PATTERNS OF OSSIFICATION IN SOUTHERN VERSUS NORTHERN PLACENTAL MAMMALS

Consensus on placental mammal phylogeny is fairly recent compared to that for vertebrates as a whole. A stable phylogenetic hypothesis enables investigation into the possibility that placental clades differ from one another in terms of their development. Here, we focus on the sequence of skeletal ossification as a possible source of developmental distinctiveness in “northern” (Laurasiatheria and Euarchontoglires) versus “southern” (Afrotheria and Xenarthra) placental clades. We contribute data on cranial and postcranial ossification events during growth in Afrotheria, including elephants, hyraxes, golden moles, tenrecs, sengis, and aardvarks. We use three different techniques to quantify sequence heterochrony: continuous method, sequence‐ANOVA (analysis of variance) and event‐paring/Parsimov. We show that afrotherians significantly differ from other placentals by an early ossification of the orbitosphenoid and caudal vertebrae. Our analysis also suggests that both southern placental groups show a greater degree of developmental variability; however, they rarely seem to vary in the same direction, especially regarding the shifts that differ statistically. The latter observation is inconsistent with the Atlantogenata hypothesis in which afrotherians are considered as the sister clade of xenarthrans. Interestingly, ancestral nodes for Laurasiatheria and Euarchontoglires show very similar trends and our results suggest that developmental homogeneity in some ossification sequences may be restricted to northern placental mammals (Boreoeutheria).     

Phosphorylation-dependent regulation of skeletogenesis in sea urchin micromere-derived cells and embryos

Sea urchin embryo micromeres when isolated and cultured in vitro differentiate to produce spicules. Although several authors have used this model, almost nothing is known about the signaling pathways responsible for initiating skeletogenesis. In order to investigate the potential involvement of phosphorylation events in spiculogenesis, the effect of inhibitors of protein kinases and phosphatases on skeleton formation was studied. Results obtained using both cultured micromeres and embryos revealed that protein tyrosine kinase and phosphatase inhibitors blocked skeleton formation, but not serine/threonine phosphatase inhibitors. The inhibitors showed a dose-dependent effect and when removed from micromere or embryo culture, spicule formation resumed. Inhibition of tyrosine phosphatases resulted in an increase in the tyrosine phosphorylation level of two major proteins and a modest decrease in the expression of the mRNA coding for type I fibrillar collagen. These findings strongly suggest that tyrosine phosphorylation and dephosphorylation is required for micromere differentiation and for normal skeletogenesis during sea urchin embryo development.     

Ontogeny and tissue differentiation of the pelvic girdle and hind limbs of anurans

We present the ontogeny of the integrated musculoskeletal complex that comprises the pelvic girdle and hind limbs of anurans. Our histological data show that the pelvic girdle originates from a single mesenchymatic condensation. The tissue differentiation sequence is cartilage, muscle and tendon. The intrusion of the ischiadic nerve into the limb bud is produced very early in ontogeny. The pre‐cartilage appears in the pre‐motile stage. Therefore, the nerve produces a movement analogous to the ‘embryonic motility’ that would induce the emergence of the pre‐cartilage. The acetabulum is the first of all cavitation processes to form, the second one being the knee. The acetabulum appears before the muscles are mature, although it has been stressed that the muscle contraction maintains joint progenitors committed to their fate. Our data indicate an explosive differentiation of all 11 muscular masses together. We provide three new characters that support the monophyly of Hyloides, Acosmanura and Neobatrachia.     

Synovial joint formation requires local Ext1 expression and heparan sulfate production in developing mouse embryo limbs and spine

Heparan sulfate proteoglycans (HSPGs) regulate a number of major developmental processes, but their roles in synovial joint formation remain unknown. Here we created conditional mouse embryo mutants lacking Ext1 in developing joints by mating Ext1^f/f and Gdf5-Cre mice. Ext1 encodes a subunit of the Ext1/Ext2 Golgi-associated protein complex responsible for heparan sulfate (HS) synthesis. The proximal limb joints did form in the Gdf5-Cre;Ext1^f/f mutants, but contained an uneven articulating superficial zone that expressed very low lubricin levels. The underlying cartilaginous epiphysis was deranged as well and displayed random patterns of cell proliferation and matrillin-1 and collagen IIA expression, indicative of an aberrant phenotypic definition of the epiphysis itself. Digit joints were even more affected, lacked a distinct mesenchymal interzone and were often fused likely as a result of local abnormal BMP and hedgehog activity and signaling. Interestingly, overall growth and lengthening of long bones were also delayed in the mutants. To test whether Ext1 function is needed for joint formation at other sites, we examined the spine. Indeed, entire intervertebral discs, normally composed by nucleus pulposus surrounded by the annulus fibrosus, were often missing in Gdf5-Cre;Ext1^f/f mice. When disc remnants were present, they displayed aberrant organization and defective joint marker expression. Similar intervertebral joint defects and fusions occurred in Col2-Cre;β-catenin^f/f mutants. The study provides novel evidence that local Ext1 expression and HS production are needed to maintain the phenotype and function of joint-forming cells and coordinate local signaling by BMP, hedgehog and Wnt/β-catenin pathways. The data indicate also that defects in joint formation reverberate on, and delay, overall long bone growth.     

Development of calcareous skeletal elements in invertebrates

Most metazoans require skeletal support systems. While the formation of bones and teeth in vertebrates has been well studied, endo- and exoskeleton development of non-vertebrates, especially calcification during terminal differentiation, has been neglected. Biomineralization of skeletons in invertebrates presents interesting research opportunities. We undertake here to survey some of the better understood examples of skeletal development in selected invertebrates. The differentiation of the skeletal spicules of euechinoid larvae and other non-vertebrate deuterostomes, the shells of molluscs, and the calcification of crustacean carapaces are surveyed. The diversity of these different kinds of animals and our present limited understanding make it difficult to identify unifying themes, but there certainly are unifying questions: How is the mineral precursor secreted? What is the nature of the interaction of mineral with the matrix proteins of the skeleton? Is there any conservation of protein domains in matrix proteins found in skeletal elements from different phyla? Are there common strategies in the development of organs that form mineralized structures?     

Role of CCN,a vertebrate specific gene family,in development

The CCN family of genes constitutes six members of small secreted cysteine rich proteins, which exists only in vertebrates. The major members of CCN are CCN1 (Cyr61), CCN2 (CTGF), and CCN3 (Nov). CCN4, CCN5, and CCN6 were formerly reported to be in the Wisp family, but they are now integrated into CCN due to the resemblance of their four principal modules: insulin like growth factor binding protein, von Willebrand factor type C, thrombospondin type 1, and carboxy‐terminal domain. CCNs show a wide and highly variable expression pattern in adult and in embryonic tissues, but most studies have focused on their principal role in osteo/chondrogenesis and vasculo/angiogenesis from the aspect of migration, growth, and differentiation of mesenchymal cells. CCN proteins simultaneously integrate and modulate the signals of integrins, bone morphogenetic protein, vascular endothelial growth factor, Wnt, and Notch by direct binding. However, the priority in the use of the signals is different depending on the cell status. Even the equivalent counterparts show a difference in signal usage among species. It may be that the evolution of the CCN family continues to keep pace with vertebrate evolution itself.