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.

How is digit identity determined during limb development?

Digit identity has been studied using the chick embryo as a model system for more than 40 years. Using this model system, several milestone findings have been reported, such as the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), the Shh gene, and the theory of morphogen and positional information. These experimental results and models provided context for understanding pattern formation in developmental biology. The focus of this review is on the determination of digit identity during limb development. First, the history of studies on digit identity determination is described, followed by descriptions of the molecular mechanisms and current models for determination of digit identity. Finally, future questions and remarkable points will be discussed.

Survival of Hoxa13 homozygous mutants reveals a novel role in digit patterning and appendicular skeletal development.

The loss of HOXA13 function severely disrupts embryonic limb development. However, because embryos lacking HOXA13 die by mid-gestation, the defects present in the mutant limb could arise as a secondary consequence of failing embryonic health. In our analysis of the mutant Hoxa13(GFP) allele, we identified a surviving cohort of homozygous mutants exhibiting severe limb defects including: missing phalanx elements, fusions of the carpal/tarsal elements, and significant reductions in metacarpal/metatarsal length. Characterization of prochondrogenic genes in the affected carpal/tarsal regions revealed significant reduction in Gdf5 expression, whereas Bmp2 expression was significantly elevated. Analysis of Gdf5 mRNA localization also revealed diffuse expression in the carpal/tarsal anlagen, suggesting a role for HOXA13 in the organization of the cells necessary to delineate individual carpal/tarsal elements. Together these results identify Gdf5 as a potential target gene of HOXA13 target gene and confirm a specific role for HOXA13 during appendicular skeletal development.

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.

Expression of cartilage oligomeric matrix protein (COMP) by embryonic and adult osteoblasts.

Cartilage oligomeric matrix protein has been implicated as an important component of endochondral ossification because of its direct effects on chondrocytes. The importance of this protein for skeletal development and growth has been recently illustrated by the identification of mutations in cartilage oligomeric protein genes in two types of inherited chondrodysplasias and osteoarthritic phenotypes: multiple epiphyseal dysplasia and pseudoachondroplasia. In the present study, we report the presence of cartilage oligomeric protein in embryonic and adult osteoblasts. A foot from a 21-week-old human fetus, subchondral bone obtained from knee replacement surgery in an adult patient, and a limb from a 19-day-postcoital mouse embryo were analyzed with immunostaining and in situ hybridization. In the human fetal foot, cartilage oligomeric protein was localized to osteoblasts of the bone collar and at the newly formed bone at the growth plate and bone diaphyses. Immunostaining was performed on the adult subchondral bone and showed positive intracellular staining for cartilage oligomeric protein of the osteoblasts lining the trabecular bone. There was no staining of the osteocytes. Immunostaining of the mouse limb showed the most intense staining for cartilage oligomeric protein in the hypertrophic chondrocytes and in the surrounding osteoblast cells of the developing bone. Cartilage oligomeric protein mRNA and protein were detected in an osteoblast cell line (MG-63), and cartilage oligomeric protein mRNA was detected from human cancellous bone RNA. These results suggest that the altered structure of cartilage oligomeric protein by the mutations seen in pseudoachondroplasia and multiple epiphyseal dysplasia may have direct effects on osteoblasts, contributing to the pathogenesis of these genetic disorders.

The different growth zones of the fetal foot.

Previous publications revealed no reliable data or models concerning the three-dimensional ontogenesis of the lower extremity. Using the method of plastination-histology in combination with 3D-computer-reconstructions we were able to produce exact, virtual 3D-specimens of 19 healthy fetal feet. The fetuses were aged between 9 to 38 weeks of gestation and age-dependently related to four defined age-groups. We compared these feet with the help of a new geometrical method. Thus, we obtained a kind of "slow-motion-picture" of the undisturbed three-dimensional development of the fetal foot. Our results show that the human fetal foot has a desultory mode of growth and that growth priorities within the foot-skeleton change dependent upon age and region. Though the growth of the fetal foot-skeleton is desultory, it is not disconnected. The result of this peculiar mode of growth is to create the foot arches and thus seems to be functionally-oriented toward the human foot's specific purposes.

[Order of appearance of the ossification centers in the foot during the period of intrauterine life in human material]. / Secuencia de aparición de los centros de osificación del pie durante el período de vida intrauterino en material humano.

Forty seven skeletons belonging to human fetuses, whose age ranged from eight to thirty eight weeks, were studied in order to establish the beginning of ossification in the bones. Red alizarin was employed in order to stain bone calcifications. Our observations agree with other authors considered as references nonetheless, we also have found differences among the beginning and the sequence of development of some of the primary centers of ossification in the fetal foot. The appearance of the ossification centers was as follows: 1) the five metatarsals: 9th. week; 2) distal phalange first digit; 11th. week; 3) a) proximal phalange 1, 2, 3, 4, 5 digits: 12th. week, b) distal phalange 2, 3, 4, 5 digits: 12th. week; 4) middle phalange second digit: 17th. week; 5) a) middle phalange fourth digit: 18th. week, b) middle phalange third digit: 18th. week, y 6) middle phalange fifth digit: 21th. week.

Charts of fetal size: limb bones.

OBJECTIVE: To construct new size charts for all fetal limb bones. DESIGN: A prospective, cross sectional study. SETTING: Ultrasound department of a large hospital. SAMPLE: 663 fetuses scanned once only for the purpose of the study at gestations between 12 and 42 weeks. METHODS: Centiles were estimated by combining separate regression models fitted to the mean and standard deviation, assuming that the measurements have a normal distribution at each gestational age. MAIN OUTCOME MEASURES: Determination of fetal limb lengths from 12 to 42 weeks of gestation. RESULTS: Size charts for fetal bones (radius, ulna, humerus, tibia, fibula, femur and foot) are presented and compared with previously published data. CONCLUSIONS: We present new size charts for fetal limb bones which take into consideration the increasing variability with gestational age. We have compared these charts with other published data; the differences seen may be largely due to methodological differences. As standards for fetal head and abdominal measurements have been published from the same population, we suggest that the use of the new charts may facilitate prenatal diagnosis of skeletal dysplasias.

Ossification sequence in infants who die during the perinatal period: population-based references.

PURPOSE: To determine population-based references for the relationships between the presence of ossification centers and gestational age and skeletal length measurements among infants who die during the perinatal period, as well as to evaluate the possible influence of intrauterine growth restriction on ossification stage. MATERIALS AND METHODS: During an 11-year period, nearly all infants who died perinatally in a well-defined geographic area routinely underwent radiography with a standardized technique. The presence of visible secondary ossification centers in the singletons (n = 495) was evaluated. Cluster analysis was used to identify stages of ossification; a sequential appearance of secondary ossification centers was assumed. Comparisons were made with Wilks lambda between male and female infants and between infants who were presumed to have growth restriction and those who were not. Reference ranges for the presence of ossification centers were calculated for interquartile ranges of femur length and gestational age. RESULTS: Eight clusters of ossification defining different stages of ossification of the pelvis, hindfeet, and knees were identified. The sequential clusters outlined well-defined intervals of femur length and gestational age. Bone lengths, birth weight, and gestational age within ossification clusters did not differ between the sexes (Wilks lambda = 0.989, P =.532) or according to whether growth restriction was presumed to exist (Wilks lambda = 0.958, P =.481). CONCLUSION: The reference diagrams calculated with this method indicate relationships between ossification sequence and both gestational age and skeletal length measurements.