Cartilage proteoglycan core protein gene expression during limb cartilage differentiation.

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.

Collagen gene expression during limb cartilage differentiation.

As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen mRNA occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II collagen mRNA occurs which parallels the progressive accumulation of cartilage matrix by cells. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen mRNA are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen.

Prostaglandin synthesis during the course of limb cartilage differentiation in vitro.

In the present study we have used radiometric thin layer chromatography (TLC) and radioimmunoassay (RIA) to examine the synthesis of various prostaglandins (PGs) during the progressive chondrogenic differentiation limb mesenchymal cells undergo in micromass culture. Throughout the 3-day culture period, [3H]arachidonic acid (AA) is metabolized to compounds which comigrate with authentic PGE2, PGF2 alpha, 6-keto-PGF1 alpha, TxB2, and PGD2. In micromass cultures prepared from the cells of whole stage-23/24 wing buds, all 3H-AA metabolites are produced in relatively small amounts during the initial period of culture, i.e. prior to the formation of extensive prechondrogenic cellular aggregates. Concomitant with maximum aggregate formation and the initiation of cartilage differentiation, there is a striking and progressive increase in the production of all the major classes of PGs from 3H-AA. PG production from 3H-AA is also at a maximum during the onset of chondrogenesis in micromass cultures prepared from the distal subridge mesenchymal cells of stage-25 wing buds in which more rapid, extensive, and homogeneous cartilage differentiation occurs. To complement these TLC studies, RIA has been used to examine the amount of various PGs synthesized from endogenous substrates by micromass culture homogenates at various times during in vitro chondrogenesis. These RIA studies also indicate that PG production is highest during periods of culture which coincide with the onset of overt chondrogenesis in both stage-23/24 whole limb and stage-25 subridge mesoderm micromass cultures. RIA indicates that PGE2 is the predominant PG produced from endogenous substrates during 1h incubations at the onset of chondrogenesis, while radiometric TLC indicates compounds which comigrate with PGF2 alpha are the major class of 3H-AA metabolites which accumulate during that time. This qualitative difference very likely reflects metabolism of parent PG compounds during the long (12h) labelling and postlabelling incubations utilized in the TLC analyses. The temporal correlation between PG production and the initiation of chondrogenesis in vitro is consistent with previous studies implicating PGs in the regulation of limb cartilage differentiation.

The effect of prostaglandins on the cyclic AMP content of limb mesenchymal cells.

We have been investigating the hypothesis that prostaglandins including prostaglandin E2 (PGE2) produced during the critical condensation phase of limb chondrogenesis are involved in the regulation of cartilage differentiation by acting as local modulators of cyclic AMP (cAMP) accumulation. The purpose of the present study was to determine directly whether PGE2 and other prostanoids which had previously been shown to stimulate in vitro chondrogenic differentiation do indeed elevate the cAMP content of limb mesenchymal cells, and to determine whether the ability of various prostanoids to increase cAMP production by these cells directly reflects the potencies of these same molecules in stimulating chondrogenesis. We have found that PGE2 does indeed elicit a striking elevation in the cAMP content of subridge mesenchymal cells, indicating that the cells possess adenylate cyclase-coupled receptors for this molecule. The effect of PGE2 on cAMP accumulation is potentiated by a phosphodiesterase inhibitor, thus paralleling the potentiating effect phosphodiesterase inhibitors have on PGE2-stimulated in vitro chondrogenesis. The effect of PGE2 on cAMP content is dose-dependent with a 3-fold increase seen at 10(-8)M, which is the lowest concentration at which PGE2 effectively stimulates chondrogenesis. PGE1, which is just as effective as PGE2 in stimulating chondrogenesis, is just as effective as PGE2 in stimulating cAMP accumulation. PGA1, which is a much less effective stimulator of chondrogenesis than PGE2 or PGE1, is less than half as potent as these molecules in elevating cAMP levels. PGF1 alpha, 6-keto PGF1 alpha, and thromboxane B2, which have little or no effect on chondrogenesis, have little or no effect on cAMP content.(ABSTRACT TRUNCATED AT 250 WORDS)

Pattern regulation along the proximodistal axis of the developing chick limb.

Transplants creating presumptively excess or deficient proximodistal segments were performed to investigate pattern regulation in stage 21 and stage 23 chick limb buds. Excess skeletal elements were not removed by regulation. Deficiencies appeared to be regulated by host tissue. The ability of the host tissue to participate in regulation declined in more proximal tissue and with increased age. These results suggest that there are two requirements for pattern regulation. First, the tissue in question must be sufficiently close to the ectodermal ridge before it can respond. Results of previous work and implications for pattern formation are discussed with these requirements in mind.