Tenascin-W inhibits proliferation and differentiation of preosteoblasts during endochondral bone formation

We identified a cDNA encoding mouse Tenascin-W (TN-W) upregulated by bone morphogenetic protein (Bmp)2 in ATDC5 osteo-chondroprogenitors. In adult mice, TN-W was markedly expressed in bone. In mouse embryos, during endochondral bone formation TN-W was localized in perichondrium/periosteum, but not in trabecular and cortical bones. During bone fracture repair, cells in the newly formed perichondrium/periosteum surrounding the cartilaginous callus expressed TN-W. Furthermore, TN-W was detectable in perichondrium/periosteum of Runx2-null and Osterix-null embryos, indicating that TN-W is expressed in preosteoblasts. In CFU-F and -O cells, TN-W had no effect on initiation of osteogenesis of bone marrow cells, and in MC3T3-E1 osteoblastic cells TN-W inhibited cell proliferation and Col1a1 expression. In addition, TN-W suppressed canonical Wnt signaling which stimulates osteoblastic differentiation. Our results indicate that TN-W is a novel marker of preosteoblasts in early stage of osteogenesis, and that TN-W inhibits cell proliferation and differentiation of preosteoblasts mediated by canonical Wnt signaling.     

Lack of either chondrocyte hypertrophy or osteogenesis in Meckel's cartilage of the embryonic chick exposed to epithelia and to thyroxine in vitro

Osteogenesis was not initiated when Meckel's cartilages from embryonic chicks of Hamburger and Hamilton (H. H.) stages 38 and 39 were recombined with mandibular epithelia obtained from embryos of H. H. stage 22 (a stage when an epithelial-mesenchymal interaction elicits osteogenesis from mandibular mesenchyme) and grafted to the chorioallantoic membranes of host embryos for 7 to 21 days. Failure of osteogenesis was not because the cartilage inhibited or blocked the osteogenesis-initiating capabilities of mandibular epithelium for mandibular epithelia could still elicit osteogenesis when removed from Meckel's cartilages and recombined with mandibular mesenchyme. Chondrocyte hypertrophy is associated with osteogenesis in other cartilages, including Meckel's cartilage from rodent embryos. However, Meckel's cartilages from chick embryos of H. H. stages 34, 38, and 39 failed to hypertrophy when cultured in the presence of 7.5 nM thyroxine (3,3',5-triiodo-L-thyroxine), although H. H. stage 28 tibial chondrocytes cocultured with Meckel's cartilage did hypertrophy. Therefore, avian Meckelian chondrocytes fail to hypertrophy or to produce osteoprogenitor cells in response to stimuli known to evoke these events in other skeletal cells.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence: II. Localization of type I and type II collagen during long bone development

The transition of type I and type II collagens during cartilage and bone development in the chick embryo was studied by immunofluorescence using antibodies against type I or type II collagens. Type II collagen was found in all cartilaginous structures which showed metachromatic staining. Type I collagen appeared in the perichondrium of the tibia at stage 28 and was also found in osteoid, periosteal and enchondral bone after decalcification, periosteum, and tendons, ligaments, and capsules.Using the immunohistological method it was possible to identify specific collagen types in areas undergoing rapid proliferation and collagen transition, such as diaphyseal and epiphyseal perichondrium, or in enchondral osteogenesis. During enchondral ossification type I collagen is deposited onto the eroded surface of cartilage. It partially diffuses into the cartilage matrix forming a “hybrid” collagen matrix with type II collagen, which is a site for subsequent ossification. During appositional growth of diaphyseal cartilage and differentiation of epiphyseal perichondrium into articular cartilage, perichondral cells switch from type I to type II collagen synthesis when differentiating into chondroblasts. In the transition zones, chondroblasts are imbedded in a “hybrid” matrix consisting of a mixture of type I and type II collagens.     

Study of differential collagen synthesis during development of the chick embryo by immunofluorescence. I. Preparation of collagen type I and type II specific antibodies and their application to early stages of the chick embryo.

The aim of this work was to prepare specific antibodies against skin and bone collagen (type I) and cartilage collagen (type II) for the study of differential collagen synthesis during development of the chick embryo by immunofluorescence. Antibodies against native type I collagen from chick cranial bone, and native pepsin-extracted type II collagen from chick sternal cartilage were raised in rabbits, rats, and guinea pigs. The antibodies, purified by cross-absorption on the heterologous collagen type, followed by absorption and elution from the homologous collagen type, were specific according to passive hemagglutination tests and indirect immunofluorescence staining of chick bone and cartilage tissues. Antibodies specific to type I collagen labeled bone trabeculae from tibia and perichondrium from sternal cartilage. Antibodies specific to type II collagen stained chondrocytes of sternal and epiphyseal cartilage, whereas fluorescence with intercellular cartilage collagen was obtained only after treatment with hyaluronidase. Applying type II collagen antibodies to sections of chick embryos, the earliest cartilage collagen found was in the notochord, at stage 15, followed by vertebral collagen secreted by sclerotome cells adjacent to the notochord from stage 25 onwards. Type I collagen was found in the dermatomal myotomal plate and presumptive dermis at stage 17, in limb mesenchyme at stage 24, and in the perichondrium of tibiae at stage 31.     

Critical periods during development as assessed by thallium-induced inhibition of growth of embryonic chick tibiae in vitro

In ovo application of thallium sulfate has been shown to produce a characteristic shortening and angulation of the tibia of the embryonic chick. The critical period for susceptibility to thallium-induced inhibition of tibial growth ends at 8 2/3 days of incubation, a time when the growth rate of the embryo declines by 55%. The aim of the present study was to expose tibiae to thallium in vitro to determine whether this response was intrinsic to the tibia. A 4-hour exposure to 400 micrograms thallium was found to be most effective. Growth of tibiae from 8-day-old embryos was inhibited, growth of tibiae from 9-day-old embryos was not, and the response of tibiae isolated from embryos of 8 2/3 days of incubation was intermediate. Therefore, the response of the tibia to thallium represents an intrinsic property and is not secondary to inhibited nerve growth as has been suggested. The critical period and its termination is also an intrinsic property of the tibia. Tibiae were exposed to thallium for 4 hours at various times after the tibiae had been established in vitro. Susceptibility to growth inhibition was shown to decline as tibiae developed beyond the critical period, a decline that could be correlated with a declining growth rate. Emphasis is placed upon critical events during development as a more useful concept than critical periods for explaining susceptibility to teratogens.     

YKL-40 protein expression in the early developing human musculoskeletal system.

YKL-40 is a growth factor for chondrocytes and fibroblasts. The aim was to evaluate YKL-40 expression in the musculoskeletal system during early human development. We studied sections from 15 human embryos [weeks 5.5-8; 7- to 31-mm crown-rump length (CRL)] and 68 fetuses (weeks 9-14; 33- to 105-mm CRL) for YKL-40 protein expression by immunohistochemistry. YKL-40 mRNA expression was evaluated in two human embryos (days 41 and 51). Initially YKL-40 is expressed in all germ layers: ecto-, meso-, and endoderm. YKL-40 mRNA and protein expression are found in tissues of the ecto-, meso-, and endoderm, and YKL-40 protein expression is present during development of cartilage, bone, joints, and muscles. At the cellular level, YKL-40 protein expression is high in tissues characterized by rapid proliferation, marked differentiation, and undergoing morphogenetic changes. Examples of rapid cell proliferation include the chondrogenic inner layer of perichondrium and the osteogenic inner layer of periosteum. Differences in YKL-40 expression during differentiation are found in the chondrogenic and osteogenic cell lineages. The initial shaping of cartilage and bone models and joints is concomitant with a strong outline of YKL-40-positive cells. This indicates that YKL-40 is associated with cell proliferation, differentiation, and tissue morphogenesis during development of the human musculoskeletal system.     

In vitro reformation of the perichondrium from perichondrial-free Meckel's cartilages of the embryonic chick

Perichondria were removed from Meckel's cartilages of chick embryos of Hamburger and Hamilton stages 34, 38, or 39 (8, 12, or 13 days of incubation) and cultured, either at the air-medium interface or submerged, under standard organ culture conditions, for 7 to 21 days. Meckel's cartilages formed a new fibrous perichondrium by the 10th day of culture. Perichondria both formed earlier and were thicker in those cartilages cultured at the air-medium interface than in those cultured submerged. Histological and ultrastructural analysis indicated that the outermost layer of Meckelian chondrocytes dedifferentiated into fibrous cells to form the new fibrous perichondrium; i.e., the fibrous perichondrium can arise from superficial chondrocytes.     

Autoradiographic localization of osteogenin binding sites in cartilage and bone during rat embryonic development

Osteogenin, a novel bone differentiation factor isolated from bone, has been recently purified and the amino acid sequence determined. Osteogenin in conjunction with a collagenous bone matrix substratum induces cartilage and bone formation in vivo. In order to understand the developmental role of osteogenin during cartilage and bone morphogenesis we examined the binding and distribution of iodinated osteogenin in developing rat embryos. Whole embryo tissue sections were made from 11, 12, 13, 15, 18, and 20 day fetuses. The specific binding of osteogenin at different stages of rat embryonic development was determined by autoradiography. Maximal binding was observed in mesodermal tissues such as cartilage, bone, perichondrium, and periosteum. During Days 11-15, peak binding was localized to perichondrium during limb and vertebral morphogenesis. By Day 18 periosteum exhibited the highest concentration of autoradiographic grains. Osteogenin was also localized in developing membranous bones of the calvarium and other craniofacial bones. Considerably less binding was observed, in decreasing order, in muscle, liver, spleen, skin, brain, heart, kidney, and intestine. The observed maximal binding during skeletal morphogenesis implies a developmental role for osteogenin.     

Teratogenic effects of retinoic acid and dimethylsulfoxide on embryonic chick wing and somite

In order to document the stage(s) at which the embryonic chick wing bud is sensitive to vitamin A teratogenesis and the kinds of defects produced by vitamin A insult to the embryonic chick wing, 1-microgram doses of retinoic acid (1 microliter RA in 90% DMSO at a concentration of 1 microgram/microliter) were locally applied to the right wing bud of chick embryos at stages 17-23 (Hamburger and Hamilton: J. Morphol., 88:49-92, '51), and the resulting limb skeleton anatomy was observed at 10 days of incubation. Local application of RA at stages 17-20 resulted in distal wing skeleton defects. There were significantly more wing skeleton defects among embryos treated at these stages with RA solution than among solvent (DMSO)-treated control embryos and than among untreated control embryos. Wings of embryos treated with RA at stages 21-23 were always normal. Scapular and vertebral defects were seen at 10 days of incubation among embryos which had been treated prior to stage 21 with both the RA solution and the solvent control. Statistical analysis and histological data suggest that scapular and vertebral defects were caused by DMSO-induced damage to somites.     

Regulation of Endochondral Cartilage Growth in the Developing Avian Limb: Cooperative Involvement of Perichondrium and Periosteum

The perichondrium and periosteum have recently been suggested to be involved in the regulation of limb growth, serving as potential sources of signaling molecules that are involved in chondrocyte proliferation, maturation, and hypertrophy. Previously, we observed that removal of the perichondrium and periosteum from tibiotarsi in organ culture resulted in an overall increase in longitudinal cartilage growth, suggesting negative regulation originating from these tissues. To determine if the perichondrium and periosteum regulate growth through the production of diffusible factors, we have tested various conditioned media from these tissues for the ability to modify cartilage growth in tibiotarsal organ cultures from which these tissues have been removed. Both negative and positive regulatory activities were detected. Negative regulation was observed with conditioned medium from (1) cell cultures of the region bordering both the perichondrium and the periosteum, (2) co-cultures of perichondrial and periosteal cells, and (3) a mixture of conditioned media from perichondrial cell cultures and periosteal cell cultures. The requirement for regulatory factors from both the perichondrium and periosteum suggests a novel mechanism of regulation. Positive regulation was observed with conditioned media from several cell types, with the most potent activity being from articular perichondrial cells and hypertrophic chondrocytes.     

Immunohistochemical detection of subunit A(M) of lactate dehydrogenase in tissues of the developing chick

Summary The distribution of A(M) subunits of lactate dehydrogenase (mainly LDH₅) in developing muscle, heart, liver, lung, kidney and cartilage tissue of chicken embryos was examined by the indirect fluorescent antibody technique. Antibodies against porcine LDH₅, purified by affinity chromatography, were used for this purpose. In special areas of newly formed myofibrils in somitic myoblasts fluorescence was already detected after 4 days of incubation, and located at the same place in muscle tissue of all advanced developmental stages examined. During the myotube stage of muscle development staining was also located in the peripheral thickened cytoplasma of the myotubes. The myocardium did not exhibit any fluorescent staining in the developmental stages examined. Endocardium, epicardium and pericardium, however, were fluorescent in young developmental stages. The liver showed fluorescence in 5- to 8-day embryos mainly in the endothelial cells of the blood sinusoids. In 9- to 12-day embryos the bile ducts became fluorescent. In lungs after 9- to 12-day development the epithelium and the surrounding tissues of bronchi exhibited strong immunofluorescence. The mesonephros exhibited faint granular fluorescence in tubule-forming cells and their membranes after 4–9 days of incubation. Advanced developmental stages only exhibited fluorescent blood cells. This latter staining is at least partly due to non-specific reactions of blood cell membranes with FITC-conjugated anti-rabbit IgG. Cartilage is characterized by non-specific fluorescence, but in embryos older than 8 days strong granular fluorescence of chondrocytes and staining of the perichondrium distinguished sections treated with anti-LDH₅ antibodies from control sections reacted only with FITC-conjugated anti-rabbit IgG. In addition, strong fluorescent staining was detectable in certain areas of the 5-day neural tube and faint staining in the mucosa of the intestine from embryos older than 10 days.     

Temporal and spatial expression of genes for cartilage extracellular matrix proteins during avian mandibular arch development

We have examined the temporal expression of genes for extracellular matrix proteins (type I collagen, type II collagen, and the cartilage specific proteoglycan core protein) during the development of the avian mandibular arch. We detected low levels of type II collagen mRNA in the mandibular arch as early as stage 15. Type II collagen mRNA remained low but increased slightly as development progressed from stage 15 to stage 25. More dramatic increases occurred after stage 25 coincident with overt chondrogenesis. In contrast, mRNA for the core protein of cartilage specific proteoglycan was not detected prior to the onset of chondrogenesis, appeared at stage 25, and increased thereafter. Type I collagen mRNA was also present as early as stage 15 and dramatically increased after stage 28/29, coincident with initiation of osteogenesis. Using in situ hybridization, we found that type II collagen mRNA became detectable in the center of the mandible around stage 24/25 coincident with the initiation of chondrogenesis. At later stages (26-32) type II collagen mRNA was localized in the cartilaginous rudiment. The pattern of hybridization observed with the proteoglycan core protein probe at later stages of development was essentially identical to that observed with the type II collagen probe. In contrast, the probe for the alpha 1 (I) collagen mRNA was localized over the perichondrium, over differentiated bone, and in areas within the mandibular arch where bone formation had been initiated.     

Early embryonic development in Thai swamp buffalo (Bubalus bubalis )

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.     

Superovulation in nonseasonal Japanese native goats, with special reference to the developmental progression of embryos

Treatment for superovulation with pregnant mare serum gonadotropin (PMSG) and follicle stimulating hormone (FSH) was carried out in nonseasonal breeder Japanese goats which are widely used as a substitute model for cattle in various studies in Japan. The proportion of females that came into estrus (93 and 99%) and the interval between PGF(2) administration and estrus (1.5 to 2.0 days) did not differ between females treated with PMSG and those treated with FSH. The number of normal embryos recovered was significantly higher (P<0.01) in FSH-treated (9.4 +/- 5.6) femals than in PMSG-treated females (5.7 +/- 4.4). The developmental stage of embryos recovered from 1.0 to 8.5 at 0.5-d intervals after mating is also described. The development to the two-cell, four-cell, eight-cell, morula, blastocyst and zona-free blastocyst stage was first observed 1.5, 2.5, 5.0 to 5.5, 6.0 and 6.5 d, respectively, after human chorionic gonadotropin (hCG) injection.     

Development of the mandibular skeleton in the embryonic chick as evaluated using the DNA-inhibiting agent 5-fluoro-2'-deoxyuridine

Mandibular development was examined in embryonic chicks following administration of 5-fluoro-2'-deoxyuridine (FUDR, 0.001-1.0 microgram/egg), an inhibitor of both DNA synthesis and of cell division. FUDR was injected in ovo at one of three developmental stages corresponding to 1) the migration of mandible-destined, midbrain-level neural crest cells (Hamburger and Hamilton [H.H.] stage 10); 2) midway through the epithelial-mesenchymal interaction required to initiate mandibular osteogenesis (H.H. stage 22), which is also after the epithelial-neural crest cell interaction required for the initiation of chondrogenesis in Meckel's cartilage; and 3) when prechondroblasts of Meckel's cartilage are beginning to differentiate (H.H. stage 25). Micromelia was induced following the administration of FUDR at either H.H. stages 22 or 25 but not when FUDR was given at H.H. stage 10. Although the micromelic mandibles were shorter than normal, Meckel's cartilage and the mandibular membrane bones both differentiated and grew along the full proximodistal length of the shortened mandibles. In contrast to the situation previously described by Ferguson for alligator embryos exposed to FUDR, the migration of neural crest cells in the embryonic chick was not inhibited by FUDR. In contrast to the situation previously described for rat embryos exposed to FUDR, differentiation of Meckel's cartilage was not inhibited in embryonic chicks exposed to FUDR. Differentiation of the membrane bones was also normal following either in ovo administration of FUDR or when mandibular processes were maintained in FUDR in vitro. Therefore, FUDR does not produce micromelia in the embryonic chick by interfering with the epithelial-mesenchymal/neural crest cell interactions, which are prerequisites or differentiation of cartilage or bone, nor by inhibiting the differentiation of chondrogenic or osteogenic mesenchymal cells after completion of these tissue interactions. Neither did the growth-inhibiting action of FUDR result from an inhibition of growth of Meckel's cartilage during the several days following initial chondrogenic differentiation. Rather, subsequent growth of the entire mandibular process was delayed. This mechanism of action differs from that in the alligator embryo, in which FUDR inhibits mandibular growth by removing mandible-destined, migrating neural crest cells, and in the rat, in which FUDR inhibits the differentiation of Meckel's cartilage but catch-up growth restores growth of the mandible to normal.     

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.     

Temporospatial patterns of apoptosis in chick embryos during the morphogenetic period of development

We examined the temporospatial pattern of naturally occurring apoptosis in chick embryos to five days of incubation (H.H. stages 1-25; Hamburger and Hamilton, 1951) using TUNEL labeling. The initial TUNEL-positive structure was the embryonic shield at stage 1. Apoptotic cells became ubiquitously present within embryos by stage 3, which is early in gastrulation. Until stage 6, TUNEL-positive cells were restricted to the headfold region. In embryos of stages 7-8, most cell death was localized at the most anterior neural plate. TUNEL-positive neural plate, notochord and somites appeared at stage 9. Otic and optic regions became TUNEL-positive at stage 11. The aggregation of cells from which the tail bud arises contains apoptotic cells from stage 11 onwards. At stage 16, scattered TUNEL-positive cells appeared in the branchial arches. Three streams of apoptotic neural crest cells in the cranial region became most clearly visible at stage 18. The secondary neural tube from which caudal structures develop contains apoptotic cells at stage 14. Apoptotic cells are present in the branchial arches and lateral body wall for extended periods, stages 16-25 and 25 respectively. At stages 24-25, intense positive regions of cell death were confined to the caudal regions of the arches, to limb and tail buds and to the lateral body wall, the latter in relation to body wall closure. The new findings in this study are discussed along with past studies to provide the temporospatial pattern of cell death during early chick development.     

The differential response of cortical and trabecular bone to aluminum administration in the rat

Osteomalacia has been noted following in vivo aluminum (Al) loading in the rat by some investigators but not by others. To determine whether the response of bone to Al differs as a function of the skeletal site examined, quantitative histology of cortical and trabecular bone was done in the tibiae from control (C, n = 10), Al-treated (AL, n = 9), nephrectomized control (NX-C, n = 7), and nephrectomized Al-treated (NX-AL, n = 8) rats given 2 mg/day of Al for 4 weeks. Bone Al content was determined by histochemical methods. In cortical bone, osteoid seam width, osteoid volume, and percent osteoid area were similar for all groups. In contrast, for trabecular bone, both forming surface (means +/- SD) (5.2 +/- 3.4 vs 1.8 +/- 1.1%, P less than 0.05) and osteoid volume (1.7 +/- 0.7 vs 1.0 +/- 0.4%, P less than 0.05) increased from control values in AL, although osteoid seam width did not differ. In NX-AL, trabecular forming surface (20.2 +/- 6.7 vs 6.2 +/- 2.4%, P less than 0.01), osteoid area (13.2 +/- 5.7 vs 3.5 +/- 0.8%, P less than 0.01), and osteoid width (18.7 +/- 5.7 vs 9.7 +/- 2.3 micron, P less than 0.01) all were greater than in NX-C. Deposits of Al were undetectable in C and NX-C, were minimal in cortical bone in AL and NX-AL, but were present at 40.5 +/- 11.5 and 71.1 +/ 6.5% of trabecular surfaces in AL and NX-AL, respectively. Osteoid area and osteoid surface each correlated with trabecular bone Al. Thus, (a) osteoid accumulates in trabecular, but not in cortical, bone after 4 weeks of Al loading; (b) the extent of osteoid accumulation correlates with the bone Al content; and (c) the histologic response to Al in cortical and trabecular bone is related to local differences in the uptake of Al into bone.