An Automated Technique for Double Staining Rat and Rabbit Fetal Skeletal Specimens to Differentiate Bone and Cartilage

Assessment of chemicals for their potential to cause developmental toxicity must include evaluation of the development of the fetal skeleton. The method described here is an improved and fully automated double staining method using alizarin red S to stain bone and alcian blue to stain cartilage. The method was developed on the enclosed Shandon PathcentreTM, and the quality of specimens reported here will be reproduced only if carried out on a similar processor under the same environmental conditions. The staining, maceration and clearing process takes approximately 6 days. The personnel time, however, is minimal since solutions are changed automatically and the fetuses are not examined or removed from the processor until the procedure is completed. Upon completion of processing, the bone and cartilage assessment of the specimens can be carried out immediately if required. Full evaluation of skeletal development in both the rat and the rabbit is necessary to meet the requirements of safety assessment studies. This method allows this to be accomplished on a large scale with consistently clear specimens and in a realistic time.     

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。     

Sequential changes in weight of the skeleton and in length of long limb bones of Macaca mulatta.

In a collection of 274 monkeys (Macaca mulatta) the relative weight of the dry, fat-free skeleton, expressed as a proportion of total body weight, increases significantly throughout the gestational period to approximately 6% with only random variation after birth. The weight of the fetal skeleton increases exponentially with age. In the postnatal period the skeletal weight increases asymptotically to adulthood, which is considered to be 6.5 years of age. Equations for estimating skeletal weight are presented. Of four subdivisions of the skeleton, the skul contributes the greatest proportion of total skeletal weight in the fetal stage with the proportion decreasing to adulthood. The contributions of the other subdivisions, postcranial axial, superior limb, and inferior limb, and inferior limb, are nearly equal in the fetal stage, with that of only the inferior limb increasing to adulthood, when it makes up the greatest proportion of total skeletal weight. Until the last third of the gestational period, the humerus is longer than the femur and the radius longer than the tibia. Thereafter, the inferior limbs grow at a faster rate than the superior limbs, resulting in an intermembral index of approximately 95% by birth and less than 90% by adulthood.     

Micro‐CT evaluation of murine fetal skeletal development yields greater morphometric precision over traditional clear‐staining methods

Traditional techniques for quantification of murine fetal skeletal development (gross measurements, clear‐staining) are severely limited by specimen processing, soft tissue presence, diffuse staining, and unclear landmarks between which to make measurements. Nondestructive microcomputed tomography (micro‐CT) imaging is a versatile, well‐documented tool traditionally used to generate high‐resolution 3‐D images and quantify microarchitectural parameters of trabecular bone. Although previously described as a tool for phenotyping fetal murine specimens, micro‐CT has not previously been used to directly measure individual fetal skeletal structures. Imaging murine fetal skeletons using micro‐CT enables the researcher to nondestructively quantify fetal skeletal development parameters including limb length, total bone volume, and average bone mineral density, as well as identify skeletal malformations. Micro‐CT measurement of fetal limb lengths correlates well with traditional clear‐staining methods (83.98% agreement), decreases variability in measurements (average standard errors: 6.28% for micro‐CT and 10.82% for clear‐staining), decreases data acquisition time by eliminating the need for tissue processing, and preserves the intact fixed fetus for further analysis. Use of the rigorous micro‐CT technique to generate 3‐D images for digital measurement enables isolation of skeletal structures based on degree of mineralization (local radiodensity), eliminating the complications of blurred stain boundaries and soft tissue inclusion that accompany clear‐staining and gross measurement techniques. Microcomputed tomography provides a facile, accurate, and nondestructive method for determining the developmental state of the fetal skeleton using not only limb lengths and identification of malformations, but total skeletal bone volume and average skeletal mineral density as well. Birth Defects Res (Part B) 2008. © 2008 Wiley‐Liss, Inc.     

Atlas of rat fetal skeleton double stained for bone and cartilage

BACKGROUND: The double staining of fetal skeleton for bone and cartilage is a very useful method to evidence skeletal abnormalities in laboratory animals. However, this method has been rarely used in routine developmental toxicity tests. One reason could be the difficulty of comparing the single skeletal pieces and of having reference points. In this paper the fetal rat skeleton double stained with Alizarin red S and Alcyan Blue is described in detail to produce an atlas for developmental toxicity laboratories.     

Pecularities of bony skeleton development in Asian clawed salamanders (Onychodactylus, Hynobiidae) related to embryonization

We studied skull, vertebral column, and limb skeleton development in Japanese clawed salamander Onychodactylus japonicus (Hynobiidae). The study is based on the ontogenetic series of embryos and larvae obtained from wild-captured adults by artificial induction of breeding using hormonal stimulation. The first stages of the skeleton formation in O. japonicus are shifted to the late embryonic period and hatching larvae already possess a well-ossified vertebral column, large number of skull ossifications and show signs of ossification in the forelimb skeleton. Compared to the primitive pattern of the skeleton development typical for other hynobiid salamanders, O. japonicus shows a number of heterochronies related to embryonization. In particular, this species is characterized by an earlier ossification of the vertebral column compared to that of the skull and by the delayed development and early reduction of the coronoid. Our results, along with the previously reported data on the skeleton development in the Fischer’s clawed salamander O. fischeri (Smirnov and Vassilieva, 2002), indicate that the genus Onychodactylus is characterized by the loss or reduction of several skeletal features typically found at early larval stages in other Hynobiidae species. In particular, provisional bones (especially the coronoid) and their dentition are underdeveloped. In addition, it is corroborated that the first tooth generation is absent in Onychodactylus, whereas such monocuspid nonpedicellate tooth generation normally develops at the early larval stages of other caudate amphibians. Since similar patterns of skeleton ontogeny are observed in other caudate groups with different extent of embryonization, it is proposed that, in different lineages of Urodela, the evolution of ontogeny followed similar pathways and was accompanied by the same changes in skeletogenesis.     

Skeletal adaptations during mammalian reproduction

Remarkable changes occur in the mammalian skeleton prior to, during and after the reproductive cycle. Skeletal changes occur with ovarian maturation and initiation of menses and estrus in adolescence, which may result in a greater accumulation of skeletal mineral in the female vs the male skeleton. There is also some evidence to suggest an excess skeletal mass in young female experimental animals. In early pregnancy, growth, modeling and perhaps suppressed remodeling promote the accumulation of calcium. Some changes may also occur with the transition from pituitary to placental control of the pregnancy. In later pregnancy, an increase in bone turnover appears to coincide with fetal skeletal mineralization. Rapid and important changes occur in the skeleton and mineral metabolism in the transition from pregnancy to lactation as the mammary gland rather than the uterus draws on the maternal calcium stores. Lactational demands are met at least partially by a temporary demineralization of the skeleton, which is associated with increased bone modeling and remodeling. Endochondral growth almost ceases during lactation, but envelope-specific bone modeling and remodeling are greatly increased. This is generally associated with a loss of skeletal mass and density, more apparent at sites with less of a mechanical role (e.g. central metaphysis regions and the endocortical envelope). The post-lactational period is profoundly anabolic with substantial increases in bone formation, but blunted resorption at almost all skeletal envelopes. Skeletal mass is increased during this period and it is associated with improved skeletal mechanical properties. There are several important observations. 1) The nulliparous animal appears to have an excess skeletal mass to perhaps compensate for maternal metabolic inefficiency of the first reproductive cycle. 2) Changes in growth, modeling and remodeling occur at different times and at different skeletal envelopes during the reproductive cycle. These site-specific, temporal changes appear to be adaptations that facilitate the use of skeletal mineral while preserving mechanical competence. 3) After the first reproductive cycle, modeling and remodeling optimize the existing skeletal mass into a structure that better accommodates the prevailing mechanical environment. 4) The post-lactational period is profoundly anabolic and may provide new strategies for preservation of skeletal mass when reproductive capacity ceases.     

Sensitive Windows of Skeletal Development in Rabbits Determined by Hydroxyurea Exposure at Different Times throughout Gestation

The critical periods of axial skeletal development in rats and mice have been well characterized, however the timing of skeletal development in rabbits is not as well known. It is important to have a more precise understanding of this timing of axial skeletal development in rabbits due to the common use of this species in standard nonclinical studies to assess embryo–fetal developmental toxicity. Hydroxyurea, a teratogen known to induce a variety of fetal skeletal malformations, was administered to New Zealand White rabbits as a single dose (500 mg/kg) on individual days during gestation (gestation day,GD 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 19) and fetal external, visceral, and skeletal morphology was examined following cesarean sections on GD 29. A wide range of fetal skeletal effects was observed following hydroxyurea treatment, with a progression of malformations from anterior to posterior structures over time, as well as from proximal to distal structures over time. The sensitive window of axial skeletal development was determined to be GD 8 to 13, while disruption of appendicular and cranio‐facial skeletal development occurred primarily from GD 11 to 16 and GD 11 to 12, respectively. The results of this study provide a better understanding of the critical developmental window for different segments of the rabbit skeleton, which will aid in the design of window studies to investigate teratogenicity in rabbits.     

The Developmental Basis of Skeletal Cell Differentiation and the Molecular Basis of Major Skeletal Defects

Vertebrate skeletal differentiation retains elements from simpler phyla, and reflects the differentiation of supporting tissues programmed by primary embryonic development. This developmental scheme is driven by homeotic genes expressed in sequence, with subdivision of skeletal primordia driven by a combination of seven transmembrane‐pass receptors responding to Wnt‐family signals, and by bone morphogenetic family signals that define borders of individual bones. In sea‐dwelling vertebrates, an essentially complete form of the skeleton adapted by the land‐living vertebrates develops in cartilage, based on type II collagen and hydrophilic proteoglycans. In bony fishes, this skeleton is mineralized to form a solid bony skeleton. In the land‐living vertebrates, most of the skeleton is replaced by an advanced vascular mineralized skeleton based on type I collagen, which reduces skeletal mass while facilitating use of skeletal mineral for metabolic homeostasis. Regulation of the mammalian skeleton, in this context, reflects practical adaptations to the needs for life on land that are related to ancestral developmental signals. This regulation includes central nervous system regulation that integrates bone turnover with overall metabolism. Recent work on skeletal development, in addition, demonstrates molecular mechanisms that cause developmental bone diseases.     

Simultaneous Differential Staining of Cartilage and Bone in Rodent Fetuses: an Alcian Blue and Alizarin Red S Procedure without Glacial Acetic Acid

Differential staining of cartilage and bone has several applications including developmental toxicology studies of new chemical candidates for pharmaceutical, industrial, and environmental use. It has been more common to stain fetal bone only using the dye alizarin red S: however, failure to evaluate the cartilaginous portion of the skeleton may result in the failure to identify toxicologically important alterations in skeletal morphology. Previously, differential staining of fetal cartilage and bone was best achieved by combining alizarin red S for staining bone with alcian blue to stain cartilage in glacial acetic acid solution: however, occupational hazards posed by the use of glacial acetic acid make these methods undesirable. Replacement of the glacial acetic acid with potassium hydrogen phthalate eliminates these hazards without compromising the quality of the stained specimen.     

Appendicular skeletal morphology in minute salamanders,genus Thorius (amphibia: Plethodontidae): Growth regulation,adult size determination,and natural variation

Patterns of growth and variation of the appendicular skeleton were examined in Thorius, a speciose genus of minute terrestrial plethodontid salamanders from southern Mexico. Observations were based primarily on ontogenetic series of each of five species that collectively span the range of adult body size in the genus; samples of adults of each of seven additional species provided supplemental estimates of the full range of variation of limb skeletal morphology. Limbs are generally reduced, i.e., pedomorphic, in both overall size and development, and they are characterized by a pattern of extreme variation in the composition of the limb skeleton, especially mesopodial elements, both within and between species. Fifteen different combinations of fused carpal or tarsal elements are variably present in the genus, producing at least 18 different overall carpal or tarsal arrangements, many of which occur in no other plethodontid genus. As many as four carpal or tarsal arrangements were observed in single population samples of each of several; five tarsal arrangements were observed in one population of T. minutissimus. Left-right asymmetry of mesopodial arrangement in a given specimen is also common. In contrast, several unique, nonpedomorphic features of the limb skeleton, including ossification of the typically cartilaginous adult mesopodial elements and ontogenetic increase in the degree of ossification of long bones, are characteristic of all species and distinguish Thorius from most related genera. They form part of a mechanism of determinate skeletal growth that restricts skeletal growth after sexual maturity. Interspecific differences in the timing of the processes of appendicular skeletal maturation relative to body size are well correlated with interspecific differences in mean adult size and size at sexual maturity, suggesting that shifts in the timing of skeletal maturation provide a mechanism of achieving adult size differentiation among species. Processes of skeletal maturation that confer determinate skeletal growth in Thorius are analogous to those typical of most amniotes – both groups exhibit ontogenetic reduction and eventual disappearance of the complex of stratified layers of proliferating and maturing cartilage in long bone epiphyses – but, unlike most amniotes, Thorius lacks secondary ossification centers. Thus, the presence of secondary ossification centers cannot be used as a criterion for establishing determinate skeletal growth in all vertebrates.     

Integration of embryonic and fetal skeletal myogenic programs at the myosin light chain 1f/3f locus

The genetic control of skeletal muscle differentiation at the onset of myogenesis in the embryo is relatively well understood compared to the formation of muscle during the fetal period giving rise to the bulk of skeletal muscle fibers at birth. The Mlc1f/3f (Myl1) locus encodes two alkali myosin light chains, Mlc1f and Mlc3f, from two promoters that are differentially regulated during development. The Mlc1f promoter is active in embryonic, fetal and adult fast skeletal muscle whereas the Mlc3f promoter is upregulated during fetal development and remains on in adult fast skeletal muscle. Two enhancer elements have been identified at the mammalian Mlc1f/3f locus, a 3′ element active at all developmental stages and an intronic enhancer activated during fetal development. Here, using transgenesis, we demonstrate that these enhancers act combinatorially to confer the spatial, temporal and quantitative expression profile of the endogenous Mlc3f promoter. Using double reporter transgenes we demonstrate that each enhancer can activate both Mlc1f and Mlc3f promoters in vivo, revealing enhancer sharing rather than exclusive enhancer-promoter interactions. Finally, we demonstrate that the fetal activated enhancer contains critical E-box myogenic regulatory factor binding sites and that enhancer activation is impaired in vivo in the absence of myogenin but not in the absence of innervation. Together our observations provide insights into the regulation of fetal myogenesis and the mechanisms by which temporally distinct genetic programs are integrated at a single locus.     

Visualizing normal and defective bone development in zebrafish embryos using the fluorescent chromophore calcein.

Zebrafish have recently become a model of choice among developmental biologists. This unique model enables both modern molecular and genetic studies to be carried out to identify genes involved in a wide variety of developmental processes. The success of the genetic approach depends largely on the application of an easy and effective screening method to identify interesting mutants. In order to develop a method for visualizing skeletal structures in zebrafish embryos that would be suitable for screening skeletal mutants, we investigated the use of the fluorescent chromophore calcein, which binds specifically to calcified skeletal structures. By using this method, we followed the development of the skeletal structures in zebrafish embryos from day 1 to day 21 postfertilization, and analyzed the effect of bone morphogenetic protein-2 (BMP2) on axial skeleton development. We found the development of the calcified skeletal structure to appear in a progressive fashion from head to tail. Calcified structures in the head (i.e., the jaw) developed first, which were then followed by the axial skeleton in the trunk. Interesting to note was that there appeared to be two domains in the calcification of vertebrae within the axial skeleton. The first three vertebrae were in the first domain; the rest being in the second domain. Compared with Alcian blue staining, we found that calcein staining indeed labels calcified skeletal structures, and, moreover, it is a more sensitive and inclusive method for visualizing skeletal structures. To determine whether calcein staining could also be used to detect abnormal bone development, we ectopically expressed BMP2 in zebrafish notochord cells. We demonstrated that ectopic expression of BMP2 in notochord cells inhibited the development of the axial skeleton. Together, these results clearly demonstrated the sensitivity of calcein staining for visualizing bone structures in developing zebrafish embryos and its effectiveness for screening for mutants that have bone structure defects.     

Skeletal form and growth in Acropora aspera (Dana) from the Pulau Seribu,Indonesia

Linear extension and calcium carbonate accretion were measured in the branching coral Acropora aspera (Dana) from shallow-water sites around Pulau Pari, Pulau Seribu, Indonesia, during both wet and dry monsoon periods. Skeletal density and corallite form were also monitored in specimens collected from sites, variously affected by wave energy resulting from the monsoonal influence. Although the reversing monsoon appeared to exert the greatest effect on skeleton growth (by influencing temperature and possibly number of “sun-hours”) wave energy was also shown to affect skeletal extension, skeletal accretion, and skeletal density. The scale of differences between growth rate measurements was greatest for weight of skeleton accreted between monsoon period (8-fold), followed by between site differences (maximum 3-fold during west monsoon) and finally between station differences (maximum 3-fold during west monsoon at an outer reef flat and reef edge station). Skeletal extension did not appear to be as sensitive to the reversing monsoon influence as skeletal accretion.     

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.     

Calcium Availability Influences Litter Size and Sex Ratio in White-Footed Mice (Peromyscus leucopus)

The production of offspring typically requires investment of resources derived from both the environment and maternal somatic reserves. As such, the availability of either of these types of resources has the potential to limit the degree to which resources are allocated to reproduction. Theory and empirical studies have argued that mothers modify reproductive performance relative to exogenous resource availability and maternal condition by adjusting size, number or sex of offspring produced. These relationships have classically been defined relative to availability of energy sources; however, in vertebrates, calcium also plays a critical role in offspring production, as a considerable amount of calcium is required to support the development of offspring skeleton(s). We tested whether the availability of calcium influences reproductive output by providing female white-footed mice with a low-calcium or standard diet from reproductive maturity to senescence. We then compared maternal skeletal condition and reproductive output, based on offspring mass, offspring number and litter sex ratio, between dietary treatments. Mothers on the low-calcium diet exhibited diminished skeletal condition at senescence and produced smaller and strongly female-biased litters. We show that skeletal condition and calcium intake can influence sex ratio and reproductive output following general theoretical models of resource partitioning during reproduction.     

Genetic regulation of osteoclast development and function

Osteoclasts are the principal, if not exclusive, bone-resorbing cells, and their activity has a profound impact on skeletal health. So, disorders of skeletal insufficiency, such as osteoporosis, typically represent enhanced osteoclastic bone resorption relative to bone formation. Prevention of pathological bone loss therefore depends on an appreciation of the mechanisms by which osteoclasts differentiate from their precursors and degrade the skeleton. The past five years have witnessed important insights into osteoclast formation and function. Many of these discoveries have been made through genetic experiments that involved the rare hereditary disorder osteopetrosis.     

Skeletal development in <Emphasis Type="Italic">Acropora palmata</Emphasis> (Lamarck 1816): a scanning electron microscope (SEM) comparison demonstrating similar mechanisms of skeletal extension in axial versus encrusting growth

Many Acropora palmata colonies consist of an encrusting basal portion and erect branches. Linear growth of the skeleton results in extension along the substrate (encrusting growth), lengthening of branches (axial growth) and thickening of branches and crust (radial growth). Scanning Electron Microscopy is used to compare the mechanisms of skeletal extension between encrusting growth and axial growth. In encrusting growth, the distal margin of the skeleton lacks corallites (which develop about 1 mm from the edge); in contrast, in axial growth, axial corallites along the branch tip form the distal portion of the skeleton. In both locations, the distal margin of the skeleton consists of a lattice-like structure composed of rods that extend from the body of the skeleton and bars that connect these rods. An actively extending skeleton is characterized by sharply pointed rods and partially developed bars. Distal growth of rods (and formation of bars) is effected by the formation of new sclerodermites. Each sclerodermite begins with the deposition of fusiform crystals (that range in length from 1 to 5 μm). These provide a surface for nucleation and growth of spherulitic tufts, clusters of short (<1 μm long) aragonite needles. The needles that are oriented perpendicular to the axis of the skeletal element (rod or bar), and perpendicular to the overlying calicoblastic epithelium, continue extension to appear on the surface of the skeleton as 10–15 μm wide bundles (of needle tips) called fasciculi. However, some crusts that abut competitors for space have a different morphology of skeletal elements (rods and bars). The distal edge of these crusts terminates in blunt coalescing rods, and bars that are fully formed. Absence of fusiform crystals, lack of sharply pointed rods and bars, and full development of sclerodermites characterize a skeletal region that has ceased, perhaps only temporarily, skeletal extension.     

Skeletal maturation of the human fetus assessed radiographically on the basis of ossification sequences in the hand and foot

In studies of postnatal human development the skeletal maturation of the hand has been found to be a better indicator of general physical maturation than attained body height. For assessment of prenatal human development the Crown-rump length (CRL) has so far been the most commonly used measure. The object of the present study is to examine the possibility of also using the skeletal maturation of the hand as a maturity indicator in fetal development. The study is based upon a radiographic and histochemical investigation of 169 human fetuses. On the basis of counting silver-impregnated diaphyses on radiographs of the hand and foot a maturity indicator (CNO = Composite Number of Ossified bones in hand and foot) was established. Owing to the marked regularity of the recorded ossification pattern, the CNO parameter can be used for evaluating fetal maturation during the early half of the prenatal period. To supplement the assessment of skeletal maturation during the later stages of development, a classification based on the shape of some bones was included in the study. In many cases fetuses of the same size (CRL) exhibited different stages of skeletal maturation (CNO). In accordance with findings from assessment of postnatal development, a more accurate evaluation of fetal development is obtained by combining the size parameter CRL with an assessment of fetal skeletal maturation, CNO.     

Ontogenic appearance of Na+ channels characterized as high affinity binding sites for tetrodotoxin during development of the rat nervous and skeletal muscle systems

The appearance of the voltage-dependent Na+ channel during the fetal and post-natal development of rat brain, cerebellum and skeletal muscle has been followed using a highly radiolabelled derivative of tetrodotoxin. The number of Na+ channels is low at the fetal stage and increases drastically during post-natal development. The time-course of this increase is different in brain, cerebellum and skeletal muscle. Changes in affinity of the Na+ channel for tetrodotoxin occur during brain and cerebellum development. The results are discussed in relation with the maturation of the three types of excitable tissues.