The teleost intervertebral region acts as a growth center of the centrum: in vivo visualization of osteoblasts and their progenitors in transgenic fish.

The vertebral column is a defined feature of vertebrates. In birds and mammals, the sclerotome yields cartilaginous material for the vertebral column. In teleosts, however, it remains uncertain whether the sclerotome participates in vertebral column formation. To investigate osteoblast development in the teleost, we established transgenic systems that allow in vivo observation of osteoblasts and their progenitors marked by fluorescence of DsRed and enhanced green fluorescent protein (EGFP), respectively. In twist-EGFP transgenic medaka, EGFP-positive cells first appeared in the ventromedial portion of respective somites corresponding to the sclerotome, migrated dorsally around the notochord, and concentrated in the intervertebral regions. Ultrastructural analysis of the intervertebral regions revealed that some of these cells were directly located on the osteoidal surface of the perichordal centrum, and enriched with rough endoplasmic reticulum in their cytoplasm. By using the double transgenic medaka of twist-EGFP and osteocalcin-DsRed, we clarified that the EGFP-positive cells in the intervertebral region differentiated into mature osteoblasts expressing the DsRed. In vivo bone labeling in fact confirmed active matrix formation and mineralization of the perichordal centrum exclusively in the intervertebral region of zebrafish larvae as well as medaka larvae. These findings strongly suggest that the teleost intervertebral region acts as a growth center of the perichordal centrum, where the sclerotome-derived cells differentiate into osteoblasts.     

The role of Bapx1 (Nkx3.2) in the development and evolution of the axial skeleton

The bagpipe-related homeobox-containing genes are members of the NK family. bagpipe ( bap ) was first identified in Drosophila and there are three different bagpipe-related genes in vertebrates. Only two of these are found in mammals, the Nkx3.1 and the Bapx1 ( Nkx3.2 ) gene. The targeted mutation in the mouse Bapx1 gene shows a vertebral phenotype in which the ventromedial elements are lacking; these are the centra and the intervertebral discs. In addition, a region of gastric mesenchyme is abnormal. This mesenchyme surrounds the posterior region of the presumptive stomach and duodenum, and in the mutant fails to support normal development of the spleen. In Drosophila, bagpipe has a role in gut mesoderm and the mutant embryos have no midgut musculature. Thus bap related genes in mouse and Drosophila have roles in patterning gut mesoderm; however, neither of the mammalian genes has a discernible role in the gut musculature. In contrast, both mammalian genes have roles in developmental processes that have appeared recently in evolution. The Bapx1 gene found in fish, amphibians, birds and mammals appears to have derived vertebrate specific functions sometime after the split between the jawless fish and gnathostomes.     

Kinetics and differentiation of somite cells forming the vertebral column: studies on human and chick embryos

We have studied the kinetics of somite cells with an antibody against proliferating cell nuclear antigen (PCNA/cyclin) in human and chick embryos, and with the BrdU anti-BrdU method in chick embryos, to investigate whether the metameric pattern of the developing vertebral column can be explained by different proliferation rates. Furthermore we applied antibodies against differentiation markers of chondrogenic and myogenic cells of the somites in order to study the correlation between proliferation and differentiation. There are no principal differences in the proliferation pattern of the vertebral column between human and chick embryos. In all stages examined, the cell density is higher in the caudal sclerotome halves than in the cranial halves. Laterally, the caudal sclerotome halves, which give rise to the neural arches, are characterized by a higher proliferative activity than the cranial halves. Although there is a high variability, the labelling indices show significant differences between the two halves with both proliferation markers. With the onset of chondrogenic differentiation, only the perichondrial cells retain a high proliferation rate. During fetal development, the neural arches and their processes grow appositionally. Even at the earliest stages, there is practically no immunostaining for PCNA or BrdU in the desmin-positive myotome cells of human and chick embryos. Axially, a higher proliferation rate is found in the condensed mesenchyme of the anlagen of the intervertebral discs than in the anlagen of the vertebral bodies. During fetal development, cells at the borders between vertebral bodies and intervertebral discs proliferate, indicating appositional growth. Our results show that local differences in the proliferation rates of the paraxial mesoderm exist, and may be an important mechanism for the establishment of the metameric pattern of the vertebral column in human and chick embryos.     

Arthrotome: a specific joint forming compartment in the avian somite.

Somitocoele cells previously have been shown to form the proximal part of the ribs, the intervertebral discs, and the intervertebral joints (synovial joints). To determine whether the somitocoele cells are necessary for the development of axial skeleton joints, we microsurgically ablated the somitocoele cells in epithelial somites of 2-day-old chick embryos. The operated embryos were analyzed after whole-mount skeletal preparations and in sections. Removal of the somitocoele cells led to two major outcomes: (1) Intervertebral joints failed to develop and resulted in the fusion of the superior articular process and the inferior articular process; (2) Adjacent vertebral bodies fused and lacked the intervertebral disc. These results demonstrate that somitocoele cells specifically give rise to intervertebral joints and discs. Furthermore, these results suggest that neighboring sclerotome cells cannot adapt to form vertebral joints in the absence of the somitocoele compartment. Thus, we provide for the first time experimental evidence for the existence of a joint forming compartment in the somites, which we term the "arthrotome."     

The development of the avian vertebral column

Segmentation of the paraxial mesoderm leads to somite formation. The underlying molecular mechanisms involve the oscillation of ”clock-genes” like c-hairy-1 and lunatic fringe indicative of an implication of the Notch signaling pathway. The cranio-caudal polarity of each segment is already established in the cranial part of the segmental plate and accompanied by the expression of genes like Delta1, Mesp1, Mesp2, Uncx-1, and EphA4 which are restricted to one half of the prospective somite. Dorsoventral compartmentalization of somites leads to the development of the dermomyotome and the sclerotome, the latter forming as a consequence of an epithelio-to-mesenchymal transition of the ventral part of the somite. The sclerotome cells express Pax-1 and Pax-9, which are induced by notochordal signals mediated by sonic hedgehog (Shh) and noggin. The craniocaudal somite compartmentalization that becomes visible in the sclerotomes is the prerequisite for the segmental pattern of the peripheral nervous system and the formation of the vertebrae and ribs, whose boundaries are shifted half a segment compared to the sclerotome boundaries. Sclerotome development is characterized by the formation of three subcompartments giving rise to different parts of the axial skeleton and ribs. The lateral sclerotome gives rise to the laminae and pedicles of the neural arches and to the ribs. Its development depends on signals from the notochord and the myotome. The ventral sclerotome giving rise to the vertebral bodies and intervertebral discs is made up of Pax-1 expressing cells that have invaded the perinotochordal space. The dorsal sclerotome is formed by cells that migrate from the dorso-medial angle of the sclerotome into the space between the roof plate of the neural tube and the dermis. These cells express the genes Msx1 and Msx2, which are induced by BMP-4 secreted from the roof plate, and they later form the dorsal part of the neural arch and the spinous process. The formation of the ventral and dorsal sclerotome requires directed migration of sclerotome cells. The regionalization of the paraxial mesoderm occurs by a combination of functionally Hox genes, the Hox code, and determines the segment identity. The development of the vertebral column is a consequence of a segment-specific balance between proliferation, apoptosis and differentiation of cells. Accepted: 25 May 2000     

New insight into progenitor/stem cells in dental pulp using Col1a1-GFP transgenes

In recent years there has been increasing progress in identifying stem cells from adult tissues and their potential application in tissue engineering. These advances provide a promising future for tooth replacement/regeneration. Essential for this approach is the identification of donor stem cells for various components of the teeth. Our studies show that pOBCol3.6GFPtpz and pOBCol2.3GFPemd transgenic animals provide a unique model to gain insight into stem cells in the dental pulp. Our in vivo studies of the developing teeth of these transgenic lines show both Col1a1-GFP transgenes are expressed in functional and fully differentiated odontoblasts. The patterns of expression of Col1a1-GFP transgenes during odontoblast differentiation correlates with the expression of DSPP. In the developing craniofacial bones both Col1a1-GFP transgenes are also expressed in osteoblasts and osteocytes of alveolar and calvarial bones. In the alveolar bones, the expression of Col1a1-GFP in osteocytes correlates with the expression of DMP1. Col1a1-3.6-GFP is expressed in the entire layer of the periosteum and in suture mesenchyme containing osteoprogenitor cells. On the other hand, Col1a1-2.3- GFP expression was limited to the osteoblastic layer of the periosteum and was not detected in the fibroblastic layer of the periosteum or in the suture mesenchyme. These observations indicate that Col1a1-3.6-GFP and Col1a1-2.3-GFP transgenes identify different subpopulations of cells during intramembranous ossification. By using the coronal portion of dental pulps isolated from postnatal transgenic mice our observations also provide direct evidence that the dental pulp contains progenitor/stem cells capable of giving rise to a new generation of odontoblast-like cells, as well as osteoblast-like cells.     

The role of the notochord in vertebral column formation

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.     

The role of the notochord in vertebral column formation

The backbone or vertebral column is the defining feature of vertebrates and is clearly metameric. Given that vertebrae arise from segmented paraxial mesoderm in the embryo, this metamerism is not surprising. Fate mapping studies in a variety of species have shown that ventromedial sclerotome cells of the differentiated somite contribute to the developing vertebrae and ribs. Nevertheless, extensive studies in amniote embryos have produced conflicting data on exactly how embryonic segments relate to those of the adult. To date, much attention has focused on the derivatives of the somites, while relatively little is known about the contribution of other tissues to the formation of the vertebral column. In particular, while it is clear that signals from the notochord induce and maintain proliferation of the sclerotome, and later promote chondrogenesis, the role of the notochord in vertebral segmentation has been largely overlooked. Here, we review the established role of the notochord in vertebral development, and suggest an additional role for the notochord in the segmental patterning of the vertebral column.     

Bapx1 homeobox gene gain-of-function mice show preaxial polydactyly and activated Shh signaling in the developing limb.

To explore Bapx1 homeobox gene function in embryonic control of development, we employed a gain-of-function approach to complement our previous loss-of-function mutant analysis. We show that transgenic mice overexpressing Bapx1 are affected by skeletal defects including hindlimb preaxial polydactyly and tibial hypoplasia. Bapx1 overexpression generates limb anteroposterior patterning defects including induction of Shh signaling and ectopic activation of functions downstream of Shh signaling into the anterior region of the autopod. Moreover, Bapx1 overexpression stimulates formation of limb prechondrogenic condensations. We also show that Shh is reciprocally able to activate Bapx1 expression in mouse embryos as the orthologous hedgehog (hh) does with the bagpipe/Bapx1 gene in Drosophila. Our results indicate that Bapx1 can modulate appendicular skeletal formation, that the genetic hierarchy between Shh/hh and Bapx1/bagpipe has been conserved during evolution, and that in mouse embryos these two genes can influence one another in a genetically reciprocal manner. We conclude that it is reasonable to expect overexpression of Bapx1 in certain forms of polydactyly.     

The generation of vertebral segmental patterning in the chick embryo

We have carried out a series of experimental manipulations in the chick embryo to assess whether the notochord, neural tube and spinal nerves influence segmental patterning of the vertebral column. Using Pax1 expression in the somite-derived sclerotomes as a marker for segmentation of the developing intervertebral disc, our results exclude such an influence. In contrast to certain teleost species, where the notochord has been shown to generate segmentation of the vertebral bodies (chordacentra), these experiments indicate that segmental patterning of the avian vertebral column arises autonomously in the somite mesoderm. We suggest that in amniotes, the subdivision of each sclerotome into non-miscible anterior and posterior halves plays a critical role in establishing vertebral segmentation, and in maintaining left/right alignment of the developing vertebral elements at the body midline.     

The binding pattern of peanut lectin associated with sclerotome migration and the formation of the vertebral axis in the chick embryo

Summary Lectins have been used extensively to detect changes in carbohydrate moieties on the surface of embryonic cells during early development. Peanut agglutinin (PNA) in particular has been used to investigate changes related to cell differentiation. PNA has also been used to differentiate between the rostral and caudal sclerotome halves which have been shown to be functionally different, with neural crest cells and neurites traversing only the rostral half during their migration. In this study, we have sectioned and stained chick embryos between 3 and 8 days of age with PNA to examine the distribution of PNA binding sites associated with the vertebral column during this period and also to determine the fates of the rostral and caudal sclerotome halves. Ultrastructural localisation of PNA-gold conjugate showed that binding sites for this lectin were present intracellularly and extracellularly both on cell surfaces and in the matrix. At the light microscope level, a clear banding pattern emerged after staining with PNA which consisted of alternating light and dark staining along the entire length of the vertebral axis of the embryo. In the younger embryos, a simple banding pattern emerged where the rostral sclerotome half of each segment stained only lightly while the caudal half stained darkly. This banding pattern was present throughout the 6 day period of development and could be traced continuously but grew more complex as the sclerotome cells migrated to surround the notochord and neural tube and as the dorsal root ganglia developed. The rostral sclerotome half was found to contribute to the caudal part of one vertebral body and its neural arch, while the caudal sclerotome half was found to contribute to the intervertebral disc, the rostral half of the next caudal vertebra, and part of its neural arch.     

Cloning and developmental expression analysis of the murine homolog of the spinocerebellar ataxia type 1 gene (Sca1)

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant neurodegenerative disorder caused by the expansion of a CAG trinucleotide repeat which encodes glutamine in the novel protein ataxin-1. In order to characterize the developmental expression pattern of SCA1 and to identify putative functional domains in ataxin-1, the murine homolog (Sca1) was isolated. Cloning and characterization of the murine Sca1 gene revealed that the gene organization is similar to that of the human gene. The murine and human ataxin-1 are highly homologous but the CAG repeat is virtually absent in the mouse sequence suggesting that the polyglutamine stretch is not essential for the normal function of ataxin-1 in mice. Cellular and developmental expression of the murine homolog was examined using RNA in situ hybridization. During cerebellar development, there is a transient burst of Sca1 expression at postnatal day 14 when the murine cerebellar cortex becomes physiologically functional. There is also marked expression of Sca1 in mesenchymal cells of the intervertebral discs during development of the spinal column. These results suggest that the normal Sca1 gene, has a role at specific stages of both cerebellar and vertebral column development.      

腰骶段椎间盘与相应椎体的应用解剖

目的　为临床诊断椎间盘疾患提供参数。方法　对 31例正常男性脊柱第 1腰椎至第 1骶椎间 15 5个椎间盘及其相应的 186个椎体上下面的矢径、横径、斜径进行了测量和比较 ,同时对其周围毗邻结构进行了观察。结果　椎间盘各径均大于其相应椎体各径 ,且自上而下逐渐增大 ,获得了腰骶段周围毗邻结构的观察结果。结论　提示结果与各椎骨间所负体重逐渐增加相适应 ,为临床应用提供解剖学依据     

Patterning spinal nerves and vertebral bones

A prominent anatomical feature of the peripheral nervous system is the segmentation of mixed (motor, sensory and autonomic) spinal nerves alongside the spinal cord. During early development their axon growth cones avoid the developing vertebral elements by traversing the anterior/cranial half of each somite‐derived sclerotome, so ensuring the separation of spinal nerves from vertebral bones as axons extend towards their peripheral targets. A glycoprotein expressed on the surface of posterior half‐sclerotome cells confines growth cones to the anterior half‐sclerotomes by contact repulsion. A closely similar glycoprotein is expressed in avian and mammalian grey matter, where we hypothesize it may have evolved to regulate neural plasticity in birds and mammals.     

Pax-1 in the development of the cervico-occipital transitional zone

The Pax-1 gene has been found to play an important role in the development of the vertebral column. The cervico-occipital transitional zone is a specialized region of the vertebral column, and malformations of this region have frequently been described in humans. The exact embryonic border between head and trunk is a matter of controversy. In order to determine a possible role of Pax-1 in the development of the cervico-occipital transitional zone we studied the expression of this gene in a series of quail embryos and murine fetuses with in situ hybridization and immunohistochemistry. Pax-1 is expressed in all somites of the embryo, including the first five occipital ones. During embryonic days 3–5 the gene is down-regulated in the caudal direction within the first five somites, whereas more caudally Pax-1 is strongly expressed in the cells of the perinotochordal tube. In 5-day-old quail embryos, the cartilaginous anlage of the basioccipital bone has developed and there is no more expression of Pax-1 in this region. The fusion of the dens axis with the body of the axis also coincides with switching off of the Pax-1 gene. More caudally, the gene is continuously expressed in the intervertebral discs of murine embryos and therefore seems to be important for the process of resegmentation. Quail embryos do not possess permanent intervertebral discs. “Hyper-” or “hyposegmentation” defects may be explained by an over- or under-expression of Pax-1 during development. We also reinvestigated the border between the head and trunk in chick embryos by performing homotopical grafting experiments of the 5^th somite between chick and quail embryos. Grafted quail cells formed mainly the caudal end of the basioccipital bone. They were also located in the cranial half of the ventral atlantic arch, and only a few cells were found in the tip of the dens axis.     

Type I collagen is a genetic modifier of matrix metalloproteinase 2 in murine skeletal development.

Recessive inactivating mutations in human matrix metalloproteinase 2 (MMP2, gelatinase A) are associated with syndromes that include abnormal facial appearance, short stature, and severe bone loss. Mmp2(-/-) mice have only mild aspects of these abnormalities, suggesting that MMP2 function is redundant during skeletal development in the mouse. Here, we report that Mmp2(-/-) mice with additional mutations that render type I collagen resistant to collagenase-mediated cleavage to TC(A) and TC(B) fragments (Col1a1(r/r) mice) have severe developmental defects resembling those observed in MMP2-null humans. Composite Mmp2(-/-);Col1a1(r/r) mice were born in expected Mendelian ratios but were half the size of wild-type, Mmp2(-/-), and Col1a1(r/r) mice and failed to thrive. Furthermore, composite Mmp2(-/-);Col1a1(r/r) animals had very abnormal craniofacial features with shorter snouts, bulging skulls, incompletely developed calvarial bones and unclosed cranial sutures. In addition, trabecular bone mass was reduced concomitant with increased numbers of bone-resorbing osteoclasts and osteopenia. In vitro, MMP2 had a unique ability among the collagenolytic MMPs to degrade mutant collagen, offering a possible explanation for the genetic interaction between Mmp2 and Col1a1(r). Thus, because mutations in the type I collagen gene alter the phenotype of mice with null mutations in Mmp2, we conclude that type I collagen is an important modifier gene for Mmp2. Developmental Dynamics 236:1683-1693, 2007. (c) 2007 Wiley-Liss, Inc.     

Calcium pyrophosphate dihydrate deposition in the intervertebral discs in a case of Wilson''s disease

The vertebral column from a known case of Wilson's disease (hepatolenticular degeneration) was examined by radiological, histological, histochemical and x-ray microanalytical techniques which demonstrated the presence of focal depositions of calcium pyrophosphate dihydrate (CPPD) in the intervertebral discs. These deposits were present in both the annulus fibrosus and the nucleus pulposus but in certain discs the deposits were concentrated near the interface between disc and vertebral body bone endplates. At these sites there was new bone formation with narrowing of the discs, irregularity and sclerosis of the bone endplates and exostosis.