The ontogenic development of the vertebrae in some gekkonoid lizards

The ontogeny of amphicoelous vertebrae was studied in Ptyodactylus hasselquistii and Hemidactylus turcicus, and that of procoelous vertebrae, in Sphaerodactylus argus. The embryos were assigned arbitrary stages, drawn to scale, and mostly studied in serial sections. Resegmentation occurs as in all amniotes. A sclerocoel divides each sclerotome into an anterior “presclerotomite” and a denser posterior “postsclerotomite.” Tissue surrounding the intersegmental boundary forms the centrum, which is intersegmental. Tissue around the sclerocoel builds the intervertebral structures, which are midsegmental. In the trunk and neck, postsclerotomites form neural arches, and presclerotomites build zygapophyses. The adult centrum consists of the perichordal primary centrum, plus neural arch bases (= secondary centrum). Between the latter and the arch proper, a neurocentral suture persists until obliterated in maturity. A dorso-ventral central canal persists on either side of the primary centrum, between the latter and the secondary centrum. The notochord becomes true cartilage midvertebrally in all vertebrae, and elastic cartilage intervertebrally in the posterior caudal region. Elsewhere its characteristic tissue persists. Intervertebrally, cervical hypapophyses, caudal chevrons and chevron-bases in the trunk are preformed early in cartilage. Directly ossifying median intercentra are added later in all regions. The first cervical presclerotomite is absent: the hypapophysis (= corpus) of the atlas consists exclusively of postsclerotomitic tissue, there is no proatlas, and the odontoid lacks the apical half-centrum present in other lepidosaurians. In the autotomous caudal region presclerotomites are as prominent as postsclerotomites. Both build neural arches, the two arches of each vertebra remaining distinct and ossifying separately, so that the intersegmental autotomy split persists between them. The last sclerotome is complete, its postsclerotomite forming a half centrum which ossifies. In Sphaerodactylus, while the vertebrae ossify, each intervertebral ring becomes concave anteriorly, convex posteriorly; it remains as a cushion between the condyle and a facet formed by differential growth of the centra. Thus these procoelous centra resemble the amphicoelous centra of Ptyodactylus and Hemidactylus, rather than the procoelus centra of other squamates. The vertebral column of Gekkonoidea closely resembles in its development and microscopical structure that of Sphenodon.     

A central role for the notochord in vertebral patterning

The vertebrates are defined by their segmented vertebral column, and vertebral periodicity is thought to originate from embryonic segments, the somites. According to the widely accepted 'resegmentation' model, a single vertebra forms from the recombination of the anterior and posterior halves of two adjacent sclerotomes on both sides of the embryo. Although there is supporting evidence for this model in amniotes, it remains uncertain whether it applies to all vertebrates. To explore this, we have investigated vertebral patterning in the zebrafish. Surprisingly, we find that vertebral bodies (centra) arise by secretion of bone matrix from the notochord rather than somites; centra do not form via a cartilage intermediate stage, nor do they contain osteoblasts. Moreover, isolated, cultured notochords secrete bone matrix in vitro, and ablation of notochord cells at segmentally reiterated positions in vivo prevents the formation of centra. Analysis of fss mutant embryos, in which sclerotome segmentation is disrupted, shows that whereas neural arch segmentation is also disrupted, centrum development proceeds normally. These findings suggest that the notochord plays a key, perhaps ancient, role in the segmental patterning of vertebrae.     

Notochord-dependent expression of MFH1 and PAX1 cooperates to maintain the proliferation of sclerotome cells during the vertebral column development.

During axial skeleton development, the notochord is essential for the induction of the sclerotome and for the subsequent differentiation of cartilage forming the vertebral bodies and intervertebral discs. These functions are mainly mediated by the diffusible signaling molecule Sonic hedgehog. The products of the paired-box-containing Pax1 and the mesenchyme forkhead-1 (Mfh1) genes are expressed in the developing sclerotome and are essential for the normal development of the vertebral column. Here, we demonstrate that Mfh1 like Pax1 expression is dependent on Sonic hedgehog signals from the notochord, and Mfh1 and Pax1 act synergistically to generate the vertebral column. In Mfh1/Pax1 double mutants, dorsomedial structures of the vertebrae are missing, resulting in extreme spina bifida accompanied by subcutaneous myelomeningocoele, and the vertebral bodies and intervertebral discs are missing. The morphological defects in Mfh1/Pax1 double mutants strongly correlate with the reduction of the mitotic rate of sclerotome cells. Thus, both the Mfh1 and the Pax1 gene products cooperate to mediate Sonic hedgehog-dependent proliferation of sclerotome cells.     

Evidence for resegmentation in the formation of the vertebral column using the novel approach of retroviral-mediated gene transfer

We have examined the somitic cell contribution to the vertebral column of the chick by genetic labeling of sclerotomal cells in early development. Single somites of embryonic Day 2 embryos were filled with retroviral particles containing the lacZ transducing vector BAG. After a further 14 or 17 days of incubation the embryos were fixed and the vertebral column was sectioned and stained histochemically for the lacZ gene product beta-galactosidase. Cells staining for the enzyme were found exclusively on the injected side of two vertebral segments; the staining was largely restricted, however, to the caudal half of the more rostral segment and the rostral half of the next more caudal segment. No embryos were observed with labeling in less than two vertebral segments. Moreover, labeled cells were not uniformly distributed within the labeled region of each vertebra; the neural arch, for example, usually contained a higher proportion of labeled cells than did the centrum. These observations support the concept of resegmentation, whereby a vertebra forms from sclerotomal cells derived from two consecutive somites resulting in a vertebral column shifted by one half segment with respect to the segmented boundaries of the somites. The quantitative distribution of labeled cells in the vertebrae also suggests that sclerotomal cells populate the region of a future vertebral segment in an orderly fashion dependent on when the cells migrate from the somite.     

The floor plate is sufficient for development of the sclerotome and spine without the notochord

Danforth'sshort-tail (Sd) mouse is a semi-dominant mutation affecting the development of the vertebral column. Although the notochord degenerates completely by embryonic day 9.5, the vertebral column exists up to the lumber region, suggesting that the floor plate can substitute for notochord function. We previously established the mutant mouse line, Skt(Gt), through gene trap mutagenesis and identified the novel gene, Skt, which was mapped 0.95cM distal to the Sd locus. Taking advantage of the fact that monitoring notochordal development and genotyping of the Sd locus can be performed using the Skt(Gt) allele, we assessed the development of the vertebra, notochord, somite, floor plate and sclerotome in +-+/+-Skt(Gt), Sd-+/+-+, Sd-Skt(Gt)/+-+, Sd-Skt(Gt)/+-Skt(Gt), Sd-+/Sd-+ and Sd-Skt(Gt)/Sd-Skt(Gt) embryos. In Sd homozygous mutants with a C57BL/6 genetic background, the vertebral column was truncated in the 6th thoracic vertebra, which was more severe than previously reported. The floor plate and sclerotome developed to the level of somite before notochord degeneration and the number of remaining vertebrae corresponded well with the level of development of the floor plate and sclerotome. Defects to the sclerotome and subsequent vertebral development were not due to failure of somitogenesis. Taken together, these results suggest that the notochord induced floor plate development before degeneration, and that the remaining floor plate is sufficient for maintenance of differentiation of the somite into the sclerotome and vertebra in the absence of the notochord.     

Histology and histochemistry of the gekkotan notochord and their bearing on the development of notochordal cartilage

The persistence of the notochord into the skeletally mature life stage is characteristic of gekkotans, but is otherwise of rare occurrence among amniotes. The taxonomic diversity of Gekkota affords the opportunity to investigate the structure and development of this phylogenetically ancestral component of the skeleton, and to determine its basic characteristics. The gekkotan notochord spans almost the entire postcranial long axis and is characterized by a moniliform morphology with regularly alternating zones of chordoid and chondroid tissue. Chordoid tissue persists in the region of intervertebral articulations and occupies the cavitations that lie between the centra of the amphicoelous vertebrae. Chondroid tissue is restricted to zones in which the diameter of the notochord is reduced, corresponding to mid‐vertebral locations. In the tail, these zones of chondroid tissue are associated with the autotomic fracture planes. Chondroid tissue first manifests during late embryogenesis, appears to differentiate from pre‐existing chordoid tissue, and has the histological and histochemical characteristics of cartilage. Our observations lend support to the hypothesis that cartilage can be derived directly from notochordal tissue, and suggest that the latter may be an evolutionary and developmental precursor to chordate cartilage. The persistence of chordoid tissue in the intervertebral regions of amphicoelous vertebrae is consistent with a suite of paedomorphic traits exhibited by gekkotans and suggests that the typical hydrostatic nature of notochordal tissue may play a role in mechanically governing patterns of displacement between adjacent amphicoelous vertebrae that lack extensive centrum‐to‐centrum contact. Morphol., 2012. © 2012 Wiley Periodicals, Inc.     

A study of the development of thoracic vertebrae in the mouse assisted by autoradiography

Evidence is accumulating that the current concept of resegmentation during vertebral formation is no longer valid. In order to shed some light on this controversial issue, in the present investigation the development of the vertebral column was studied in a graded series of mouse embryos by conventionally stained serial sections and methyl thymidine 3H autoradiography. It was found that the vertebral bodies do not originate from the upper and lower halves of sclerotomes but from a continuous central tissue mass surrounding the notochord, the perichordal tube. The caudal sclerotome half gives rise to the neural arch and the transverse processes, whereas the cranial half forms the connective tissue of the interneural arch space. An intrasclerotomic cleft supposed to form the intervertebral cleft was not found to exist, in accordance with previous studies. The inferred intrasclerotomic cleft is merely the interface between the loose tissue of the cranial sclerotome half and the densely packed cells of the caudal half.     

Epimorphin acts extracellularly to promote cell sorting and aggregation during the condensation of vertebral cartilage

Formation of vertebrae occurs via endochondral ossification, a process involving condensation of precartilaginous cells. Here, we provide the first molecular evidence of mechanism that underlies initiation of this process by showing that the extracellular factor, Epimorphin, plays a role during early steps in vertebral cartilage condensation. Epimorphin mRNA is predominantly localized in the vertebral primordium. When provided exogenously in ovo, it causes precocious differentiation of chondrocytes, resulting in the formation of supernumerary vertebral cartilage in chicken embryos. To further analyze its mode of action, we used an in vitro co-culture system in which labeled 10T1/2 or sclerotomal prechondrogenic cells were co-cultured with unlabeled Epimorphin-producing cells. In the presence of Epimorphin, the labeled cells formed tightly packed aggregates, and sclerotomal cells displayed augmented accumulation of NCAM and other early markers of chondrocyte differentiation. Finally, we found that the Epimorphin expression is initiated during vertebrogenesis by Sonic hedgehog from the notochord mediated by Sox 9. We present a model in which successive action of Epimorphin in recruiting and stacking sclerotomal cells leads to a sequential elongation of a vertebral primordium.     

A culture system for the live analysis of successive developmental processes and the morphological control of mammalian vertebral cartilage

The mesoderm-derived segmental somite differentiates into dermomyotome and sclerotome, the latter of which undergoes vertebrogenesis to spinal cartilage and ultimately to vertebral bones. However, analysis and manipulation of the developing mammalian vertebrae in the same embryo has been infeasible because of their placental-dependent embryogenesis. Here, we report a novel culture system of the mouse embryonic tailbud, by which the developmental processes of mammalian vertebral cartilage are traceable and manipulatable in the same sample. The anaplastic segmental somites/sclerotomes in the tailbud of 13 gestational day (g.d.) embryo that are structurally continuous to the vertebral column underwent progressive vertebrogenesis when (1) the ectoderm-derived nascent epidermis was microsurgically removed prior to cultivation, and (2) the sample was incubated at the air-medium interface. After cultivation for 5 days, the size and shape of the instructed vertebral cartilage showed features comparable to well-differentiated body vertebra along with the expression of the cartilage marker collagen type II, suggesting that aggressive differentiation of the sclerotomal cell lineage was achieved. In the presence of recombinant bone morphogenic protein (BMP) and Noggin, or adenoviral particles for extracellular epimorphin, dramatic alteration of the vertebral morphology ensued in the explants. Thus, this model system provides an approach to study the detailed molecular mechanisms of mammalian vertebrogenesis and enables pretreatment strategies of precartilagious fragments for improving the efficacy of subsequent transplantation.     

Embryonic development of the axial column in the little skate,Leucoraja erinacea

The morphological patterns and molecular mechanisms of vertebral column development are well understood in bony fishes (osteichthyans). However, vertebral column morphology in elasmobranch chondrichthyans (e.g., sharks and skates) differs from that of osteichthyans, and its development has not been extensively studied. Here, we characterize vertebral development in an elasmobranch fish, the little skate, Leucoraja erinacea , using microCT, paraffin histology, and whole‐mount skeletal preparations. Vertebral development begins with the condensation of mesenchyme, first around the notochord, and subsequently around the neural tube and caudal artery and vein. Mesenchyme surrounding the notochord differentiates into a continuous sheath of spindle‐shaped cells, which forms the precursor to the mineralized areolar calcification of the centrum. Mesenchyme around the neural tube and caudal artery/vein becomes united by a population of mesenchymal cells that condenses lateral to the sheath of spindle‐shaped cells, with this mesenchymal complex eventually differentiating into the hyaline cartilage of the future neural arches, hemal arches, and outer centrum. The initially continuous layers of areolar tissue and outer hyaline cartilage eventually subdivide into discrete centra and arches, with the notochord constricted in the center of each vertebra by a late‐forming “inner layer” of hyaline cartilage, and by a ring of areolar calcification located medial to the outer vertebral cartilage. The vertebrae of elasmobranchs are distinct among vertebrates, both in terms of their composition (i.e., with centra consisting of up to three tissues layers—an inner cartilage layer, a calcified areolar ring, and an outer layer of hyaline cartilage), and their mode of development (i.e., the subdivision of arch and outer centrum cartilage from an initially continuous layer of hyaline cartilage). Given the evident variation in patterns of vertebral construction, broad taxon sampling, and comparative developmental analyses are required to understand the diversity of mechanisms at work in the developing axial skeleton of vertebrates. J. Morphol. 278:300–320, 2017. © 2017 Wiley Periodicals, Inc.     

Aspects of vertebral development in lizards and snakes

The embryonic and postnatal development of vertebrae of the mid-trunk and mid-tail in Lacerta vivipara, Anguis fragilis and Natrix natrix have been studied. Centra and neural arches develop independently after the perichordal tube has been formed, the neurocentral suture representing a consistent boundary between them. The condyle and cup of each centrum are formed by differential growth of cartilage; in Anguis and Natrix the joints between centra are synovial. The zygosphenial joints of Lacerta are continuous with the zygapophysial joints and not separate from them as in snakes. The autotomy splits in the tail vertebrae of Lacerta are derived from four distinct components which become continuous in postnatal life. The chordal cartilage is not involved in the split formation.     

Compressed vertebrae in Atlantic salmon Salmo salar: evidence for metaplastic chondrogenesis as a skeletogenic response late in ontogeny

Anterior/posterior (a/p) compression of the vertebral column, referred to as 'short tails', is a recurring event in farmed Atlantic salmon. Like other skeletal deformities, the problem usually becomes evident in a late life phase, too late for preventive measures, making it difficult to understand the aetiology of the disease. We use structural, radiological, histological, and mineral analyses to study 'short tail' adult salmon and to demonstrate that the study of adult fish can provide important insights into earlier developmental processes. 'Short tails' display a/p compressed vertebrae throughout the spine, except for the first post-cranial vertebrae. The vertebral number is unaltered, but the intervertebral space is reduced and the vertebrae are shorter. Compressed vertebrae are characterized by an unchanged central part, altered vertebral end plates (straight instead of funnel-shaped), an atypical inward bending of the vertebral edges, and structural alterations in the intervertebral tissue. The spongiosa is unaffected. The growth zones of adjacent vertebrae fuse and blend towards the intervertebral space into chondrogenic tissue. This tissue produces different types of cartilage, replacing the notochord. The correspondence in location of intervertebral cartilage and deformed vertebral end plates, and the clearly delimited, unaltered, central vertebral parts suggest that the a/p compression of vertebral bodies is a late developmental disorder that may be related to a metaplastic shift of osteogenic tissue into chondrogenic tissue in the vertebral growth zone. Given the lack of evidence for infections, metabolic disorders and/or genetic disorders, we propose that an altered mechanical load could have caused the transformation of the bone growth zones and the concomitant replacement of the intervertebral (notochord) tissue by cartilaginous tissues in the 'short tails' studied here. This hypothesis is supported by the role that notochord cells are known to play in spine development and in maintaining the structure of the intervertebral disk.     

A col10a1:nlGFP transgenic line displays putative osteoblast precursors at the medaka notochordal sheath prior to mineralization

In teleosts, such as medaka, ossification of the vertebral column starts with the mineralization of the notochordal sheath in a segmental pattern. This establishes the chordal centrum, which serves as the basis for further ossifications by sclerotome derived osteoblasts generating the vertebral body. So far, it is unclear which cells produce the notochordal sheath and how a segmental pattern of mineralization is established in teleosts. Here, we use a transgenic medaka line that expresses nlGFP under the control of the col10a1 promoter for in vivo analysis of vertebral body formation. We show that col10a1:nlGFP expression recapitulates endogenous col10a1 expression. In the axial skeleton, col10a1:nlGFP cells appear prior to the mineralization of the notochordal sheath in a segmental pattern. These cells remain on the outer surface of the chordal centra during mineralization as well as subsequent perichordal ossification of the vertebral bodies. Using twist1a1:dsRed and osx:mCherry transgenic lines we show that a subset of col10a1:nlGFP cells is derived from sclerotomal precursors and differentiates into future osteoblasts. For the first time, this shows a segmental occurrence of putative osteoblast precursors in the vertebral centra prior to ossification of the notochordal sheath. This opens the possibility that sclerotome derived cells in teleosts are implicated in the establishment of the mineralized vertebral column in a similar manner as previously described for tetrapods.     

Chondrogenic gradients in the developing vertebral body.

Since in literature the question of the spatio-temporal sequence of the cartilage maturation in the developing vertebra is still controversial, the authors studied by light and electron microscope, the chondrification of the vertebral body in chick embryo from the 6th to the 13th incubation days, in order to define the correlations between morphology and distribution of the cartilage cells in this phase of vertebral development. The results show that the chondrogenesis follows spatio-temporal gradients, starting at about the 8th incubation day from a zone located between notochord and neural tube, slightly cranially to the midvertebral level. From this starting point the chondrification proceeds with dorso-lateral and radial progression, and at the same time extends towards the cranial and caudal plates of the developing vertebra. These data are compared to the findings obtained by other authors on the ultrastructural and biochemical aspects of the vertebral development.     

Fusion of the cervical vertebrae of mammals

Morphogenesis of the cervical vertebrae has been investigated in Dipis sagitt. and in Rattus norvegicus. The main distinctive feature of the Dipis embryos at the mesenchymal stage is their very thin perichordal intervertebral rings. As a result, short cartilaginous vertebral bodies and thin intervertebral discs develop, cervical segments lengthen more slowly than those of the Rattus. Because of the small length of the Dipis cervical segments, the cartilaginous neural arches and the transverse processes of 2-6 vertebrae draw nearer and fuse. Owing to the insufficient development of the Dipis intervertebral discs and the nuclei pulposae, the normal formation of the vertebral epiphyses is disturbed, this results in fusion of the neighbouring osseous vertebral bodies.     

Vertebral development and amphibian evolution

Amphibians provide an unparalleled opportunity to integrate studies of development and evolution through the investigation of the fossil record of larval stages. The pattern of vertebral development in modern frogs strongly resembles that of Paleozoic labyrinthodonts in the great delay in the ossification of the vertebrae, with the centra forming much later than the neural arches. Slow ossification of the trunk vertebrae in frogs and the absence of ossification in the tail facilitate the rapid loss of the tail during metamorphosis, and may reflect retention of the pattern in their specific Paleozoic ancestors. Salamanders and caecilians ossify their centra at a much earlier stage than frogs, which resembles the condition in Paleozoic lepospondyls. The clearly distinct patterns and rates of vertebral development may indicate phylogenetic separation between the ultimate ancestors of frogs and those of salamanders and caecilians within the early radiation of ancestral tetrapods. This divergence may date from the Lower Carboniferous. Comparison with the molecular regulation of vertebral development described in modern mammals and birds suggests that the rapid chondrification of the centra in salamanders relative to that of frogs may result from the earlier migration of sclerotomal cells expressing Pax1 to the area surrounding the notochord.     

Evolving possibilities: postembryonic axial elongation in salamanders with biphasic (Eurcyea cirrigera,Eurycea longicauda,Eurycea quadridigitata) and paedomorphic life cycles (Eurycea nana and Ambystoma mexicanum)

Vaglia, JL., White, K, and Case, A. 2012. Evolving possibilities: postembryonic axial elongation in salamanders with biphasic (Eurcyea cirrigera, Eurycea longicauda, Eurycea quadridigitata) and paedomorphic life cycles (Eurycea nana and Ambystoma mexicanum). —Acta Zoologica (Stockholm) 93 : 2–13. Typically, the number of vertebrae an organism will have postembryonically is determined during embryogenesis via the development of paired somites. Our research investigates the phenomenon of postembryonic vertebral addition in salamander tails. We describe body and tail growth and patterns of postsacral vertebral addition and elongation in context with caudal morphology for four plethodontids (Eurycea) and one ambystomatid. Eurycea nana and Ambystoma mexicanum have paedomorphic life cycles; Eurcyea cirrigera, Eurycea longicauda and Eurycea quadridigitata are biphasic. Specimens were collected, borrowed and/or purchased, and cleared and stained for bone and cartilage. Data collected include snout‐vent length (SVL), tail length (TL), vertebral counts and centrum lengths. Eurycea species with biphasic life cycles had TLs that surpassed SVL following metamorphosis. Tails in paedomorphic species elongated but rarely exceeded body length. Larger TLs were associated with more vertebrae and longer vertebrae in all species. We observed that rates of postsacral vertebral addition varied little amongst species. Regional variation along the tail becomes prominent following metamorphosis in biphasic developers. In all species, vertebrae in the posterior one‐half of the tail taper towards the tip. We suggest that a developmental link might exist between the ability to continually add vertebrae and regeneration in salamanders.