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.     

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

Cell condensation in chondrogenic differentiation.

Reduction of intercellular spaces in the areas of prospective cartilage and bone formation (precartilage condensation) precedes chondrogenesis and may represent an important step in the process of cartilage differentiation during limb skeletogenesis. We have attempted to clarify the role of the microenvironment established during cell condensation, taking advantage of a tissue culture model system that allows condensation (i.e., increased cell density due to cell aggregation) and chondrogenic differentiation (i.e., synthesis of cartilage-specific extracellular matrix proteins, such as type II collagen and acquisition of a chondrocyte morphology) of chick embryo cartilage-derived undifferentiated cells. To prevent condensation cells were grown in carboxymethylcellulose and changes in the differentiation pathway were evaluated. In another series of experiments, we have separated single cells from the aggregated cells and analyzed their differentiation properties. Morphological analyses and the evaluation of type II collagen expression, at both the protein and the mRNA level, show that a reduced rate of cell clustering and cell to cell contact parallels a reduction of cell recruitment into the differentiation program. On the basis of our results, we suggest that the following cascade of events regulates the early stages of chondrocyte differentiation: (a) the acquisition of the ability to establish cell to cell contacts, (b) the formation of a permissive environment capable of activating the differentiation program, and (c) the expression of differentiation markers.     

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.     

Fibronectin gene expression during limb cartilage differentiation

A critical event in limb cartilage differentiation is a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely juxtaposed and interact with one another prior to initiating cartilage matrix deposition. Fibronectin (FN) has been suggested to be involved in regulating the onset of condensation and chondrogenesis by actively promoting prechondrogenic aggregate formation during the process. We have performed a systematic quantitative study of the expression of the FN gene during the progression of chondrogenesis in vitro and in vivo. In high-density micromass cultures of limb mesenchymal cells, FN mRNA levels increase about 5-fold coincident with the crucial condensation process, and remain relatively high during the initial deposition of cartilage matrix by the cells. Thereafter, FN mRNA levels progressively decline to relatively low levels as the cultures form a virtually uniform mass of cartilage. The changes in FN mRNA levels in vitro are paralleled closely by changes in the relative rate of FN synthesis as determined by pulse-labeling and immunoprecipitation analysis. The relative rate of FN synthesis increases 4- to 5-fold at condensation and the onset of chondrogenesis, after which it progressively declines to low levels as cartilage matrix accumulates. High levels of FN gene expression also occur at the onset of chondrogenesis in vivo. In the proximal central core regions of the limb bud in which condensation and cartilage matrix deposition are being initiated, FN mRNA levels and the relative rates of FN synthesis become progressively about 4-fold higher than in the distal subridge region, which consists of undifferentiated mesenchymal cells that have not yet initiated condensation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor

The regulatory role of parathyroid hormone (PTH)/PTH-related peptide (PTHrP) signaling has been implicated in embryonic skeletal development. Here, we studied chondrogenic differentiation of the mouse embryonal carcinoma-derived clonal cell line ATDC5 as a model of chondrogenesis in the early stages of endochondral bone development. ATDC5 cells retain the properties of chondroprogenitor cells, and rapidly proliferate in the presence of 5% FBS. Insulin (10 micrograms/ml) induced chondrogenic differentiation of the cells in a postconfluent phase through a cellular condensation process, resulting in the formation of cartilage nodules, as evidenced by expression of type II collagen and aggrecan genes. We found that differentiated cultures of ATDC5 cells abundantly expressed the high affinity receptor for PTH (Mr approximately 80 kD; Kd = 3.9 nM; 3.2 x 10(5) sites/cell). The receptors on differentiated cells were functionally active, as evidenced by a PTH-dependent activation of adenylate cyclase. Specific binding of PTH to cells markedly increased with the formation of cartilage nodules, while undifferentiated cells failed to show specific binding of PTH. Northern blot analysis indicated that expression of the PTH/PTHrP receptor gene became detectable at the early stage of chondrogenesis of ATDC5 cells, preceding induction of aggrecan gene expression. Expression of the PTH/PTHrP receptor gene was undetectable in undifferentiated cells. The level of PTH/PTHrP receptor mRNA was markedly elevated parallel to that of type II collagen mRNA. These lines of evidence suggest that the expression of functional PTH/PTHrP receptor is associated with the onset of chondrogenesis. In addition, activation of the receptor by exogenous PTH or PTHrP significantly interfered with cellular condensation and the subsequent formation of cartilage nodules, suggesting a novel site of PTHrP action.     

穗花牡荆花芽分化过程中形态和生理指标变化

以穗花牡荆为研究材料,通过探究其花芽分化进程和生理特性,为花期调控技术提供成花机理。采用物候期观察和石蜡切片相结合的方法并测定花芽分化过程中相关生理指标,研究花发育过程中的形态和生理变化。结果表明,穗花牡荆花芽分化为一年多次分化型,其进程可划分为七个时期：未分化期、总轴花序原基分化期、初级分轴花序原基分化期、次级分轴花序原基分化期、小花原基分化期、花器官分化前期和花器官分化后期。同一植株不同位置花芽及同一花序中不同单花分化的进程不同,第一季花期后各阶段的花芽分化形态常存在重叠。花芽分化过程中不同时期叶片和花芽的可溶性糖和可溶性蛋白质含量均有上升下降的变化,总体上叶片中营养物质含量高于花芽保证营养供应。花芽分化过程中,IAA、ABA、CTK和GA3整体水平上先升后降有利于花芽分化进行。研究认为,花芽中大量的可溶性糖和蛋白质积累及较高的碳氮比,有利于穗花牡荆花芽形态分化顺利完成。低水平的GA3/ABA和IAA/CTK有利于花序的形成,ABA/CTK和ABA/IAA比值升高促进小花原基和小花萼片原基的分化, GA3/CTK、GA3/ABA和GA3/IAA比值升高促进花瓣原基、雄雌蕊原基发育。     

Comparative morphological examination of vertebral bodies of teleost fish using high-resolution micro-CT scans

Vertebral bodies of teleost fish are formed by the sclerotomal bone covering the chordacentrum. The internal part of the sclerotomal bone is composed of an amphicoelous hourglass shaped autocentrum, which is common in most fish species. In contrast, the external shape of the sclerotomal bone varies extensively among species. There are multiple hypotheses regarding the composition and formation of the external structure. However, as they are based on studies of few extant or extinct species, their applicability to other species remains to be clarified. To understand the morphology, formation, and composition of vertebral bodies in teleosts, we performed a comparative analysis using micro-CT scans of 32 species from 10 orders of Teleostei and investigated the detailed morphology of the sclerotomal bone, especially its plate-like ridge and trabeculae. We discovered two structural characteristics that are shared among most of the examined species. One was the sheet-like trabeculae that extend radially from the center of the vertebral body with a constant thickness. The other was the presence of hollow spaces on the internal parts of the lateral ridge and trabeculae. The combination of different arrangements of sheet-like trabeculae and internal hollow spaces formed different shapes of the lateral structure of the vertebral body. The properties of these two characteristics suggest that the external part of the sclerotomal bone grows outward by deposition at the bone tip, and that, concurrently, bone absorption occurs in the internal part of the sclerotomal bone. The vertebral arches were also formed by the sheet-like trabeculae, indicating that both, the vertebral body and the arches, are formed by the same component. The micro-CT scanning data were uploaded to a public database so they can be used for future studies on fish vertebrae.     

Temporal and spatial analysis of hyaluronidase activity during development of the embryonic chick limb bud

The glycosaminoglycan hyaluronate (HA) appears to play an important role in limb cartilage differentiation. The large amount of extracellular HA accumulated by prechondrogenic mesenchymal cells may prevent the cell-cell and/or cell-matrix interactions necessary to trigger chondrogenesis, and the removal of extracellular HA may be essential to initiate the crucial cellular condensation process that triggers cartilage differentiation. It has generally been assumed that HA turnover during chondrogenesis is controlled by the activity of the enzyme hyaluronidase (HAase). In the present study we have performed a temporal and spatial analysis of HAase activity during the progression of limb development and cartilage differentiation in vivo. We have separated embryonic chick wing buds at several stages of development into well-defined regions along the proximodistal axis in which cells are in different phases of differentiation, and we have examined HAase activity in each region. We have found that HAase activity is clearly detectable in undifferentiated wing buds at stage 18/19, which is shortly following the formation of a morphologically distinct limb bud rudiment, and remains relatively constant throughout subsequent stages of development through stage 27/28, at which time well-differentiated cartilage rudiments are present. Moreover, HAase activity in the prechondrogenic distal subridge regions of the limb at stages 22/23 and 25 is just as high as, or even slightly higher than, it is in proximal central core regions where condensation and cartilage differentiation are progressing. We have also found that limb bud HAase is active between pH 2.2 and 4.5 and is inactive above pH 5.0. This suggests that limb HAase is a lysosomal enzyme and that extracellular HA would have to be internalized to be degraded. These results indicate that the onset of chondrogenesis is not associated with the appearance or increase in activity of HAase. We suggest that possibility that HA turnover may be regulated by the binding and endocytosis of extracellular HA in preparation for its intracellular degradation by lysosomal HAase. Finally, we have found that the apical ectodermal ridge (AER)-containing distal limb bud ectoderm possesses a relatively high HAase activity. We suggest the possibility that a high HAase activity in the AER may ensure a rapid turnover and remodeling of the disorganized HA-rich basal lamina of the AER that might be essential for limb outgrowth.     

The spatial and temporal expression of HNK-1 by myogenic and skeletogenic cells in the embryonic rat

The existence of phenotypic differences within a population of cells provides evidence for discrete stages in cellular differentiation and/or identifies subsets of cells with unique functional properties. The monoclonal antibody HNK-1 has been widely shown to identify subpopulations of cells in the developing nervous system. In this paper we focus on the developmental expression of HNK-1 immunoreactivity by derivatives of somitic (paraxial) mesoderm. We show that between embryonic day 12 and 14 (E12–E14) the HNK-1 epitope is transiently expressed by postmitotic myotomal cells. In E14–E17 developing vertebral columns (which are derived from somitic sclerotomal cells), HNK-1 immunolabeling was expressed by subpopulations of skeletogenic cells, including perinotochordal cells associated with the forming annulus fibrosus and cells within or adjacent to the perichondrium. Chondrocytes within forming centra and vertebral arches did not exhibit HNK-1 immunoreactivity. These results, taken together, show that the expression of the HNK-1 epitope can be used to identify subsets of myogenic and skeletogenic cells both spatially and temporally in the developing rat.     

山鸡椒雄花花芽发育形态解剖特征观察

采用体视显微镜、扫描电镜和石蜡切片技术对山鸡椒（Litsea cubeba（Lour.） Pers.）雄花花芽分化发育的外部形态和内部解剖结构进行了观察研究。结果显示:（1）山鸡椒雄花花芽分化发生可分为5个时期,即未分化期、花序原基分化期、苞片原基分化期、花原基分化期和花器官分化期,其中花器官分化期又可细分为花被原基分化期、雄蕊原基分化期和雌蕊原基分化期;各相邻分化时期存在一定重叠现象;花期从翌年1月上旬至3月下旬。（2）雄花成熟结构中具有独特的雄蕊蜜腺,蜜腺绿色且形态不规则,着生于内轮雄蕊基部,分布于花丝两侧,夹在内外轮雄蕊的花丝之间,与内轮花丝紧密相连。（3）雄蕊花药四室,花药壁发育属于基本型;腺质绒毡层;小孢子母细胞减数分裂过程中胞质分裂属于连续型;成熟花粉为2-细胞花粉粒;成熟花粉粒外壁刺突较多,刺突基部膨大,外壁露出部分粗糙,无薄壁区,有少数小穿孔。（4）山鸡椒雄花中绝大多数雌蕊发育至腹缝线卷合形成子房室时停止,柱头发育不良或者败育,花柱缩短或缺失,不能受精,直到开花结束,即发生退化。本研究明确了山鸡椒雄花花芽发育发生各个阶段时间、形态变化特点及外部形态变化特征,山鸡椒小孢子发生、雄配子体发育至散粉期变化特点和规律以及雄花中退化雌蕊发育的进程,可为山鸡椒优良品种选育、调控花期和提高结实率提供一定的参考。     

The effects of dexamethasone on the differentiation and the fertilisation of the germinal primordium in the chick embryo

We showed that, in the chick embryo, the fertilisation of the attractive germinal epithelium by primary germ cells can be represented by a three-dimensional diagram in which the space and time co-ordinates are graduated in terms of the segmentation of the axial and paraxial mesoderm. We thus established that the differentiation of the coelomic epithelium into an attractive germinal epithelium and the fertilisation of the gonadal primordium both occur by mechanisms that are tightly linked to somitogenesis. In the continuous presence of a constant concentration of Dexamethasone, a marked inhibition of the rate of fertilisation of the gonadal primordium was observed. A mathematical analysis of the mode of action of the inhibitor revealed the progressive establishment of a competition between Dexamethasone and the molecule(s) responsible for the process of attraction. Given the chemical nature of the inhibitor, we propose that the endogenous factor that triggers the first step of the differentiation of the germinal primordium is a steroid-containing complex.     

Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development

Hedgehog (Hh) signaling plays multiple roles in the development of the anterior craniofacial skeleton. We show that the earliest function of Hh is indirect, regulating development of the stomodeum, or oral ectoderm. A subset of post-migratory neural crest cells, that gives rise to the cartilages of the anterior neurocranium and the pterygoid process of the palatoquadrate in the upper jaw, condenses upon the upper or roof layer of the stomodeal ectoderm in the first pharyngeal arch. We observe that in mutants for the Hh co-receptor smoothened (smo) the condensation of this specific subset of crest cells fails, and expression of several genes is lost in the stomodeal ectoderm. Genetic mosaic analyses with smo mutants show that for the crest cells to condense the crucial target tissue receiving the Hh signal is the stomodeum, not the crest. Blocking signaling with cyclopamine reveals that the crucial stage, for both crest condensation and stomodeal marker expression, is at the end of gastrulation--some eight to ten hours before crest cells migrate to associate with the stomodeum. Two Hh genes, shh and twhh, are expressed in midline tissue at this stage, and we show using mosaics that for condensation and skeletogenesis only the ventral brain primordium, and not the prechordal plate, is an important Hh source. Thus, we propose that Hh signaling from the brain primordium is required for proper specification of the stomodeum and the stomodeum, in turn, promotes condensation of a subset of neural crest cells that will form the anterior neurocranial and upper jaw cartilage.     

BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes

Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes.