Studies on skeleton formation in reptiles: Patterns of ossification in the skeleton of Chelydra serpentina (Reptilia, Testudines)

Patterns and sequence of ossification are described throughout the skeleton of Chelydra serpentina Linnaeus. Evidence is adduced documenting the decoupling of ossification processes from sequence and patterns of chondrification. Convergence of ontogenetic repatterning in the ossification of the axial skeleton in Chelydra and Squamata is discussed, as are problems of adaptive modification of ossification patterns. The development of a carapace may be correlated with changes of ossification patterns in the postcranial axial skeleton of turtles, but the most striking evidence for the adaptive modification of ossification sequence obtains from a comparison of the limb skeleton and its ossification in Chelydra and in sea turtles     

Postnatal skeletal ontogeny in Callisaurus draconoides and Uta stansburiana (Iguania: Phrynosomatidae).

The mineralization of the skeleton from hatching to near maximum size in two phrynosomatid lizards, Callisaurus draconoides and Uta stansburiana, is described in detail. Observed patterns in the appearance of epiphyseal secondary centers, ossification centers, apophyseal ossifications, and calcifications, the distribution of sesamoids, and the timing of fusions, are compared and contrasted with observations of other squamates available in the literature. Overall, Callisaurus and Uta exhibit an advanced state of ossification in the hatchling relative to other squamate neonates and share a similar sequence of braincase fusions and appearance of secondary centers. Preliminary observations suggest that patterns of postnatal skeletal development are highly conserved and independent of patterns of prenatal morphogenesis, and thus a potentially rich source of character data for systematic investigations.     

Immunohistochemical studies of the extracellular matrix in the condylar cartilage of the human fetal mandible: collagens and noncollagenous proteins.

This study provides data concerning the cells and their extracellular matrix in prenatal human mandibular condylar cartilage. The latter cartilage represents a secondary type of cartilage since it develops late in the morphogenesis of the craniofacial skeleton. The cartilage of the mandibular condyle is actively involved in endochondral ossification, thus showing all the phases of cartilage growth, maturation, and mineralization that precedes de novo bone formation. The present study focused on the localization and distribution of the major macromolecules that are normally encountered in cartilage and bone, including collagens, proteoglycans, fibronectin, osteonectin, osteocalcin, alkaline phosphatase, and anchorin CII. It became clear that the mineralized zone of the cartilage already contained bone-specific antigens; thus the above zone might serve as an essential propagative predecessor in the ossification process.     

Craniofacial growth of fetal Macaca nemestrina: A cephalometric roentgenographic study

The prenatal growth of the macaque craniofacial skeleton is described using lateral radiographs of 82 fetal and 25 neonatal Macaca nemestrina whose known gestational ages range from 50 to 186 days. The ossification sequence of the craniofacial bones resembles that in the human fetus. During gestation, the macaque neurocranium loses its round, globular shape, becoming flattened and elongated in an anteroposterior direction. In contrast, the morphologic pattern of the face is established early in fetal life, and little change takes place during the remaining prenatal period. The macaque craniofacial dimensions develop along the general skeletal growth pattern, unlike the human craniofacial dimensions, which follow an intermediate pattern between the neural and general skeletal patterns. However, despite minor differences, the macaque and human fetal faces follow the same basic patterns of growth.     

Studies on skeleton formation in reptiles, v. Patterns of ossification in the skeleton of Alligator mississippiensis DAUDIN (Reptilia, Grocodylia)

Patterns of ossification are described in the endo-and exoskeleton of Alligator mississippiensis. The occurrence of a dermo-supraoccipital is discussed in light of the independence of dermal and endochondral bone. The development of the bony secondary palate is discussed in light of Haeckelian recapitulation. The sequence of ossification in the limb skeleton is shown to differ from the sequence of chondrification of the cartilaginous precursors. Patterns of ossification in Alligator are compared to lepidosaurs in terms of sequence and timing. Important differences relate to ossification patterns in the limb skeleton: lepidosaurs show a dominance of digit III > IV > II > I > V, whereas Alligator shows a dominance of digits III > II> IV > I > V in the ossification process. Ontogenetic repatterning in the ossification of the axial skeleton is discussed as it bears on the serial homology of dorsal ribs, sacral ribs and caudal ribs (transverse processes).     

Large-scale double-staining of rat fetal skeletons using Alizarin Red S and alcian blue

A critical component in the conduct of a prenatal developmental toxicity study is the evaluation of fetal skeletal development. As the developing rodent fetus is typically evaluated at gestation day 20, at a time when ossification of the skeleton is incomplete, a thorough assessment of skeletal development would include both ossified and cartilaginous structures. Current methods to double-stain the fetal skeleton using Alizarin Red S and Alcian Blue are typically described for small sample sizes or using time allotments for each processing step that are unsuitable for industry. In an industrial setting, there is a need for an effective means to double-stain fetal skeletons on a large scale (i.e., hundreds of fetuses simultaneously). This article describes a method used in our laboratory to stain both fetal bone and cartilage using solutions and procedures on an industrial scale.     

A new rapid radiological procedure for routine teratological use in bone ossification assessment: a supplement for staining methods

BACKGROUND: Presently, bone ossification is assessed by the study of single-stained fetal bones (alizarin red-S) or double-stained bones and cartilaginous structures (alcian blue followed by alizarin red-S). Both methods, especially double-staining, are labor-intensive, time-consuming, and provide qualitative information regarding skeleton ossification. Quantitative evaluation of ossification is more difficult and is usually based on determination of calcium and other minerals in the bone by means of atomic absorption spectrometry. Here we introduce a simple new method that allows quantitative determination of skeleton ossification before routine staining examination. METHODS: Fetuses delivered by laparotomy on the 16th and 21st day of gestation as well as 1-day-old rat pups were examined. The fetuses and pups were prenatally subcutaneously exposed to sodium valproate or to physiological saline. Lateral, prone, and supine digital radiograms of each fetus were taken using the Digora-Soredex digital radiography system and the Planmeca Intra intraoral X-ray machine. According to the best visualization, the data concerning vertebra were analyzed. All the fetuses were then routinely double-stained using alcian blue and alizarin red-S. RESULTS: Malformations of axial skeleton (rib, sternum, and thoracic and sacral vertebra) were found in valproate-treated groups. Unlike cartilage malformations, the bone changes were detected in similar frequency in radiological and staining methods. Differences in densities according to the degree of ossification in the vertebral arches and bodies at different levels of the vertebral column, between drug-treated and negative control groups were noted. CONCLUSIONS: The preliminary results suggest that digital radiography examination is a useful method in determining delaying of skeleton ossification not detectable by other methods. It balances qualitative and quantitative aspects of the presently used methods and is also simple, objective, fast, and relatively inexpensive.     

Ossification patterns in the tetrapod limb – conservation and divergence from morphogenetic events

Two different patterns of the condensation and chondrification of the limbs of tetrapods are known from extensive studies on their early skeletal development. These are on the one hand postaxial dominance in the sequential formation of skeletal elements in amniotes and anurans, and on the other, preaxial dominance in urodeles. The present study investigates the relative sequence of ossification in the fore‐ and hindlimbs of selected tetrapod taxa based on a literature survey in comparison to the patterns of early skeletal development, i.e. mesenchymal condensation and chondrification, representing essential steps in the late stages of tetrapod limb development. This reveals the degree of conservation and divergence of the ossification sequence from early morphogenetic events in the tetrapod limb skeleton. A step‐by‐step recapitulation of condensation and chondrification during the ossification of limbs can clearly be refuted. However, some of the deeper aspects of early skeletal patterning in the limbs, i.e. the general direction of development and sequence of digit formation are conserved, particularly in anamniotes. Amniotes show a weaker coupling of the ossification sequence in the limb skeleton with earlier condensation and chondrification events. The stronger correlation between the sequence of condensation/chondrification and ossification in the limbs of anamniotes may represent a plesiomorphic trait of tetrapods. The pattern of limb ossification across tetrapods also shows that some trends in the sequence of ossification of their limb skeleton are shared by major clades possibly representing phylogenetic signals. This review furthermore concerns the ossification sequence of the limbs of the Palaeozoic temnospondyl amphibian Apateon sp. For the first time this is described in detail and its patterns are compared with those observed in extant taxa. Apateon sp. shares preaxial dominance in limb development with extant salamanders and the specific order of ossification events in the fore‐ and hindlimb of this fossil dissorophoid is almost identical to that of some modern urodeles.     

Ossification communalities of the hand and other body parts: their implication to skeletal assessment

Correlating the age at appearance of 71 postnatal ossification centers (OC's) with every other OC, 4,970 correlations in all, then grouping correlations by body part, the hand does not exhibit usefully higher communality (mean r) than the foot, shoulder, hip, elbow or knee. While low communality round bones and ossification sequence polymorphisms together account for the fact that no one body part adequately represents the entire skeleton, it is also true that ossification communality throughout the skeleton is low unless OC's of maximum predictive value are separately employed.     

二噁英致大鼠先天骨骼发育畸形中软骨细胞的病理学改变

目的探讨大鼠骨骼发育过程中环境类致畸因子对大鼠骨骼发育的先天性致畸作用。方法应用不同剂量的环境类致畸因子-二噁英（2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD）构建先天性Wistar大鼠骨骼发育畸形动物模型;茜素红染色法制作并观察透明骨骼标本;采用光镜和透射电镜观察胎鼠趾骨骨化中心的软骨细胞病理学变化及细胞超微结构的改变。结果TCDD在5～15μg／kg浓度下诱导了大鼠骨骼发育畸形,畸形包括：内翻足、脊柱裂、腭裂、无尾畸形等,并存在剂量依赖性生物学效应;光镜下可见在畸形胎鼠的肢端骨化中心软骨发生带缩小,软骨细胞变性。透射扫描电镜下见软骨细胞核内粗面内质网扩张,核基质降解,线粒体嵴不规则。结论在高雌激素水平下,TCDD可以诱导大鼠骨骼发育畸形;TCDD可能通过干扰软骨细胞的形态和功能代谢,引起原发性骨化中心的结构紊乱而发挥骨骼致畸效应。     

Ossification sequence of the avian order anseriformes,with comparison to other precocial birds

Ossification sequences are poorly known for most amniotes, and yet they represent an important source of morphogenetic, phylogenetic, and life history information. Here, the author describes the ossification sequences of three ducks, the Common Eider Somateria mollissima dresseri, the Pekin Duck Anas platyrhynchos, and the Muscovy Duck Cairina moschata. Sequence differences exist both within and among these species, but are generally minor. The Common Eider has the most ossified skeleton prior to hatching, contrary to what is expected in a subarctic migrant species. This may be attributed to a tradeoff between growth rate and locomotory performance. Growth rate is higher in hatchlings with more cartilaginous skeletons, but this may compromise locomotion. No major ossification sequence differences were observed in the craniofacial skeleton when compared with Galliformes, which suggests that the influence of adult morphology on ossification sequence might be relatively minor in many taxa. Galliformes and Anseriformes, while both highly ossified at hatching, differ in the location of their late‐stage ossification centers. In Anseriformes, these are most often located in the appendicular skeleton, whereas in Galliformes they are in the thoracic region and form the ventilatory apparatus. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

The structure and prenatal morphogenesis of the nasal septum in the rat

One hundred fetuses of the Sprague-Dawley rat were used: ten for each prenatal day, beginning with the twelfth day of gestation. Pregnant animals were sacrificed, fetuses removed and subsequently fixed in buffered formalin solution. Fetal heads were dehydrated, embedded in paraffin, and sectioned serially in the rostrocaudal direction at 10 to 15 μ. Serial sections from fetuses representing each day of gestation were stained with either H and E, Mallory's trichrome procedure, Gomori's reaction for alkaline phosphatase, or Steedman's alcian blue reaction. At the twelfth day, the primary nasal cavities were first observed. One day later, the nasobuccal membrane was established, and the vomeronasal organ invaginated into the nasal septum. Following the rupture of the membrane, at the fourteenth day, the nasal and buccal cavities remained in communication until the palatal shelves fused with the septum, at the seventeenth day. Prior to the thirteenth day, the septal skeleton is mesenchymal. The ossification in the vomer started at the sixteenth day and expanded progressively throughout prenatal life. First glandular primordia, one on each side of the septum, were observed during the sixteenth day, the number increased to five at term. The ducts ended in single blind sacs, before the eighteenth day, afterwards, the ducts presented an increasing number of collateral and terminal branches. There was no evidence of mucigen secretion from the septal glands during prenatal life. The initial stratified olfactory epithelium differentiated morphologically into a vestibular, respiratory, and an olfactory epithelium prior to the sixteenth prenatal day.     

Studies on skeleton formation in reptiles. I. The postembryonic development of the skeleton in Cyrtodactylus pubisulcus (Reptilia: Gekkonidae)

The study of ossification during postembryonic development of the lizard Cyrtodactylus pubisulcus reveals consistent patterns in the skeleton of the body axis and of the limbs. The vertebral column shows a distinct antero-posterior gradient in ossification; the serial homology of sacral ribs and caudal transverse processes with dorsal ribs requires further scrutiny. The sequence of ossification of carpal and tarsal elements is constant, yet different from the pattern of chondrification as described in the literature. The homology of a separate 'intermedium' in the ossified lizard carpus requires further discussion. The development of the lizard astragalus is discussed in detail, as is the ossification of epiphyses in the limbs.     

Skeletal ossification and sequence heterochrony in xenarthran evolution

Previous analyses of how mammals vary in their ossification sequences have focused on monotremes, marsupials, and boreoeutherian placentals. Here, we focus on the sequence of cranial and postcranial ossification events during growth in the xenarthran skull and skeleton, including armadillos, anteaters, and sloths. We use two different methods to quantify sequence heterochrony: sequence analysis of variance (ANOVA) and event‐paring/Parsimov. Our results indicate that Parsimov is conservative and does not detect clear heterochronic shifts between xenarthran and boreoeutherian placentals. Sequence‐ANOVA performs better, but both methods exhibit sensitivity to the artifactual accumulation of ties. By controlling for ties and taking into account results that the methods have in common, our analysis suggests that xenarthrans significantly differ from other placentals by a late ossification of the sternum and an early ossification of the phalanges and pubis. We interpret these differences as showing that heterochrony plays a role in the skeletal development of xenarthrans, a change from previous studies that have emphasized the developmental homogeneity of the skeleton across placental mammals.     

Cleft palate following prenatal food restriction in mice: association with elevated maternal corticosteroids.

Following food restriction of pregnant CD-1 mice, the maternal serum corticosteroid levels and incidence of skeletal and visceral abnormalities of the progeny were determined. Dams from the groups receiving restricted food on gestation days 6 to 15 had increased resorptions, and the progeny had delayed ossification of the skeleton with cleft palate accompanied by delayed ossification of the presphenoid bone. The incidence and intensity of these changes was correlated with the level of food restriction. The incidence of cleft palate for progeny from dams receiving 4.0 to 2.5 g of feed per day ranged from 6.3% to 100.0%, respectively. Serum corticosteroid levels of pregnant control and food restricted dams were similar on gestation day 10, but the levels of food restricted dams were 6 to 10 times greater than controls on gestation day 15. Thus cleft palate, delayed skeletal ossification, and increased resorptions are predictably produced by food restriction of CD-1 mice and were highly correlated with the elevated endogenous serum corticosteroid levels.     

Radiographic determination of prenatal basicranial ossification

In a previous investigation on prenatal development of the human cranial base, the sequence in which the bones develop in the midsagittal region was elucidated. The purpose of the present study was to identify fetal ossification on horizontal plane roentgenograms of the occipital and sphenoid bones in the central part of the cranial base, and establish stages in bone appearance related to general fetal developmental parameters. This study is based upon roentgenograms of the cranial base of 145 human fetuses from the first half of the prenatal period. Two different maturation patterns of the sphenoid bone were observed. The first, most common pattern is characterized by a midsagittal centre of ossification and the second by bilateral centres of ossification in the corpus of the sphenoid bone. These bilateral centres might in some cases be connected by a slight bony bridge. It appears that these different maturation patterns are maintained throughout the period investigated. The material was divided into five well-defined developmental stages for both maturation patterns and general parameters of fetal development. Mapping different aspects of ossification in normal cranial development is necessary for understanding deviations of cranial maturation and growth.     

Comparative ossification sequence and skeletal development of the postcranium of palaeognathous birds (Aves: Palaeognathae)

Palaeognaths constitute one of the most basal lineages of extant birds, and are also one of the most morphologically diverse avian orders. Their skeletal development is relatively unknown, in spite of their important phylogenetic position. Here, we compare the development of the postcranial skeleton in the emu (Dromaius novaehollandiae), ostrich (Struthio camelus), greater rhea (Rhea americana) and elegant crested‐tinamou (Eudromia elegans), focusing on ossification. All of these taxa are characterized by element loss in the appendicular skeleton, but there are several developmental mechanisms through which this loss occurs, including failure to chondrify, failure to ossify and fusion of cartilages prior to ossification. Further evidence is presented here to support a reduction in size of skeletal elements resulting in a delay in the timing of ossification. This study provides an important first look at the timing and sequence of postcranial ossification in palaeognathous birds, and discusses the influence of changes in the pattern of skeletal development on morphological evolution.     

Matrix remodeling during endochondral ossification

Endochondral ossification, the process by which most of the skeleton is formed, is a powerful system for studying various aspects of the biological response to degraded extracellular matrix (ECM). In addition, the dependence of endochondral ossification upon neovascularization and continuous ECM remodeling provides a good model for studying the role of the matrix metalloproteases (MMPs) not only as simple effectors of ECM degradation but also as regulators of active signal-inducers for the initiation of endochondral ossification. The daunting task of elucidating their specific role during endochondral ossification has been facilitated by the development of mice deficient for various members of this family. Here, we discuss the ECM and its remodeling as one level of molecular regulation for the process of endochondral ossification, with special attention to the MMPs.