The development of acellularity of the vertebral bone of the Japanese medaka,Oryzias latipes (Teleostei; Cyprinidontidae)

Among vertebrates, some teleosts are unique in having bone which lacks osteocytes embedded in the matrix. The fate of cells that secrete the matrix of these acellular bones has not been investigated thoroughly. Histological and fluorescent microscopic analysis of the vertebral bone of Oryzias latipes demonstrated that acellularity is not a secondary appearance of an early cellular bone during ontogeny. Vertebral bone is devoid of cells embedded in the matrix throughout development. Cells that secrete bone matrix do not become trapped in their own secretion. Instead, they always remain as a surface layer over the outer surface of the bone. Fluorescent microscopic visualization of tetracycline injected into growing fish demonstrated that bone was only deposited by osteoblasts lining the outer surface of the bone; no deposition of bone took place on the inner surface.     

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.     

Exposure to pro-inflammatory cytokines upregulates MMP-9 synthesis by mesenchymal stem cells-derived osteoprogenitors

An intimate interplay exists between the bone and the immune system, which has been recently termed osteoimmunology. The activity of immune cells affects the intrinsic balance of bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts. The aim of this study was to determine the possible interaction between inflammatory-induced conditions and matrix metalloproteinases-2,-9 (MMP-2,-9) synthesis and secretion by bone marrow-derived osteoprogenitor cells during advanced stages of osteogenesis. Rat bone marrow-derived mesenchymal stem cells (MSCs) were cultured in the presence of osteogenic supplements in order to direct the cells towards the osteogenic differentiation lineage. At the late stages of osteogenesis, assessed by histochemistry, immunohistochemistry and RT-PCR, cultures were exposed to pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin-1alpha (IL-1α). Biochemical, histochemical and molecular biology techniques were used to discern the influence of pro-inflammatory cytokines on MMP-2,-9 synthesis and secretion. Results indicated that MMP-9 synthesis and secretion were significantly induced after exposure to the cytokines (TNF-α, IL-1α) treatment, while MMP-2 levels remained unchanged. These results indicate that in response to inflammatory processes, osteoblasts, in addition to osteoclasts, can also be involved and contribute to the process of active bone resorption by secretion and activation of MMPs.     

IL-6 Contributes to the Defective Osteogenesis of Bone Marrow Stromal Cells from the Vertebral Body of the Glucocorticoid-Induced Osteoporotic Mouse

Osteoporosis is one of the most prevalent skeletal system diseases. It is characterized by a decrease in bone mass and microarchitectural changes in bone tissue that lead to an attenuation of bone resistance and susceptibility to fracture. Vertebral fracture is by far the most prevalent osteoporotic fracture. In the musculoskeletal system, osteoblasts, originated from bone marrow stromal cells (BMSC), are responsible for osteoid synthesis and mineralization. In osteoporosis, BMSC osteogenic differentiation is defective. However, to date, what leads to the defective BMSC osteogenesis in osteoporosis remains an open question. In the current study, we made attempts to answer this question. A mouse model of glucocorticoid-induced osteoporosis (GIO) was established and BMSC were isolated from vertebral body. The impairment of osteogenesis was observed in BMSC of osteoporotic vertebral body. The expression profiles of thirty-six factors, which play important roles in bone metabolisms, were compared through antibody array between normal and osteoporotic BMSC. Significantly higher secretion level of IL-6 was observed in osteoporotic BMSCs compared with normal control. We provided evidences that IL-6 over-secretion impaired osteogenesis of osteoporotic BMSC. Further, it was observed that β-catenin activity was inhibited in response to IL-6 over-secretion. More importantly, in vivo administration of IL-6 neutralizing antibody was found to be helpful to rescue the osteoporotic phenotype of mouse vertebral body. Our study provides a deeper insight into the pathophysiology of osteoporosis and identifies IL-6 as a promising target for osteoporosis therapy.     

Matrix vesicle heterogeneity: possible morphogenetic functions for matrix vesicles.

Extracellular membranous matrix vesicles were localized and described using electronmicroscopy during chondrogenesis, osteogenesis, and dentinogenesis. Evidence indicates that matrix vesicles in each of these specific tissue types function to concentrate and transport ions and enzymes which serve as nucleation sites for the mineralization of hydroxylapatite. We have examined different developmental stages of Meckel's cartilage, alveolar bone and epithelial-mesenchymal interactions associated with tooth formation in newborn mice. These ultrastructural studies indicate matrix vesicle heterogeneity. Whereas most matrix vesicles contain alkaline phosphatase activity during cartilage, bone and dentine mineralization, in earlier developmental stages matrix vesicles contain acid phosphatase activities and little, if any, alkaline phosphatase. Tissue type, specific developmental stage, and ultrastructural criteria indicate various "classes" of matrix vesicles. During epithelial-mesenchymal interactions in tooth development, mesenchymal cells (preodontoblasts) appear to be the source of matrix vesicles as indicated by the complementarity between H-2 histocompatibility alloantigen specificity on the cell surface and that of the matrix vesicle outer surface; matrix vesicles are limited by a trilaminar membrane derived from the mesenchymal cells. Some of the vesicles located adjacent to dividing inner enamel epithelial cells contain RNA's as determined by electron microscopic autoradiography in situ, as well as by direct biochemical assays. We postulate that matrix vesicles have many different and important biological functions, one of which may be to mediate developmental information from mesenchyme to epithelia during "instructive" stages of tooth development.     

Static osteogenesis does not precede dynamic osteogenesis in periosteal ossification of Pleurodeles (Caudata,Amphibia) and Pogona (Squamata,Lepidosauria)

Two successive mechanisms have been described in perichondral ossification: (1) in static osteogenesis, mesenchymal cells differentiate into stationary osteoblasts oriented randomly, which differentiate into osteocytes in the same site; (2) in dynamic osteogenesis, mesenchymal cells differentiate into osteoblasts that are all oriented in the same direction and move back as they secrete collagen fibers. This study is aimed at testing the hypothesis that the ontogenetic sequence static then dynamic osteogenesis observed in the chicken and in the rabbit is homologous and was acquired by the last common ancestor of amniotes or at a more inclusive node. For this we analyze the developmental patterns of Pleurodeles (Caudata, Amphibia) and those of the lizard Pogona (Squamata, Lepidosauria). We processed Pleurodeles larvae and Pogona embryos, prepared thin and ultrathin sections of appendicular bones, and analyzed them using light and transmission electron microscopy. We show that static osteogenesis does not precede dynamic osteogenesis in periosteal ossification of Pleurodeles and Pogona . Therefore, the null hypothesis is rejected and according to the parsimony method the ontogenetic sequence observed in the chicken and in the rabbit are convergent. In Pleurodeles and Pogona dynamic osteogenesis occur without a previous rigid mineralized framework, whereas in the chicken and in the rabbit dynamic osteogenesis seems to take place over a mineralized support whether bone (in perichondral ossification) or calcified cartilage (in endochondral ossification). Interestingly, in typical dynamic osteogenesis, osteoblasts show an axis (basal nucleus—distal endoplasmic reticulum) perpendicular to the front of secreted unmineralized bone matrix, whereas in Pleurodeles and Pogona this axis is parallel to the bone matrix.     

Osteogenic cell lineage analysis is facilitated by organ cultures of embryonic chick periosteum

Monoclonal antibodies against the surface of embryonic osteogenic cells (SB-1, SB-2, SB-3, and SB-5) have been used to characterize the sequence of transitions involved in the osteogenic cell lineage. In the present study, immunohistochemical analyses of the expression of osteogenic cell surface antigens in organ cultures of folded chick periosteum were performed. Unlike traditional culture methods using isolated osteoblastic cells, periosteal explants form a mineralized bone tissue in 4 to 6 days which is virtually identical to the in vivo counterpart. Examination of fresh explants confirm that no mature osteoblastic cells were present, although a discontinuous layer of preosteoblasts was evident. As the wave of osteodifferentiation swept through the cultured tissue, antibody SB-1 reacted with the surface of a large family of cells associated with the developing bone. Antibodies SB-3 and SB-2 reacted with progressively smaller subsets of cells, namely those in successively closer physical association with the newly formed and mineralizing bone. Cells recently encased in bone matrix were stained by both SB-2 and SB-5 antibodies, while those cells deep within the matrix reacted only with antibody SB-5. Analysis of this culture model allows dissection of the discrete cellular transition steps of osteogenesis, and reveals that osteogenic precursor cells proceed through the unique lineage stages which have been documented for in vivo osteogenesis. This culture system has furthermore provided evidence which is used to refine our understanding of the osteogenic cell lineage.     

Using human neural crest-derived progenitor cells to investigate osteogenesis: An in vitro study

Human tooth contains a distinct population of neural crest-derived progenitor cells (dNC-PCs) which are known to give rise to specialized daughter cells of an osteogenic lineage. We hypothesised that dNC-PCs could develop into neural crest-derived bone in a self-propagating and extracorporal culture system. Thus, we examined the three-dimensional structure obtained from osteogenic-stimulated dNC-PCs by morphological, biochemical and spectroscopic methods. After the onset of stimulation, cells formed a multilayer with outer cells covering the surface and inner cells secreting a hyaline matrix. With prolonged culture, multilayers contracted and formed a three-dimensional construct which subsequently converted to a calcified mass. Differentiation of progenitor cells was associated with apoptosis. Cell types which survived were smooth muscle actin-positive cells and bone-like cells. The expression of osteoblastic markers and the secretion of a collagenous matrix indicate that the bone cells had acquired their functional phenotype. Furthermore, these cells produced and secreted membrane-bound vesicles into the newly forming matrix. Consequently, an early biomineralized extracellular matrix was found with calcium phosphate deposits being associated with the newly formed collagen matrix framework. The molar calcium–phosphorus-ratio of the mineralized collagen indicated that amorphous calcium phosphate was present within this matrix. The data suggest that stimulated cultures of dNC-PCs are able to recapitulate some processes of the early phase of osteogenesis.     

Age-related changes in bone formation,osteoblastic cell proliferation,and differentiation during postnatal osteogenesis in human calvaria

We have determined the age-related changes in the growth characteristics and expression of the osteoblast phenotype in human calvaria osteoblastic cells in relation with histologic indices of bone formation during postnatal calvaria osteogenesis. Histomorphometric analysis of normal calvaria samples obtained from 36 children, aged 3 to 18 months, showed an age-related decrease in the extent of bone surface covered with osteoblasts and newly synthesized collagen, demonstrating a progressive decline in bone formation during postnatal calvaria osteogenesis. Immunohistochemical analysis showed expression of type I collagen, bone sialoprotein, and osteonectin in the matrix and osteoblasts, with no apparent age-related change during postnatal calvaria osteogenesis. Cells isolated from human calvaria displayed characteristics of the osteoblast phenotype including alkaline phosphatase (ALP) activity, osteocalcin (OC) production, expression of bone matrix proteins, and responsiveness to calciotropic hormones. The growth of human calvaria osteoblastic cells was high at 3 months of age and decreased with age, as assessed by (³H)-thymidine incorporation into DNA. Thus, the age-related decrease in bone formation is associated with a decline in osteoblastic cell proliferation during human calvaria osteogenesis. In contrast, ALP activity and OC production increased with age in basal conditions and in response to 1,25(OH)₂, vitamin D₃, suggesting a reciprocal relationship between cell growth and expression of phenotypic markers during human postnatal osteogenesis. Finally, we found that human calvaria osteoblastic cells isolated from young individuals with high bone formation activity in vivo and high growth potential in vitro had the ability to form calcified nodular bone-like structures in vitro in the presence of ascorbic acid and β-glycerophosphate, providing a new model to study human osteogenesis in vitro. J. Cell. Biochem. 64:128–139. © 1997 Wiley-Liss, Inc.     

Effect of extracellular matrix on prolactin secretion and mRNA accumulation in GH3 cells

The matrix upon which cells grow affects their morphology, growth rate, response to external stimuli, and protein synthesis. GH3 cells, a well-characterized rat pituitary tumor cell line, synthesize and secrete growth hormone and prolactin (Prl). These cells are rounded, attach loosely, and form clumps when plated on plastic. GH3 cells plated on an extracellular matrix (ECM) from bovine corneal endothelial cells become flattened and strongly adherent to the culture dish, and have an initial increased rate of proliferation. Cells cultured on plastic have a 48-hr lag period before the start of cell division; this can be shortened by increasing the concentration of serum in the medium. Since GH3 cells store little Prl, hormone release is a good index of Prl synthesis. Prl secretion from cells cultured on extracellular matrix is twice as great as from cells cultured on plastic. The increase in Prl secretion from cells grown on extracellular matrix paralleled by a concomitant increase in the accumulation of prolactin mRNA. Cells cultured on plastic secrete more Prl in response to TRH stimulation than do cells cultured on ECM. Cells grown on either surface were unresponsive to dopamine. Thus, culturing cells on ECM may change their morphology and affect the synthesis and regulation of specific cellular proteins and their mRNAs.     

Late skeletal development at the articulation between upper pharyngeal jaws and neurocranial base in the fish, Astatotilapia elegans, with the participation of a chondroid form of bone

This paper presents light-microscopical details of the late development of skeletal tissues at the joint between upper pharyngeal jaws (UPJs) and neurocranial base (parasphenoid and basioccipital bones) in the acellular-boned teleost Astatotilapia elegans. On each of the supporting elements, a bone tissue (AB) is deposited that is anomalous because of its retention of cells within the matrix. Later, this layer is gradually replaced by the anomalous large-celled chondroid kind of bone (CB). Both AB and CB probably grow by apposition from the overlying fibrous layer. Osteoblastlike cells secrete osteoid, which soon calcifies and traps the cells. As in young cellular membrane bone, cells in the AB have a wide, elongate shape and lie amidst sparse, calcified, bonelike matrix but lack a canalicular system. Later generations of enclosed cells have a more vesicular shape, with at least some cells remaining alive in the calcified matrix. Appositional growth of the chondroid bone at its articular side is matched from a certain stage onward by erosion at its basal side. On the upper pharyngeal jaws this resorption is clearly related to the development of new teeth. Although in older stages and adults the chondroid tissue resembles a secondary cartilage, the term chondroid bone (CB) was preferred because of (1) the continuing formation by osteoblastlike cells; (2) the staining affinities of its matrix with that of bone; and (3) its formation both on cartilage bone (the infrapharyngobranchials III-IV and basioccipital bone) and on membrane bone (the parasphenoid bone).     

Pulsed electromagnetic fields accelerate proliferation and osteogenic gene expression in human bone marrow mesenchymal stem cells during osteogenic differentiation

Osteogenesis is a complex series of events involving the differentiation of mesenchymal stem cells to generate new bone. In this study, we examined the effect of pulsed electromagnetic fields (PEMFs) on cell proliferation, alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and gene expression in bone marrow mesenchymal stem cells (BMMSCs) during osteogenic differentiation. Exposure of BMMSCs to PEMFs increased cell proliferation by 29.6% compared to untreated cells at day 1 of differentiation. Semi‐quantitative RT‐PCR indicated that PEMFs significantly altered temporal expression of osteogenesis‐related genes, including a 2.7‐fold increase in expression of the key osteogenesis regulatory gene cbfa1, compared to untreated controls. In addition, exposure to PEMFs significantly increased ALP expression during the early stages of osteogenesis and substantially enhanced mineralization near the midpoint of osteogenesis. These results suggest that PEMFs enhance early cell proliferation in BMMSC‐mediated osteogenesis, and accelerate the osteogenesis. Bioelectromagnetics 31:209–219, 2010. © 2009 Wiley‐Liss, Inc.     

Analysis of fruiting body development in the aggregative ciliate Sorogena stoianovitchae (Ciliophora, Colpodea)

The fruiting body-forming ciliate Sorogena stoianovitchae is a protist that is multicellular in one stage of its life cycle. When nutrient levels are depleted, a number of Sorogena cells aggregate beneath the water surface to form an aerial fruiting body. Based on morphologies and the inhibition of protein synthesis, fruiting body development is divided into five distinct stages: (1) aggregation before sunrise, (2) compact aggregation after sunrise, (3) secretion of mucous matrix, (4) stalk-elongation, and (5) completion of the fruiting-body. In the aggregation stage, the cells were trapped in a matrix material that stained orange with 4',6-diamino-2-phenylindole (DAPI), but differed from the mucous matrix in the later stage. A short interruption of the dark period, at 6-8 h after the onset of dark, inhibited fruiting body development. Irrespective of the length of the dark period (10-16 h), the cells remained in the aggregation stage until the beginning of the light period. Therefore, an uninterrupted dark period of more than 8 h is critical for the initial aggregation of cells, but subsequent development is triggered by light.     

Collagen, type V, alpha1 (COL5A1) is regulated by TGF-beta in osteoblasts.

Bone matrix contains high concentrations of growth factors that are known to play important regulatory roles during osteogenesis, particularly transforming growth factor-beta (TGF-beta). Divergent effects of TGF-beta on bone formation have been reported both in vitro and in vivo depending upon experimental conditions, cells employed and their stage of maturation. In this study, we have used a clonal osteoblastic cell line MC3T3-E1, derived from newborn mouse calvaria, as an in vitro model of bone development. These cells undergo an ordered, time-dependent developmental sequence characterized by three stages (proliferation, differentiation and mineralization), over a 30-35-day period. In this study, cDNA microarray technology was used to study the expression profile of 8470 genes, in the presence of TGF-beta1 during osteoblast development. Microarray analysis revealed 120 cDNAs to be differentially expressed in MC3T3-E1 osteoblasts that had been treated with TGF-beta1. From the 120 differentially expressed genes, we selected Collagen, type V, alpha1 (COL5A1) {differential expression=+4.9} for further studies since it represents a previously uncharacterized component of the bone matrix. Using Northern blotting, we found that, when MC3T3-E1 cells were treated with TGF-beta1, COL5A1 was up-regulated during the proliferation and differentiation phases of osteogenesis. Furthermore, by a combination of RNA in situ hybridization and Northern blotting, we found COL5A1 mRNA to be expressed in the calvaria and developing bone of the E17.5 mouse embryos. Lastly, significant COL5A1 protein expression was observed by immunohistochemistry in the developing bone of the E17.5 mouse embryos. In conclusion, by the use of in vitro and in vivo approaches, we have discovered that the COL5A1 gene is a target of TGF-beta during osteogenesis.     

Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage.

We have developed methodology that enables alkaline phosphatase (ALP) to be histochemically stained reproducibly in decalcified paraffin-embedded bone and cartilage of rodents. Proximal tibiae and fourth lumbar vertebrae were fixed in periodate-lysine-paraformaldehyde (PLP) fixative, decalcified in an EDTA-G solution, and embedded in paraffin. In the articular cartilage of the proximal tibia, ALP activity was localized to the hypertrophic chondrocytes and cartilage matrix of the deep zone and the maturing chondrocytes of the intermediate zone. The cells and matrix in the superficial zone did not exhibit any enzyme activity. In tibial and vertebral growth plates, a progressive increase in ALP expression was seen in chondrocytes and cartilage matrix, with activity being weakest in the proliferative zone, higher in the maturing zone, and highest in the hypertrophic zone. In bone tissue, ALP activity was detected widely in pre-osteoblasts, osteoblasts, lining cells on the surface of trabeculae, some newly embedded osteocytes, endosteal cells, and subperiosteal cells. In areas of new bone formation, ALP activity was detected in osteoid. In the bone marrow, about 20% of bone marrow cells expressed ALP activity. In adult rats, the thickness of the growth plates was less and ALP activity was enhanced in maturing and hypertrophic chondrocytes, cartilage matrix in the hypertrophic zone, and primary spongiosa. This is the first time that ALP activity has been successfully visualized histochemically in decalcified, paraffin-embedded mineralized tissues. This technique should prove to be a very convenient adjunct for studying the behavior of osteoblasts during osteogenesis.     

Conditional ablation of osteoblasts in medaka

Different from tetrapods, teleost vertebral centra form without prior establishment of a cartilaginous scaffold, in two steps: First, mineralization of the notochord sheath establishes the vertebral centra. Second, sclerotome derived mesenchymal cells migrate around the notochord sheath. These cells differentiate into osteoblasts and deposit bone onto the mineralized notochord sheath in a process of intramembranous bone formation. In contrast, most skeletal elements of the cranial skeleton arise by chondral bone formation, with remarkably similar mechanisms in fish and tetrapods. To further investigate the role of osteoblasts during formation of the cranial and axial skeleton, we generated a transgenic osx:CFP-NTR medaka line which enables conditional ablation of osterix expressing osteoblasts. By expressing a bacterial nitroreductase (NTR) fused to Cyan Fluorescent Protein (CFP) under control of the osterix promoter these cells become sensitive towards Metronidazole (Mtz). Mtz treatment of stable osx:CFP-NTR transgenic medaka for several consecutive days led to significant loss of osteoblasts by apoptosis. Live staining of mineralized bone matrix revealed reduced ossification in head skeletal elements such as cleithrum and operculum, as well as in the vertebral arches. Interestingly in Mtz treated larvae, intervertebral spaces were missing and the notochord sheath was often continuously mineralized resulting in the fusion of centra. We therefore propose a dual role for osx-positive osteoblasts in fish. Besides a role in bone deposition, we suggest an additional border function during mineralization of the chordal centra. After termination of Mtz treatment, osteoblasts gradually reappeared, indicating regenerative properties in this cell lineage. Taken together, the osx:CFP-NTR medaka line represents a valuable tool to study osteoblast function and regeneration at different stages of development in whole vertebrate specimens in vivo.     

Enhanced Healing of Rat Calvarial Bone Defects with Hypoxic Conditioned Medium from Mesenchymal Stem Cells through Increased Endogenous Stem Cell Migration via Regulation of ICAM-1 Targeted-microRNA-221

The use of conditioned medium from mesenchymal stem cells may be a feasible approach for regeneration of bone defects through secretion of various components of mesenchymal stem cells such as cytokines, chemokines, and growth factors. Mesenchymal stem cells secrete and accumulate multiple factors in conditioned medium under specific physiological conditions. In this study, we investigated whether the conditioned medium collected under hypoxic condition could effectively influence bone regeneration through enhanced migration and adhesion of endogenous mesenchymal stem cells. Cell migration and adhesion abilities were increased through overexpression of intercellular adhesion molecule-1 in hypoxic conditioned medium treated group. Intercellular adhesion molecule-1 was upregulated by microRNA-221 in mesenchymal stem cells because microRNAs are key regulators of various biological functions via gene expression. To investigate the effects in vivo, evaluation of bone regeneration by computed tomography and histological assays revealed that osteogenesis was enhanced in the hypoxic conditioned medium group relative to the other groups. These results suggest that behavioral changes of endogenous mesenchymal stem cells through microRNA-221 targeted-intercellular adhesion molecule-1 expression under hypoxic conditions may be a potential treatment for patients with bone defects.     

Studies on H-Y antigen in different cell fractions of the testis during pubescence

Summary Various cell types of the rat testis during pubescence, including germ, Sertoli, and Leydig cells, were partially enriched. The fractions were tested for the presence, binding, and secretion of H-Y antigen. The main results are: Immature germ cells are H-Y antigen-negative until the late diploid stages, and late primary spermatocytes or spermatids become positive; the somatic cells of the gonad are positive at all ages examined (18 days old to adulthood). Secretion of H-Y antigen is restricted to the Sertoli cell fraction. Binding of externally supplied antigen takes place on Leydig cells; the Sertoli cell surface will be saturated because of active secretion; there is no binding to germ cells. Thus, immature germ cells seem to be the only H-Y antigen-negative cells of the male organism, and the Sertoli cells seem to be the only ones to secrete H-Y antigen.     

Ascidian Larvae: The Role of Test Cells in Preventing Hydrophobicity

Abstract Ascidian test cells co-differentiate on the surface of each ovarian oocyte beneath the vitelline coat. They become vacuolated and later occupy the perivitelline compartment of each egg and embryo. In some species, their vacuoles contain submicroscopic granules or filaments called ‘ornaments’ and acidic glycosaminoglycans. These test cells deposit their products on the surface of the larval tunic in late embryogenesis. In these species, the test cells are lost at hatching. In other species, the test cell vacuoles contain acidic glycosaminoglycans, but no ornaments. In these species, the test cells attach to the larval tunic and probably secrete acidic glycosaminoglycans. We deprived the embryos of seven species of ascidians of their test cells and vitelline coats during midembryogenesis. After completing their development, the larvae of both kinds of species were hydrophobic. They easily become trapped on the surface of sea water in cultures. Normal larvae (controls), bearing test cell secretions, are hydrophilic and never become trapped. We infer that negatively charged secretions of the test cells make normal larvae hydrophilic. Some molgulids with direct development have no test cells, no fins and no swimming larva. We reason that the test cells of these species may have been lost during evolution because they no longer had an important role in preventing hydrophobicity.