Cranial Ossification Research Articles

Ossification of Atlantic cod (Gadus morhua) – Developmental stages revisited

In studies of marine larvae, it is common to use days post-hatch as a developmental reference point. We show that age is a poor measure of morphological and physiological development in Atlantic cod. Therefore, we propose a set of five developmental stages of Atlantic cod from start-feeding until the juvenile stage, based on cranial ossification as previously done in Atlantic halibut. Cod follows a sequence of cranial ossification that is to a large extent preserved in most fish species examined. These stages are therefore tools to standardize sampling and to reduce growth dependent variation in the analysis of larvae during development. We show that several developmental stages are present in the same rearing unit at a given time. We also demonstrate that nutrition during early development is a vital foundation for robust skeletal development. Cod larvae supplied with copepods instead of rotifers followed by Artemia, develop less skeletal deformities at 10cm standard length, despite given the same formulated feed from 1.8cm standard length and onwards. Statement of relevanceThis paper provides developmental stages that are vital for best practice protocols in aquaculture. By relating farming practices to developmental stages and not age, the right treatment ect may be provided.This manuscript does also highlight the importance of nutrition during live feed stages on events that may occur late in the production cycle.

Prenatal cranial ossification of the humpback whale (Megaptera novaeangliae).

Being descendants of small terrestrial ungulate mammals, whales underwent enormous transformations during their evolutionary history, that is, extensive changes in anatomy, physiology, and behavior were evolved during secondary adaptations to life in water. However, still only little is known about whale ontogenetic development, which help to identify the timing and sequence of critical evolutionary events, such as modification of the cetacean ear. This is particularly true for baleen whales (Mysticeti), the group including the humpback whale Megaptera novaeangliae. We use high-resolution X-ray computed tomography to reinvestigate humpback whale fetuses from the Kükenthal collection at the Museum für Naturkunde, Berlin, thus, extending historic descriptions of their skeletogenesis and providing for the first time sequences of cranial ossification for this species. Principally, the ossification sequence of prenatal Megaptera follows a typical mammalian pattern with the anterior dermal bones being the first ossifying elements in the skull, starting with the dentary. In contrast to other mammals, the ectotympanic bone ossifies at an early stage. Alveolar structure can be observed in both the maxillae and dentaries in these early prenatal specimens but evidence for teeth is lacking. Although the possibility of obtaining new embryological material is unlikely due to conservation issues, our study shows that reexamination of existing specimens employing new technologies still holds promise for filling gaps in our knowledge of whale evolution and ontogeny.

Assessment of modularity in the urodele skull: an exploratory analysis using ossification sequence data.

The potential presence of developmental modules is studied in the urodele skull using several classical statistical methods that have not previously been used in this context. Principal component analysis (PCA) of ossification sequence data on 21 bones in 21 extant urodele species suggests the presence of up to four developmental modules, but examination of statistically significant correlations using phylogenetic independent contrasts (PIC) and correcting for multiple tests using the false discovery rate suggests the presence of only two modules of uneven size and of two bones that may not be part of these modules. Thus, PCA does not appear to be a reliable method to investigate modularity; direct investigation of statistically significant correlations using PIC or other phylogeny-informed methods is recommended. A binomial test of the distribution of significant correlations between characters shows significant heterogeneity, which suggests that modularity is indeed present in the data. A cluster analysis gives inconsistent results that apparently do not reflect developmental modules. The data include a phylogenetic signal, as shown by a permutation-based test with squared change parsimony, but this is detectable only when the whole matrix is analyzed, and a plot of the tree onto developmental space through Evolutionary PCA shows that homoplasy is pervasive. Evolutionary rates between characters vary about 90-fold. Canonical variates analyses suggest that obligatorily neotenic urodeles may be discriminated from other urodeles on the basis of cranial ossification sequence data.

Comparative skeletogenesis of the Oriental Tree Frog Hyla orientalis (Anura: Hylidae)

The ontogeny of skeletal development of the Oriental Tree Frog, Hyla orientalis (Bedriaga, 1890), is followed from tadpole to froglet and compared with four other hylid species i.e., Hypsiboas lanciformis, Hypsiboas pulchellus, Phyllomedusa vaillanti and Pseudis platensis. Our analysis and comparison is based on cleared and double-stained specimens. The parasphenoid is the first skeletal element that ossified in Hyla orientalis is followed by the exoocipital, prootic, axial and appendicular skeletal elements, frontoparietal, nasal, upper jaw, squamosal, pterygoid, lower jaw, vomer and quadratojugal. Ossification initiated at early Stage 33 in Hyla orientalis whereas in other hylids it begins after Stage 37. Major transformations of the larval jaw were visible after Stage 42. Cranial ossifications were completed after appendicular and axial ossifications of Hyla orientalis. All skeletal elements ossified before the end of the metamorphosis, a unique feature of Hyla orientalis in comparison to the other hylids studied here.

Ultrasonic diagnosis of hip dysplasia in rachitic pathology in infants (1180.14)

Objective: to identify the signs of hip dysplasia in a large clinical material along with the symptoms of rickets (sweating, alopecia of the occipital region of the head, muscular hypotonia, irritability child).Materials: 935 children were observed at the CCH No 2 in Astana for 2010 with hip joint pathology, of whom up to 6 months, 56.6% after 6 months ‐ 32,6% to be conservative treatment and 10.8% children received surgical treatment.Results: Ultrasonography in 35.8% children 2‐3 months of age revealed a sharp flattening of the bone and bone bay window roof (angle d ranged between 34 ‐39), cartilaginous portion of the acetabulum has been shifted cranial ossification nucleus head femur was missing. At the age of 4‐5 months during treatment in the stirrups Pavlik appeared signs of the formation of the femoral head in the form of separate small “clumps” in 46.5% children in the remaining 36.5%, these terms are shifted to 6‐7 months of age (the angle d 48‐52). Only by 9‐10 months gradually underwent a process of mergers and the formation of a homogeneous core of ossification center of the femoral head (the angle d approaching 56‐58), cartilaginous portion of the roof of the acetabulum was relatively short. By the year 33% of patients the size of the hip bone formation were reduced, and only 67% of patients taking shape hip well‐formed, bone bay took a rectangular shape, cartilaginous part of the femoral head is completely covered).

Statistical support for the hypothesis of developmental constraint in marsupial skull evolution

BackgroundIn contrast to placental neonates, in which all cranial bones are ossified, marsupial young have only the bones of the oral region and the exoccipital ossified at birth, in order to facilitate suckling at an early stage of development. In this study, we investigated whether this heterochronic shift in the timing of cranial ossification constrains cranial disparity in marsupials relative to placentals.MethodsWe collected three-dimensional (3D) landmark data about the crania of a wide range of extant placentals and marsupials, and from six fossil metatherians (the clade including extant marsupials and their stem relatives), using a laser scanner and a 3D digitizer. Principal components analysis and delta variance tests were used to investigate the distribution and disparity of cranial morphology between different landmark sets (optimizing either number of landmarks or number of taxa) of the whole skull and of individual developmental or functional regions (neurocranium, viscerocranium, oral region) for extant placentals and marsupials. Marsupial and placental data was also compared based on shared ecological aspects including diet, habitat, and time of peak activity.ResultsWe found that the extant marsupial taxa investigated here occupy a much smaller area of morphospace than the placental taxa, with a significantly (P<0.01) smaller overall variance. Inclusion of fossil taxa did not significantly increase the variance of metatherian cranial shape. Fossil forms generally plotted close to or within the realm of their extant marsupial relatives. When the disparities of cranial regions were investigated separately, significant differences between placentals and marsupials were seen for the viscerocranial and oral regions, but not for the neurocranial region.ConclusionThese results support the hypothesis of developmental constraint limiting the evolution of the marsupial skull, and further suggest that the marsupial viscerocranium as a whole, rather than just the early-ossifying oral region, is developmentally constrained.

Comparative skull morphology of uropeltid snakes (Alethinophidia: Uropeltidae) with special reference to disarticulated elements and variation.

Uropeltids form a diverse clade of highly derived, fossorial snakes that, because of their phylogenetic position among other alethinophidian lineages, may play a key role in understanding the early evolution of cranial morphology in snakes. We include detailed osteological descriptions of crania and mandibles for eight uropeltid species from three nominal genera (Uropeltis, Rhinophis, and Brachyophidium) and emphasize disarticulated elements and the impact of intraspecific variation on previously proposed morphological characters used for phylogenetic analysis. Preliminary analysis of phylogenetic relationships strongly supports a clade composed exclusively of species of Plectrurus, Uropeltis, and Rhinophis. However, monophyly of each of those genera and Melanophidium is not upheld. There is moderate support that Sri Lankan species (e.g., Rhinophis and Uropeltis melanogaster) are monophyletic with respect to Indian uropeltids. Previously proposed characters that are phylogenetically informative include the shape of the nasals, length of the occipital condyle, level of development of the posteroventral process of the dentary, and participation of the parietal in the optic foramen. Additionally, thirty new features that may be systematically informative are identified and described, but were not verified for their utility. Such verification must await availability of additional disarticulated cranial material from a larger sample of taxa. All characters require further testing through increased focus on sources and patterns of intraspecific variation, inclusion of broader taxonomic samples in comparative studies, and exploration of skeletal development, sexual dimorphism, and biogeographic patterns. Additionally, trends in the relative enlargement of the sensory capsules, reduction in cranial ossification and dentition, fusion of elements, and the appearance of novel morphological conditions, such as the structure and location of the suspensorium, may be related to fossoriality and miniaturization in some uropeltid taxa, and may complicate analysis of relationships within Uropeltidae and among alethinophidian snakes.

Principles of cranial base ossification in humans and rats

Conclusions: 1. The principle of bilateral symmetry depends on the chordal cartilage that is the keystone in cranial base ossification in rats and humans, due to its anatomical situation and for the production of the chordin protein that regulates the bone morphogenetic protein BMP-7. 2. In humans and in rats, foramen lacerum closure follows a line of intramembranous ossification that depends on BMP-7, regulated by the first branchial pouch. 3. The cranial base ossification patterns and centres are similar in humans and in rats, except in the otic capsule, palate and the lateral pterygoid plate. 4. The neural crest may induce cranial ossification through the cranial nerves. Objectives: To study the patterns of cranial base ossification in humans and in rats, considering the chordal cartilage, and the otic, nasal and orbit capsules, as well as the participation of the branchial arches and pouches. Methods: This was a light microscopy study of human fetal specimens obtained from spontaneous abortions with the following crown-rump-lengths (crl) 45, 74, 90, 134, 145 and 270 mm, and a 1-day-old neonate (360 mm crl), who had died of sudden death syndrome. We also examined Webster albino rat embryos of 16, 18 and 20 days of gestation and a postnatal series of rats 8 h and 1, 3, 4, 6, 7, 10 and 13 days old, as well as adult animals. Results: In the 45 mm human fetus, the chordal cartilage with the nasal, otic and orbit capsules initiates cranial base ossification. Foramen lacerum closure begins in the 16-day-old rat embryo, following a line of membranous ossification between the external pterygoid process and the lateral alisphenoidal wing at ovalis foramen level. This is not a timing symmetrical process, which may persist until the 10th postnatal day in the rat. In the human fetus of 74 mm, the foramen lacerum space is closed by a membranous fusion ossification between the chordal cartilage and otic capsule, finishing at the 270 mm specimen. Endochondral ossification of the human otic capsule first appeared in the 145 mm (18 weeks) fetal specimen with four ossifying centres. The rat otic cartilaginous capsule showed rapid endochondral ossification, in the third and fourth postnatal day specimens.

Potential For Intracranial Movements in Pterosaurs

Based on comparative anatomical, morphological, and phylogenetic considerations the potential of pterosaurs for cranial kinesis is assessed. Our investigation shows that whereas skeletally mature derived pterodactyloids have completely fused, rigid and doubtlessly akinetic skulls, skeletally immature derived pterodactyloids and more basal pterosaurs possess key features in the morphology of their otic and basal joints that are suggestive of cranial kinesis, namely streptostyly. In addition, pterosaurs exhibit an evolutionarily informative trend in the degree of cranial ossification, where it is low in most nonpterodactyloids (here named bifenestratans), intermediate in Rhamphorhynchus and Archaeopterodactyloidea, and high in derived pterodactyloids. Incomplete fusion could also indicate loose connections between skull elements. However, another crucial anatomical requirement of a kinetic skull, the permissive kinematic linkage is absent in all pterosaurian taxa. The fact, that the presence of permissive kinematic linkages in the skull is also a prerequisite of all types of cranial kinesis, provides hard evidence that all members of Pterosauria had akinetic skulls. Thus, the presence of the morphological attributes indicative of intracranial movements in some pterosaurs must be explained on grounds other than real potential for cranial kinesis. It could either be of mechanical or ontogenetic importance, or both. Alternatively, it might be considered as the morphological remnant of a real, kinetic skull possessed by the diapsid ancestors of pterosaurs.

Occipital Ossification of Balaenopteroid Mysticetes

The bones of the posterior portion of the mammalian skull often exhibit incomplete ossification of the joints between the bones at the time of birth, with complete ossification at some point after birth. The sequence of ossification of these joints in mysticetes can be used to characterize the relative age in the calf and early juvenile ontogenetic stages. This study examined occipital joints ossification of 38 dry prepared neonate specimens in four mysticete species from two families (Eschrichtiidae: Eschrichtius robustus; Balaenopteridae: Balaenoptera acutorostrata, Balaenoptera physalus, and Megaptera novaeangliae). Each of the joints responsible for the fusion of the occiput were examined and rated for degree of ossification. The cranial ossification analysis indicates that E. robustus calves have open occipital joints until ~6 months of age and are born at a less mature stage than closely related balaenopterids. All of the species followed the same sequence of ossification: basioccipital/exoccipital joint, followed by the basioccipital/basisphenoid joint, and completed by the supraoccipital/exoccipital joint.

Heterochrony and developmental modularity of cranial osteogenesis in lipotyphlan mammals.

BackgroundHere we provide the most comprehensive study to date on the cranial ossification sequence in Lipotyphla, the group which includes shrews, moles and hedgehogs. This unique group, which encapsulates diverse ecological modes, such as terrestrial, subterranean, and aquatic lifestyles, is used to examine the evolutionary lability of cranial osteogenesis and to investigate the modularity of development.ResultsAn acceleration of developmental timing of the vomeronasal complex has occurred in the common ancestor of moles. However, ossification of the nasal bone has shifted late in the more terrestrial shrew mole. Among the lipotyphlans, sequence heterochrony shows no significant association with modules derived from developmental origins (that is, neural crest cells vs. mesoderm derived parts) or with those derived from ossification modes (that is, dermal vs. endochondral ossification).ConclusionsThe drastic acceleration of vomeronasal development in moles is most likely coupled with the increased importance of the rostrum for digging and its use as a specialized tactile surface, both fossorial adaptations. The late development of the nasal in shrew moles, a condition also displayed by hedgehogs and shrews, is suggested to be the result of an ecological reversal to terrestrial lifestyle and reduced functional importance of the rostrum. As an overall pattern in lipotyphlans, our results reject the hypothesis that ossification sequence heterochrony occurs in modular fashion when considering the developmental patterns of the skull. We suggest that shifts in the cranial ossification sequence are not evolutionarily constrained by developmental origins or mode of ossification.

A large-scale survey of heterochrony in anuran cranial ossification patterns

Most anurans have a biphasic life cycle, which includes metamorphosis from a tadpole stage to an adult frog. This process involves extensive transformations of the cranial skeleton, which have been of long-standing interest with respect to anuran skeletal evolution and taxonomy. In this study, large-scale patterns of anuran skeletal ossification are assessed by collecting the most comprehensive data set on anuran cranial ossification to date from the literature, including data for 45 anuran and one caudate outgroup species. Ossification sequences were translated into event-pair matrices for explorative phylogenetic analysis and phylogenetically informed parsimony search for heterochrony using the Parsimov algorithm. Rank variability of single bones across species was also analysed. Little phylogenetic signal was retrieved from a parsimony-based phylogenetic analysis of event-pairs, and only a few species that are generally agreed to be closely related are placed close to each other (e.g. some Pipidae and Costata). Parsimov analysis revealed some clade-specific heterochrony in anuran clades of varying inclusiveness. Our results show that relating heterochronic changes in anuran cranial ontogeny to parameters such as direct development or miniaturization is problematic because of the high evolvability of cranial ossification sequences. Rank variation analysis suggests that anuran cranial bones are highly variable in their sequence positioning, possibly because tadpole and adult cranial morphology do not co-evolve. Elements which are lost in some species ossify at the end of the sequence, providing evidence for the notion that failure of anuran cranial elements to ossify is due to processes of paedomorphosis. Die meisten Anura (Froschlurche) zeichnen sich durch einen biphasischen Lebenszyklus aus, während dessen sie eine Metamorphose vom Kaulquappenstadium zum adulten Frosch durchlaufen. Die Metamorphose geht mit weitreichenden Umformungen im kraniellen Skelett einher. Dieses Phänomen stößt seit langem im Rahmen von Fragestellungen mit Bezug auf Skelettevolution und taxonomische Klassifizierung auf Interesse. In der vorliegenden Studie werden anhand der bisher umfassendsten Datenerhebung, basierend auf der Literatur der letzten 50 Jahre, die Reihenfolge der Verknöcherung im Kranialskelett der Anura untersucht. Einbezogen wurden kranielle Verknöcherungs-sequenzen von 45 Vertretern der Anura und einem Vertreter der Caudata (Schwanzlurche). Die Sequenzen wurden als ‘Event-pairs’ kodiert, um eine informelle phylogenetische Analyse nach dem Sparsamkeitsprinzip sowie phylogenetisch basierte Heterochronieanalysen unter Anwendung des Parsimov-Algorithmus durchzuführen. Außerdem wurde die zwischenartliche Variabilität des Verknöcherungszeitpunktes innerhalb der Sequenz für die einzelnen Knochen untersucht. Die phylogenetische Analyse gruppierte einige Arten, welche generell als nahverwandt angesehen werden, z.B. einige Vertreter der Pipiden und Costata. Die Parsimov-Analyse zeigte, daß sich nur wenige Anurengruppen unterschiedlicher Diversität durch Heterochronien ausweisen. Unsere Ergebnisse zeigen, dass eine kausale Verbindung von Heterochronien mit Eigenschaften wie Direktentwicklung problematisch ist. Die Analyse der Verknöcherungsvariabilität weist auf große Variabilität des Verknöcherungs-Zeitpunkt der einzelnen Knochen hin; dies kann möglicherweise durch die relativ unabhängige Evolution von Larval- und Adultschädelmorhpologie erklärt werden. Elemente, die in einigen Arten nicht vorkommen, verknöchern generell am Ende der Sequenz. Dies spricht für die Annahme, daß der Verlust von Schädelknochen bei Anuren auf Pädomorphose zurückzuführen ist.

Looking closer at the determining of a phenotype? Compare by stages or size, not age

Summary During ontogeny, individual fish larvae and juveniles will develop at different paces despite apparently identical age and environment, and even genetic background. The urgent need to elucidate mechanisms critical to juvenile production, recruitment or even lifelong growth patterns implies that measures and results are compared by a meaningful standard such as developmental stage of the individual fish. Characterizations of developmental stages based on relatively invariable characteristics such as the sequence of cranial ossification have proven to be not only commercially and scientifically applicable in Atlantic halibut, but also point to a practical use for a character which is preserved in most fishes examined. Once described, useful proxies for stage determinants may be size parameters such as standard length, or preferably myotome height (particularly for flatfishes). The worst proxy for development of a batch of fish is age, exacerbated by the expense of analyzing irrelevant intervals.

A tale of two deletions: A report of two novel 20p13 → pter deletions

We report on two patients with 1.7 and 1.2 Mb terminal 20p deletions, which have apparently not been reported previously. Both individuals exhibit certain similar features including large fontanelles, ear abnormalities, and seizures. However, even though the deletions are of similar size, there were many disparate features between the two. The deletions in each patient encompass at least 28 genes that may provide useful candidates for ear development and cranial ossification.

Chondrogenic and ossification patterns and sequences in White's skink Liopholis whitii (Scincidae, Reptilia)

The prenatal patterns and sequences of chondrogenesis and ossification are described for the skink Liopholis whitii; eight embryos and one adult specimen were examined. Cranial ossification begins in elements of the braincase and the palate, followed by areas of the lower jaw and the cranial roof. Ossification proceeds anteroposteriorly along the axial skeleton and from proximal to distal in the forelimbs and hind limbs. In the zeugopods, the ossification progresses from posterior to anterior, while there is also a strict postaxial order in the autopods with the predominance of digit III in the forelimb and digit IV in the hind limb. Ossification of the femur and tibia starts before the corresponding elements of the forelimb, but simultaneously with the onset in clavicles. Elements of the hind limb display advanced ossification with respect to their forelimb counterparts, with earlier uptake of Alizarin Red in the tarsals, metatarsals and phalanges. The preaxial area of the fused astragalocalcaneum-complex is first to begin ossification among the tarsals. The sequence of ossification is mostly conserved in the skeleton of squamates, as shown in a comparative analysis of heterochrony, but some shifts were found in cranial, vertebral and autopodial regions. (© 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)

Comparative ossification and development of the skull in palaeognathous birds (Aves: Palaeognathae)

Ratites and tinamous are a morphologically diverse group of flightless and weakly flighted birds. As one of the most basal clades of extant birds, they are frequently used as an outgroup for studies discussing character evolution within other avian orders. Their skeletal development is not well known in spite of their important phylogenetic position, and studies have historically been plagued with small sample sizes and limited anatomical and temporal scope. Here, I describe the ossification of the skull in the emu (Dromaius novaehollandiae), ostrich (Struthio camelus), greater rhea (Rhea americana), and elegant crested-tinamou (Eudromia elegans). Skeletal development is remarkably consistent within palaeognaths, in spite of large differences in absolute size and incubation period. Adult morphology appears to play a role in interordinal differences in the sequence and timing of ossification of certain bones. Neither the timing of cranial ossification events relative to stage nor the sequence of ossification events provides any evidence in support of a paedomorphic origin of the palaeognathous palate. This study provides an important first look at the timing and sequence of skull development in palaeognathous birds, providing data that can be compared to better-studied avian systems in order to polarize ontogenetic characters. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 156, 184–200.

Cranial Osteogenesis and Suture Morphology in Xenopus laevis: A Unique Model System for Studying Craniofacial Development

BackgroundThe tremendous diversity in vertebrate skull formation illustrates the range of forms and functions generated by varying genetic programs. Understanding the molecular basis for this variety may provide us with insights into mechanisms underlying human craniofacial anomalies. In this study, we provide evidence that the anuran Xenopus laevis can be developed as a simplified model system for the study of cranial ossification and suture patterning. The head structures of Xenopus undergo dramatic remodelling during metamorphosis; as a result, tadpole morphology differs greatly from the adult bony skull. Because of the extended larval period in Xenopus, the molecular basis of these alterations has not been well studied.Methodology/Principal FindingsWe examined late larval, metamorphosing, and post-metamorphosis froglet stages in intact and sectioned animals. Using micro-computed tomography (μCT) and tissue staining of the frontoparietal bone and surrounding cartilage, we observed that bone formation initiates from lateral ossification centers, proceeding from posterior-to-anterior. Histological analyses revealed midline abutting and posterior overlapping sutures. To determine the mechanisms underlying the large-scale cranial changes, we examined proliferation, apoptosis, and proteinase activity during remodelling of the skull roof. We found that tissue turnover during metamorphosis could be accounted for by abundant matrix metalloproteinase (MMP) activity, at least in part by MMP-1 and -13.ConclusionA better understanding of the dramatic transformation from cartilaginous head structures to bony skull during Xenopus metamorphosis may provide insights into tissue remodelling and regeneration in other systems. Our studies provide some new molecular insights into this process.

A new species of Trachycephalus (Amphibia, Anura, Hylidae) from the State of Goiás, Brazil

A new species of Trachycephalus is described for the Cerrado biome of Goiás, Brazil. Trachycephalus mambaiensis sp. nov. is distinguished from the other ten species of the genus by the skin co-ossified with the skull, heavy cranial ossification, frontoparietal that fails to articulate with squamosal, absence of a crista occipitalis and secreting glands of milky and viscous substances. The skull of the new species shows an intermediary condition between species of Trachycephalus with a well ossified skull (Casque-headed frogs) and those without cranial ossification.

Number of Ossification Centers in the Anuran Cranial Bones Depends upon the Rate of Development: Experimental Evidence

Cranial development was compared in Rana temporaria (Anura; Ranidae) and Bombina variegata (Anura; Discoglossidae) specimens reared at normal, high, and low level of thyroid hormone (TH). Sequence and timing of cranial ossification as well as rate of a particular bone’s differentiation and growth depend upon TH level. Also, bones which normally develop from a single ossification center (frontoparietal, parasphenoid, vomer, squamosal, angulosplenial, septomaxilla, pterygoid, and premaxilla) were revealed to form from two or more ossification centers in specimens reared at high and low TH level. This indicates them to develop from several early precalcified anlagen which normally fuse prior to the beginning of calcification thus appearing as a single ossification center. Changes in the rate of a particular bone’s differentiation and growth caused by manipulation with a TH level may cause precocious beginning of their calcification or their delayed fusion. As a result, they begin to calcify prior to their fusion thus appearing as several separate ossification centers. Number of ossification centers depends upon the rate of cranial and overall somatic development. Interspecies and individual differences in the rate of cranial and overall ontogeny are responsible for the inter- and intraspecific variability in the number of ossification centers recorded in anurans.

Effects of water hardness on skeletal development and growth in juvenile fathead minnows

Skeletal growth in vertebrates is partially controlled by the availability of minerals. The correlation between aqueous calcium content and fish skeletal growth (“ossification”) is especially important as calcium absorption from water is an essential route to acquire this mineral. In this study, we test the hypothesis that a calcium-abundant aquatic environment will expedite larval skeletal development in fathead minnows beyond the skeletal development in a low-calcium environment. Fathead minnows were chosen for this study as they are a widely used toxicological model organism with well established rearing protocols. We divided three batches each of fathead minnow eggs from a colony supplied with calcium-abundant water (Ca = 65 ± 1.5 mg/L as CaCO 3; hardness > 175 mg/L) and one with a low, yet adequate calcium content (Ca = 15 ± 0.11 mg/L as CaCO 3; hardness < 50 mg/L) to rear fish. Half of each egg batch was exchanged between laboratories and fish were then reared to 60 days post-hatch under similar conditions. At 60 days, juveniles were sacrificed, cleared and stained. Ossification patterns for cranial (jaw) and caudal (fin) structures were quantified using NIH Image ( http://rsb.info.nih.gov/nih-image/). Fish reared in calcium-abundant water had significantly increased survival but lower whole body mass when compared to their conspecifics in low-calcium water. Cranial ossification patterns did not vary. Caudal fin ossification was statistically significant, but slightly and surprisingly enhanced in juveniles reared in low-calcium water. All observed differences were independent of the origin of fish and imply differences in fish growth among cultures maintained under similar rearing conditions for the purpose of this study. Results of this investigation have implications for the rearing of model organisms for toxicological studies and need to be amended by physiological explorations.