Thomas F. Linsenmayer

Developmental acquisition of type × collagen in the embryonic chick tibiotarsus☆

The temporal and spatial distribution of short chain skeletal (Type X) collagen was immunohistochemically examined in the chick tibiotarsus from 6 days of embryonic development to 1 day posthatching. The monoclonal antibody employed (AC9) was recently produced and characterized as being specific for an epitope located within the helical domain of the type X collagen molecule (T. M. Schmid and T. F. Linsenmayer, J. Cell Biol., in press). The earliest detectable appearance of type X collagen was at 7.5 days, at which time it was restricted to a middiaphyseal location (i.e., in the primary center of ossification). This was in marked contrast to type II collagen, which appears earlier and is distributed throughout the cartilaginous anlagen. With increasing embryonic age, the reactivity with the type X antibody progressively extended toward the epiphyses, lagging somewhat behind the progression of chondrocyte hypertrophy. The anti-type X collagen antibody also reacted with the bony matrix itself, but the immunofluorescent signal produced by this source was considerably less than that produced by cartilage. At 19 days of development, a new small site of type X deposition was initiated in an epiphyseal location, which subsequently enlarged in circumference. These results are consistent with our previous biochemical studies suggesting that, in cartilage, type X collagen is specifically a product of that population of chondrocytes which have undergone hypertrophy.

Temporal and spatial transitions in collagen types during embryonic chick limb development.

Abstract To determine whether transitions occur in the types of collagen synthesized during embryonic chick limb development, the α chain composition of the collagens produced by whole limbs and various anatomical regions of limbs was analyzed at different stages (23–24 to 40). The tissues were incubated in the presence of 3 H-proline and 3 H-lysine and the α chain distribution of the purified, labeled collagens was determined by chromatography on carboxymethyl cellulose columns. We found that the stage 23–24 leg mesenchyme is producing predominantly, if not solely, an (α1) 2 α2 type collagen (chain type as yet undetermined). At about stage 25–26 the limb core begins synthesizing detectable amounts of (α1) 3 collagen, which we presume to be cartilage type collagen, [α1 (II)] 3 , while the outer portion of the limb largely continues to produce (α1) 2 α2. The production of (α1) 3 collagen in the core progressively increases until, by stage 33 it is the only species detectable in the tibial diaphysis. Shortly thereafter (by stage 35 + –36) (α1) 2 α2 type collagen reappears in the tibial diaphysis signifying the production of bone collagen, [α1 (I)] 2 α2. During the next several days of incubation, the relative proportion of (α1) 2 α2 increases in the bony diaphysis while (α1) 3 remains the predominant species synthesized in the cartilaginous epiphysis.

Dual origin of glomerular basement membrane.

The histogenesis of renal basement membranes was studied in grafts of avascular, 11-day-old mouse embryonic kidney rudiments grown on chick chorioallantoic membrane (CAM). Vessels of the chick CAM invade the mouse tissue during an incubation period of 7-10 days and eventually hybrid glomeruli composed of mouse epithelium and chick endothelium form. Formation of basement membranes during this development was followed by immunofluorescence and immunoperoxidase stainings using polyclonal and monoclonal antibodies against mouse and chick collagen type IV and against mouse laminin. These antibodies were species-specific as shown in immunochemical and immunohistologic analyses. The glomerular basement membrane contained both mouse and chick collagen type IV, demonstrating its dual cellular origin. All other basement membranes were either exclusively of chick origin (mesangium, vessels) or of mouse origin (tubuli, Bowmans capsule).

Chondrogenesis from single limb mesenchyme cells

Abstract It is believed that cell-cell interaction between mesenchyme cells is involved in the initiation of chondrogenesis, based largely on the inability of limb mesenchyme cells to differentiate into cartilage unless cultures are inoculated at densities greater than confluency. The present study describes a culture situation in which single limb mesenchyme cells either in or on type I collagen gels are shown to differentiate into cartilage, as defined by the appearance of a pericellular alcian blue staining matrix, intracellular type II collagen (demonstrated by indirect immunofluorescence with monoclonal antibody), and clonable cartilage cells. Because the differentiation of cartilage cells from single mesenchyme cells occurs only when the cells are in a round configuration, it is proposed that cell shape changes are one factor that can mediate effects of cell-cell interaction on differentiation.

The collagen of chick embryonic notochord

Abstract Notochords, isolated from 2 1 2 day chick embryos, were cultured in the presence of 3H proline and the labeled proteins co-purified with chick skin carrier collagen. The purified material, most of which eluted from CM-cellulose as a single peak in the region of the carrier collagen α1 chain, contained 41% of the incorporated proline as hydroxyproline and from gel filtration measurements had a molecular weight of approximately 100,000 daltons. When the material was chromatographed on DEAE-cellulose with carrier α1 chains from both skin [α1 (I)] and cartilage [α1 (II)], it eluted predominantly with the cartilage chains.

Identification and characterization of up-regulated genes during chondrocyte hypertrophy

Chondrocyte hypertrophy involves de novo acquisition and/or increased expression of certain gene products including, among others, type X collagen, alkaline phosphatase, and matrix metalloproteinases. To analyze further the genetic program associated with chondrocyte hypertrophy, we have employed a modification of the polymerase chain reaction‐mediated subtractive hybridization method of Wang and Brown (Wang and Brown [1991] Proc. Natl. Acad. Sci 88:11505). Cultures of hypertrophic tibial chondrocytes and nonhypertrophic sternal cells were used for poly A+ RNA isolation. Among 50 individual cDNA fragments isolated for up‐regulated hypertrophic genes, 18 were tentatively identified by their similarities to entries in the GenBank database, whereas the other 32 showed no significant similarity. The identified genes included translational and transcriptional regulatory factors, ribosomal proteins, the enzymes transglutaminase and glycogen phosphorylase, type X collagen (highly specific for hypertrophic cartilage matrix), gelsolin, and the carbohydrate‐binding protein galectin. Two of these, transglutaminase and galectin, were cloned and were further characterized. The chondrocyte transglutaminase revealed previously in hypertrophic cartilage by immunochemical methods appears to be the chicken equivalent of mammalian factor XIIIa (showing 75% overall protein similarity). The chicken chondrocyte galectin is a variant of mammalian galectin‐3. Galectins are known to bind to components found in hypertrophic cartilage, and factor XIIIa is known to crosslink some of the same components, possibly modifying them for calcification and/or removal.

Hypertrophic cartilage matrix. Type X collagen, supramolecular assembly, and calcification.

During endochondral bone formation, individual chondrocytes undergo a progression from young cells undergoing rapid division, to mature cells having the greatest capacity for synthesizing matrix components, to hypertrophic cells, becoming greatly enlarged and eventually removed. Concomitant with, and subsequent to, the process of cellular hypertrophy, major changes occur in the cartilage matrix. These include: (a) enlargement of lacunae to accommodate the great increase in volume of the individual chondrocytes; ’ (b) calcification of the cartilage matrix, providing a substratum for bone deposition; and (c) removal of both calcified and uncalcified cartilage matrix, resulting in formation of a marrow cavity. In young, prehypertrophic cartilage the chondrocytes synthesize a mixture of collagen types 11, IX, and XI, which become coassembled into heterotypic fibrils. Once the cells have initiated hypertrophy, they add type X to their biosynthetic repertoire. Quantitatively, type X collagen may be a minor component of the growth plate; but during the latter stages of hypertrophy, chondrocytes devote much of their biosynthetic potential to its synthesis. As the hypertrophic program progresses, the synthesis of this collagen increases, with a concomitant decrease in synthesis of the others.* This, coupled with the morphological events occurring in the cells and matrix during hypertrophy, have prompted us to hypothesize that: (a) the molecule may represent a component added to the matrix to facilitate turnover; or (b) it may participate in the calcification of the cartilage matrix.

Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes

We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckels cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS)

Control of integumentary patterns in the chick

To elucidate some of the parameters of pattern formation, experiments were done using chick skin from feather, large-scale, and small-scale forming regions. Pieces of feather-forming thigh tract skin, isolated 2 days before feathers appear in ovo, develop normally when explanted to the chorioallantoic membrane. The first row forms near the edge of the explant, and the secondary primordia form sequentially, establishing the normal pattern of diagonally intersecting rows. Thus, both the sequential (temporal) appearance and the spatial arrangement of feathers are controlled within the skin itself. Normal development also occurs in each half of the tract when these are separated before explantation. Thus, no tract-specific “initiator row” (which would normally be contained only in the lateral half) is necessary for development. To determine whether a pattern forms early and remains latent until primordia appear, explants of equal size were cultured from young and old embryos. If a latent pattern exists, the explants from the younger embryos should produce a predictably larger number of primordia, which should also be spaced closer together. Neither prediction held, indicating that no latent pattern exists. To identify which tissue controls the temporal sequence of initiation, pieces were separated by trypsinization into dermis and epidermis. The tissues were then rotated 180° with respect to one another and recombined. On these explants, the first primordia appear in the region expected for dermal control. Finally, to determine which tissue controls spatial arrangements, dermal-epidermal recombinations were made between components from different regions and different ages of skin. In all cases the dermis controls spatial arrangement.

Chondrocyte-derived transglutaminase promotes maturation of preosteoblasts in periosteal bone.

During endochondral development, elongation of the bone collar occurs coordinately with growth of the underlying cartilaginous growth plate. Transglutaminases (TGases) are upregulated in hypertrophic chondrocytes, and correlative evidence suggests a relationship between these enzymes and mineralization. To examine whether TGases are involved in regulating mineralization/osteogenesis during bone development, we devised a coculture system in which one cellular component (characterized as preosteoblastic) is derived from the nonmineralized region of the bone, and the other cellular component is hypertrophic chondrocytes. In these cocultures, mineralization is extensive, with the preosteoblasts producing the mineralized matrix, and the chondrocytes regulating this process. Secreted regulators are involved, as conditioned medium from chondrocytes induces mineralization in preosteoblasts, but not vice versa. One factor is TGase. In the cocultures, inhibition of TGase reduces mineralization, and addition of the enzyme enhances it. Exogenous TGase also induces markers of osteoblastic differentiation (i.e., bone sialoprotein and osteocalcin) in the preosteoblasts, suggesting their differentiation into osteoblasts. Two possible signaling pathways may be affected by TGase and result in increased mineralization (i.e., TGF-beta and protein kinase A pathways). Addition of exogenous TGF-beta2 to the cocultures increases mineralization; though, when mineralization is induced by TGase, there is no detectible elevation of TGF-beta, suggesting that these two factors stimulate osteogenesis by different pathways. However, an interrelationship seems to exist between TGase and PKA-dependent signaling. When mineralization of the cocultures is stimulated through the addition of TGase, a concomitant reduction (50%) in PKA activity occurs. Consistent with this observation, addition of an activator of PKA (cyclic AMP) to the cultures inhibits matrix mineralization, while known inhibitors of PKA (H-89 and a peptide inhibitor) cause an increase in mineralization. Thus, at least one mechanism of TGase stimulation probably involves inhibition of the PKA-mediated signaling.