hypothesized for a possible variability in the presence or absence of their secondary (= epiphyseal) centers of ossification linked to the unique morphology of the human foot within primates. The aim of this study was thus to provide original and detailed data on the occurrence of these centers. Classically, the big toe or hallux (I) presents two secondary centers and the lateral toes (II-V) three centers, and consequently the five toes present a total of 14 secondary centers. The material studied consisted of 261 foot radiographs from 261 young individuals of European origin (202 males and 59 females; 6-16 years). The presence (or absence) of the secondary centers of the phalanges of the toes was assessed for each foot. Feet presenting a biphalangeal variant in one or more lateral toes were studied separately. The theoretical possibilities of association of the three secondary centers in a given lateral toe (II-V) are eight in number; these eight patterns were studied and coded in the present study by types A-H. An exceptional variability in the occurrence of the secondary centers in lateral toes (II-V) was observed, and the classic pattern of phalangeal ossification was never observed. The absence of one or more secondary centers seems to be observed only in the human species, and we suggest that this could be a derived pattern specific to the human species, i.e., autapomorphic pattern. These results are of interest in the characterization and understanding of the reduction in size of the lateral toes which characterizes the specific evolution of the human foot.

Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development.

In the developing limb, Bmp4 is expressed in the apical ectodermal ridge (AER) and underlying mesoderm. Insight into the function of Bmp4 in limb development has been hampered by the early embryonic lethality of Bmp4 null embryos. We directly investigated Bmp4 using a conditional null allele of Bmp4 and the Prx1(cre) transgene to inactivate Bmp4 in limb bud mesoderm. The limb bud mesoderm of Prx1(cre);Bmp4 mutants was defective in production of Bmp4 but still competent to respond to Bmp signaling. Prx1(cre);Bmp4 mutant embryos had defective digit patterning including hindlimb preaxial polydactyly with posterior digit transformations. The Prx1(cre);Bmp4 mutants also had postaxial polydactyly with digit five duplications. Bmp4 mutant limbs had delayed induction and maturation of the AER that resulted in expanded Shh signaling. Moreover, the AER persisted longer in the Bmp4 mutant limb buds exposing the forming digits to prolonged Fgf8 signaling. Our data show that Bmp4 in limb mesoderm regulates AER induction and maturation and implicate signaling from the AER in regulation of digit number and identity.

The duplicated longitudinal epiphysis or "kissing delta phalanx": evolution and variation in three different disorders.

BACKGROUND: The delta phalanx, also known as the delta bone, or longitudinal epiphyseal bracket (LEB), has been described in a variety of syndromes and dysplasias. However, the "kissing delta phalanx" is not as well recognized in the literature; it consists of a duplicated longitudinal bracketed epiphysis, or a complex of two adjacent delta bones, with opposing convex portions facing each other. Magnetic resonance imaging of the kissing delta phalanx has not been previously described. OBJECTIVE: To describe the evolution, variation and associated osseous anomalies of the kissing delta phalanx in three patients with distinct distal limb malformations using both plain films and magnetic resonance imaging. RESULTS: Patient 1 had Rubinstein-Taybi syndrome and, in addition to a kissing delta phalanx in both feet, had corresponding delta metatarsals (MT1). Patient 2 had Cenani-Lenz syndactyly with distinct variation of the kissing delta phalanx in each hand. He had a disorganized appearance to the phalanges, metacarpal fusions and carpal coalitions. Patient 3 had an isolated oligosyndactyly of the left hand with metacarpal fusions and carpal coalitions. All three patients were followed over time. We describe two types of kissing delta phalanges: separated (without fusion of the corresponding epiphyseal brackets) and nonseparated (with fusion of the corresponding epiphyseal brackets). Both types can be seen in the same patient and are a reflection of the relative degree of segmentation of the two delta bones. CONCLUSION: The appearance of this rare osseous abnormality changes with time and can be found in a limited number of uncommon disorders. It can also be found in association with other osseous anomalies of the distal extremities; therefore magnetic resonance imaging early in life can greatly assist in surgical planning. Recognition of the kissing delta phalanx may be an important radiological clue to diagnosis of these rare disorders.

Developmental morphology of limb reduction in Hemiergis (Squamata: Scincidae): chondrogenesis, osteogenesis, and heterochrony.

Digit loss is a common theme in tetrapod evolution that may involve changes in several developmental processes. The skink genus Hemiergis provides an ideal model to study these processes in closely related taxa: within three Western Australian Hemiergis species, digit quantity ranges between two and five. For three consecutive reproductive seasons, gravid females of Hemiergis were collected in the field and their embryos prepared for histological analysis of limb skeletal development (chondrogenesis and osteogenesis). Comparative studies of skeletal developmental morphology demonstrate that limbs with fewer than five digits do not result from a simple truncation of a putative ancestral (five-digit) developmental program. The developmental and adult morphologies in two-, three-, and four-digit Hemiergis are neither predicted nor explained by a simple model of heterochrony involving either chondrogenesis or osteogenesis. In postnatal Hemiergis, digit number and relative limb length do not correlate in a simple linear fashion. Instead, limb size and digit reduction may correlate with substrate conditions and burrowing behavior.

Radiographic analysis of growth in pediatric microsurgical toe-to-hand transfers.

Microsurgical toe-to-hand transfers may provide improved hand function in children with absent digits. To date, documentation of the growth potential of these transferred digits has not been performed. This study reviewed the authors' series of pediatric toe-to-hand transfers, with specific attention paid to measuring growth by radiographic analysis. From 1995 to 2000, 23 toe-to-hand transfers were performed in 18 children. Age at time of transfer ranged from 2.8 to 13 years. Indications included constriction band syndrome, transverse deficiency, longitudinal deficiency, traumatic amputation, and vascular malformation. The transfers were successful in 22 of 23 procedures (96 percent success rate). Radiographic analysis of growth was performed using three criteria: (1) appearance of open epiphyseal plates, (2) comparison with preoperative radiographs, and (3) comparison with radiographs of the contralateral control toe. Epiphyseal plates remained open on postoperative radiographs in 27 of 28 phalanges (96 percent) at a mean of 12 months' follow-up (range, 1 to 36 months). The preoperative foot radiographs were compared with serial radiographs of the transferred toe over time. In 10 toe transfers with follow-up greater than 6 months (mean, 21 months), nine patients had increased bony length in the transferred digit. In four patients, radiographs of the toe transfer were compared with radiographs of the corresponding toe on the opposite foot. With a mean follow-up of 29 months, all patients had equal length measurements of the toe transfer with the contralateral toe control. These data provide objective evidence that digital growth potential is preserved in toe-to-hand transfers. Furthermore, this bone growth is comparable with that of the corresponding toe on the contralateral foot. Therefore, microsurgical toe-to-hand transfers may provide children with extra digits that maintain growth and improve hand function.

Hormonal induction of thumb pads and the evolution of secondary sexual characteristics of the Southeast Asian fanged frog, Rana blythii.

The fanged frogs of Southeast Asia do not express most of the hormone-dependent secondary sexual characteristics such as thumb pads that are common to other ranid frogs. At the same point in the evolutionary history of the group that these androgen-mediated characteristics are lost, male parental care first evolves. This behavior is often correlated with low androgen levels. Prior work indicates that in one of the fanged frogs, Rana blythii, adult males have low androgen levels compared to North Temperate species of Rana. This leads to the question of whether these low androgen levels are related to the unusual male parental care and the lack of expression of the thumb pad and other hormone-dependent secondary sexual characteristics in this species. We tested that hypothesis by examining the effects of exogenous dihydrotestosterone supplements on the expression of thumb pads in Rana blythii. Dihydrotestosterone injections appear to stimulate the expression of the thumb pad in R. blythii. These results support the hypothesis that low androgen levels are involved in the loss of the thumb pad in R. blythii. This work provides an example of how mapping characters on phylogenies can be a powerful approach for gaining insights into proximate physiological mechanisms of selection at the evolutionary level.

A mutational analysis of the 5' HoxD genes: dissection of genetic interactions during limb development in the mouse.

Using gene targeting in mice, we have undertaken a systematic mutational analysis of the homeobox-containing 5' HoxD genes. In particular, we have characterized the limb defects observed in mice with independent targeted disruptions of hoxd-12 and hoxd-13. Animals defective for hoxd-12 are viable, fertile, and appear outwardly normal yet have minor autopodal defects in the forelimb which include a reduction in the bone length of metacarpals and phalanges, and a malformation of the distal carpal bone d4. The limb phenotypes observed in hoxd-13 mutant mice are more extensive, including strong reductions in length, complete absences, or improper segmentations of many metacarpal and phalangeal bones. Additionally, the d4 carpal bone is not properly formed and often produces an extra rudimentary digit. To examine the genetic interactions between the 5' HoxD genes, we bred these mutant strains with each other and with our previously characterized hoxd-11 mouse to produce a series of trans-heterozygotes. Skeletal analyses of these mice reveal that these genes interact in the formation of the vertebrate limb, since the trans-heterozygotes display phenotypes not present in the individual heterozygotes, including more severe carpal, metacarpal and phalangeal defects. Some of these phenotypes appear to be accounted for by a delay in the ossification events in the autopod, which lead to either the failure of fusion or the elimination of cartilaginous elements. Characteristically, these mutations lead to the overall truncation of digits II and V on the forelimb. Additionally, some trans-animals show the growth of an extra postaxial digit VI, which is composed of a bony element resembling a phalange. The results demonstrate that these genes interact in the formation of the limb. In addition to the previously characterized paralogous interactions, a multitude of interactions between Hox genes is used to finely sculpt the forelimb. The 5' Hox genes could therefore act as a major permissive genetic milieu that has been exploited by evolutionary adaptation to form the tetrapod limbs.