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 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.     

维甲酸对斑马鱼中枢神经系统及软骨发生的影响

脂溶性的维甲酸可通过固醇类激素作用途径启动ＨＯＸ族及ＬＩＭ族调节基因,进而影响脊椎动物胚胎的形态发生、再生组织的分化。本文通过不同浓度系列表明,维甲酸对早期鱼胚发育的影响程度依赖于其作用浓度与作用时间,１０＾－６￣１０＾－７Ｍ的浓度范围为作用敏感区。维甲酸对中枢神经系统前后轴上的影响尤其显著,主要表现为小头畸形、无眼或无心脏。作者首次用Ｂｒｄｕ－Ａｎｔｉ－Ｂｎｄｕ标记处于Ｓ期的细胞核表明,ＲＡ作用     

Bmp inhibition is necessary for post-gastrulation patterning and morphogenesis of the zebrafish tailbud

Intricate interactions between the Wnt and Bmp signaling pathways pattern the gastrulating vertebrate embryo using a network of secreted protein ligands and inhibitors. While many of these proteins are expressed post-gastrula, their later roles have typically remained unclear, obscured by the effects of early perturbation. We find that Bmp signaling continues during somitogenesis in zebrafish embryos, with high activity in a small region of the mesodermal progenitor zone at the posterior end of the embryo. To test the hypothesis that Bmp inhibitors expressed just anterior to the tailbud are important to restrain Bmp signaling we produced a new zebrafish transgenic line, allowing temporal cell-autonomous activation of Bmp signaling and thereby bypassing the effects of the Bmp inhibitors. Ectopic activation of Bmp signaling during somitogenesis results in severe defects in the tailbud, including altered morphogenesis and gene expression. We show that these defects are due to non-autonomous effects on the tailbud, and present evidence that the tailbud defects are caused by alterations in Wnt signaling. We present a model in which the posteriorly expressed Bmp inhibitors function during somitogenesis to constrain Bmp signaling in the tailbud in order to allow normal expression of Wnt inhibitors in the presomitic mesoderm, which in turn constrain the levels of canonical and non-canonical Wnt signaling in the tailbud.     

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.     

Vertebral Abnormalities Following Heat Shock in Xenopus Embryos

Xenopus embryos were heat-shocked at several stages of development and vertebral abnormalities were examined by means of in situ staining. A high incidence of vertebral abnormalities was evident in larvae treated at the neurula stages (stages 15 and 20) and at the tailbud stages (stages 32 and 35). Heat shock at the neurula stages led to malformed vertebrae and their fusion following an altered arrangement of the arcualia. Heat shock at the tailbud stages induced an asymmetric arrangement of the sacrum and a change in the number of vertebrae, but the arrangement of the arcualia was not affected. Early events of the development of the vertebral column are discussed in relation to somitogenesis.     

CHANGES IN THE CAPACITY FOR CLONAL GROWTH AND DIFFERENTIATION IN VITRO OF THE VERTEBRAL CARTILAGE CELLS WITH EMBRYONIC DEVELOPMENT. 1. CULTURE IN THE STANDARD MEDIUM

When culturing the vertebral cartilage cells of the chick embryo, the youngest embryo from which the dissociated single cell of the cartilage gives rise to the differentiated cartilage colony (CMC) in the given conditions of the clonal cell culture is at st.26. At this stage, percentage of such cells to make CMC in vitro is less than 0.1% of the inoculated single cells. With development, percentage of such “stabilized” cells increases, finally reaching to 35% at st.40. In embryos from st.29 to st.36, many of the cultured single cells give rise to the regressing colonies (RGC) which show some proliferation at the early stage in vitro, but in due time cease to proliferate and regress.     

An experimental study of the somitomeric organization of the avian segmental plate

The segmental plate mesoderm of chicken and Japanese quail embryos HH stages 9 to 16 was studied with scanning electron microscopy (SEM) imaging. The segmental plates were found to exhibit a metameric pattern consisting of tandemly stacked somitomeres. It was found that the numbers of somitomeres in segmental plates removed from the same embryo were nearly identical. Furthermore, the number of somitomeres in a segmental plate was found to be quite consistent (10.0 ± 1.5) and independent of the length of the segmental plate. These results are very similar to those obtained in previous experimental studies in which “prospective somites” were detected in avian segmental plates. Further experiments showed that for each somite that is formed by a cultured segmental plate-containing explant, the somitomere complement of the segmental plate is reduced by one. It was concluded that the segmental plate mesoderm is already organized into a metameric pattern consisting of somitomeres and that the somitomeres undergo further morphogenesis to become somites. The specification of the somite pattern in birds may occur at the level of Hensen's node and the cephalic primitive streak.     

Segmental expression of aggrecan in the non-segmented perinotochordal sheath underlies normal segmentation of the vertebral column.

The embryonic vertebral column is derived from the unsegmented axial mesenchyme surrounding the notochord, and its development and differentiation are influenced by the notochord. The role of cartilage in determining the ultimate pattern of the segmental skeleton has been well documented, but a gene whose segmental expression corresponds to the pattern of the developing skeleton has yet to be identified. We show that chick aggrecan is initially expressed within the entire length of the notochord, and as development proceeds, aggrecan expression becomes restricted to the surrounding perinotochordal sheath in a segmental pattern, mirroring the differentiated somite pattern.     

SOMITE CHONDROGENESIS : A Structural Analysis

Light and electron microscopy are used in this study to compare chondrogenesis in cultured somites with vertebral chondrogenesis These studies have also characterized some of the effects of inducer tissues (notochord and spinal cord), and different nutrient media, on chondrogenesis in cultured somites Somites from stage 17 (54–60 h) chick embryos were cultured, with or without inducer tissues, and were fed nutrient medium containing either horse serum (HS) and embryo extract (EE), or fetal calf serum (FCS) and F12X Amino acid analyses were also utilized to determine the collagen content of vertebral body cartilage in which the fibrils are homogeneously thin (ca. 150 Å) and unbanded. These analyses provide strong evidence that the thin unbanded fibrils in embryonic cartilage matrix are collagen. These thin unbanded collagen fibrils, and prominent 200–800 Å protein polysaccharide granules, constitute the structured matrix components of both developing vertebral cartilage and the cartilage formed in cultured somites Similar matrix components accumulate around the inducer tissues notochord and spinal cord. These matrix components are structurally distinct from those in embryonic fibrous tissue The synthesis of matrix by the inducer tissues is associated with the inductive interaction of these tissues with somitic mesenchyme. Due to the deleterious effects of tissue isolation and culture procedures many cells die in somitic mesenchyme during the first 24 h in culture. In spite of this cell death, chondrogenic areas are recognized after 12 h in induced cultures, and through the first 2 days in all cultures there are larger accumulations of structured matrix than are present in equivalently aged somitic mesenchyme in vivo. Surviving chondrogenic areas develop into nodules of hyaline cartilage in all induced cultures, and in most non-induced cultures fed medium containing FCS and F12X There is more cell death, less matrix accumulation, and less cartilage formed in cultures fed medium containing HS and EE. The inducer tissues, as well as nutrient medium containing FCS and F12X, facilitate cell survival, the synthesis and accumulation of cartilage matrix, and the formation of cartilage nodules in cultured somites.     

The first cleavage furrow demarcates the dorsal-ventral axis in Xenopus embryos

To determine the relationship between the first cleavage furrow and the dorsal-ventral axis of the Xenopus embryo, a heritable intracellular marker was injected into one blastomere at the two-cell stage. Embryos were selected in which the cleavage furrow bisected the crescent-shaped region of pale pigmentation or in which it formed 45-90 degrees from this region. This region, which is located in the animal hemisphere of the Xenopus embryo, meets the criteria of the grey crescent as defined in other amphibian species. At tailbud stages the interface between the labeled and unlabeled halves was always coincident with the midsagittal plane. This correlation shows that the first cleavage furrow demarcates the dorsal-ventral axis. The labeling pattern was the same whether the first cleavage furrow bisected the region of pale pigmentation or whether it formed 90 degrees from it. However, when this region was bisected (70% of embryos) it always was located on the dorsal side of the embryo. Thus the region of pale pigmentation indicates the dorsal side of the embryo only when it is bisected by the first cleavage furrow.     

The determination of myogenic and cartilage cells in the early chick embryo and the modifying effect of retinoic acid

Summary The time of determination of cartilage and skeletal muscle was studied by making chimeric grafts or explants of small tissue pieces from several stages of early chick or quail embryos. Chondrogenesis was assessed by histology or with antibodies directed against type II collagen or cartilage proteoglycan, while myogenesis was detected immunohistochemically with antibodies directed against 3 different muscle markers, including muscle myosin. Grafts from Hensen's node, primitive streak and segmental plate of donor embryos of Stage 3–5 (Hamburger and Hamilton) were transplanted under the ectoderm in the extraembryonic area of Stage 12 host embryos. In addition, explants and mesodermal cells were cultured on glass in DMEM+F12 medium supplemented with 10% FCS. The results showed that determined myogenic cells could first be detected in Hensen's node and the primitive streak at Stage 3⁺–4 and that they developed from mesodermal cells located between the epiblast and hypoblast. Myogenic cells also appeared in grafted and explanted segmental plate with or without notochord from Stage 5 embryos. On the other hand, cartilage cells only formed in grafted and explanted segmental plate that also contained notochord. RA (1 ng/ml) could induce the formation of cartilage cells in the explanted primitive streak without Hensen's node or notochord taken from Stage 3–5 embryos and could also promote the differentiation of myogenic cells in primitive streak from Stage 3 embryo. Thus RA can substitute for Hensen's node or the notochord in the induction of cartilage cells and has some stimulatory effects on the differentiation of myogenic cells. Additional evidence indicates that the hypoblast might play an inductive role in the formation of the notochord which may subsequently promote the differentiation of cartilage cells. Offprint requests to: M. Solursh     

The fate of cells in the tailbud of Xenopus laevis

The vertebrate tailbud and trunk form very similar tissues. It has been a controversial question for decades whether cell determination in the developing tail proceeds as part of early axial development or whether it proceeds by a different mechanism. To examine this question more closely, we have used photoactivation of fluorescence to mark small neighborhoods of cells in the developing tailbud of Xenopus laevis. We show that, in one region of the tailbud, very small groups of adjacent cells can contribute progeny to the neural tube, notochord and somitic muscle, as well as other identified cell types within a single embryo. Groups averaging three adjacent cells at a later stage can contribute progeny with a similar distribution. Our data suggest that the tailbud contains multipotent cells that make very late germ-layer decisions.     

Development of the Synarcual in the Elephant Sharks (Holocephali; Chondrichthyes): Implications for Vertebral Formation and Fusion

The synarcual is a structure incorporating multiple elements of two or more anterior vertebrae of the axial skeleton, forming immediately posterior to the cranium. It has been convergently acquired in the fossil group ‘Placodermi’, in Chondrichthyes (Holocephali, Batoidea), within the teleost group Syngnathiformes, and to varying degrees in a range of mammalian taxa. In addition, cervical vertebral fusion presents as an abnormal pathology in a variety of human disorders. Vertebrae develop from axially arranged somites, so that fusion could result from a failure of somite segmentation early in development, or from later heterotopic development of intervertebral bone or cartilage. Examination of early developmental stages indicates that in the Batoidea and the ‘Placodermi’, individual vertebrae developed normally and only later become incorporated into the synarcual, implying regular somite segmentation and vertebral development. Here we show that in the holocephalan Callorhinchus milii, uniform and regular vertebral segmentation also occurs, with anterior individual vertebra developing separately with subsequent fusion into a synarcual. Vertebral elements forming directly behind the synarcual continue to be incorporated into the synarcual through growth. This appears to be a common pattern through the Vertebrata. Research into human disorders, presenting as cervical fusion at birth, focuses on gene misexpression studies in humans and other mammals such as the mouse. However, in chondrichthyans, vertebral fusion represents the normal morphology, moreover, taxa such Leucoraja (Batoidea) and Callorhinchus (Holocephali) are increasingly used as laboratory animals, and the Callorhinchus genome has been sequenced and is available for study. Our observations on synarcual development in three major groups of early jawed vertebrates indicate that fusion involves heterotopic cartilage and perichondral bone/mineralised cartilage developing outside the regular skeleton. We suggest that chondrichthyans have potential as ideal extant models for identifying the genes involved in these processes, for application to human skeletal heterotopic disorders.     

Programming neural Hoxd10: in vivo evidence that early node-associated signals predominate over paraxial mesoderm signals at posterior spinal levels

Studies of the programming of Hox patterns at anterior spinal levels suggest that these events are accomplished through an integration of Hensen's node-derived and paraxial mesoderm signaling. We have used in vivo tissue manipulation in the avian embryo to examine the respective roles of node- derived and other local signals in the programming of a Hox pattern at posterior spinal levels. Hoxd10 is highly expressed in the lumbosacral (LS) spinal cord and adjacent paraxial mesoderm. At stages of LS neural tube formation (stages 12-14), the tailbud contains the remnants of Hensen's node and the primitive streak. Hoxd10 expression was analyzed after transposition of LS neural segments with and without the tailbud, after isolation of normally positioned LS segments from the stage 13 tailbud, and after axial displacement of posterior paraxial mesoderm. Data suggest that inductive signals from the tailbud are primarily responsible for the programming of Hoxd10 at neural plate and the earliest neural tube stages. After these stages, the LS neural tube appears to differ from more anterior neural segments in its lack of dependence on Hox-inductive signals from local tissues, including paraxial mesoderm. Our data also suggest that a graded system of repressive signals for posterior Hox genes is present at cervical and thoracic levels and likely to originate from paraxial mesoderm.     

Fates of the blastomeres of the 16-cell stage Xenopus embryo

The fate of each of the blastomeres in the 16-cell stage Xenopus embryo which had been carefully selected for stereotypic cleavages was determined by intracellularly marking a single blastomere with horseradish peroxidase and identifying the labeled progeny in the tailbud embryo by histochemistry. Each blastomere populated all three primary germ layers. The progeny of each blastomere were distributed characteristically both in phenotype and in location. For example, most organs were populated by the descendants of particular sets of blastomeres. Furthermore, within an organ the progeny of a single blastomere were restricted to defined spatial addresses. This study describes the fates of identified 16-cell stage blastomeres and demonstrates that they are distinct and predictable if embryos are preselected for stereotypic cleavages.     

Molecular aspects of vertebral chondrogenesis]

In this article, we review the data showing that dorso-ventral polarization of the vertebral cartilage involves two distinct molecular pathways, acting sequentially during development. The first one is mediated by the notochord and the ventral part of the neural tube, by the action of the Sonic Hedghehog protein (SHH): it directs the formation of the cartilaginous pieces located in a deep position within the embryo. The second one is responsible for the differentiation of the dorsal parts of the vertebra, differentiating under the ectoderm; it is initiated by the roof plate of the neural tube, by the action of the secreted protein BMP4. Those two pathways cannot be exchanged, rather they are antagonists. These data allow us to draw a new model for vertebral chondrogenesis.     

Nuclear reorganisation and chromatin decondensation are conserved, but distinct, mechanisms linked to Hox gene activation

The relocalisation of some genes to positions outside chromosome territories, and the visible decondensation or unfolding of interphase chromatin, are two striking facets of nuclear reorganisation linked to gene activation that have been assumed to be related to each other. Here, in a study of nuclear reorganisation around the Hoxd cluster, we suggest that this may not be the case. Despite its very different genomic environment from Hoxb, Hoxd also loops out from its chromosome territory, and unfolds, upon activation in differentiating embryonic stem (ES) cells and in the tailbud of the embryo. However, looping out and decondensation are not simply two different manifestations of the same underlying change in chromatin structure. We show that, in the limb bud of the embryonic day 9.5 embryo, where Hoxd is also activated, there is visible decondensation of chromatin but no detectable movement of the region out from the chromosome territory. During ES cell differentiation, decondensed alleles can also be found inside of chromosome territories, and loci that have looped out of the territories can appear to still be condensed. We conclude that evolutionarily conserved chromosome remodelling mechanisms, predating the duplication of mammalian Hox loci, underlie Hox regulation along the rostrocaudal embryonic axis. However, we suggest that separate modes of regulation can modify Hoxd chromatin in different ways in different developmental contexts.