Type IIB Procollagen Research Articles

Influence of digits, ectoderm, and retinoic acid on chondrogenesis by mouse interdigital mesoderm in culture

We have cultured tissues isolated from the interdigital zones (IDZ) of the mouse footplate in the presence of the digits, ectoderm, and all-trans retinoic acid. The objective was to understand how these various factors influence the developmental fate of the interdigital tissues. Neutral red staining showed that these tissues normally differentiate by dying between day 12.5-14.5. However, if they were isolated from the footplate between day 12.5-13.5 (when cell death is not overtly obvious in the IDZ) and maintained in organ culture, these tissues would develop into cartilage and soft connective tissues. In culture, chondrogenesis is initiated very rapidly in the interdigital explants as revealed by in situ hybridization with riboprobes specific for type IIA and IIB procollagen mRNAs. The ability of interdigital tissues to form cartilage is not attributed to factors present in the serum of the culture medium as this phenomenon is also observed in serumless cultures. We have found that if all-trans retinoic acid, at concentrations of 10-50 ng/ml culture medium, were added to the explants it could inhibit chondrogenesis and promote cell death. Moreover, in some of the cultures, a single digit was left attached to the interdigital tissue. This also dramatically reduced the incidence of chondrogenesis. We have tried to determine whether the digits and ectoderm can produce a diffusible factor that can prevent cartilage from developing by culturing day 12.5 interdigital tissues in ectoderm and digit conditioned media. The ectoderm conditioned medium had no effects on interdigital growth or chondrogenesis. In contrast, the size of interdigital explants cultured in the presence of digit conditioned medium was shown to be significantly smaller than the control. These explants also produced a smaller quantity of cartilage as revealed by Alcian blue binding assay. In sum, our results showed that the fate of the interdigital tissues are not fully determined until after day 13.5. These tissues have the potentials to form cartilage and soft connective tissues. We tentatively propose that these interdigital tissues do not normally realize their histogenetic potentials because of the antichondrogenic influence of the digits and retinoic acid.

In situ expression of collagen and proteoglycan genes in notochord and during skeletal development and growth.

Cartilage is an important tissue in skeletogenesis, in the growth of long bones, and as a flexible component of the mature skeleton. The extracellular matrix proteins type II collagen and aggrecan comprise 90% of the matrix and are characteristic of cartilage. Type II collagen provides structural integrity to the tissue, while aggrecan confers resiliency. The quantity of type II procollagen is controlled at the level of transcription of mRNA from the COL2A1 gene. In addition, type II procollagen can be expressed in two isoforms by differential splicing of the primary gene transcript, a post-transcriptional control mechanism. The two mRNAs either include exon 2 (type IIA) or exclude exon 2 (type IIB) which encodes the major portion of the amino (NH2)-propeptide [Ryan and Sandell (1990), J. Biol. Chem., 265:10334-10339]. The aggrecan gene also encodes alternative splice forms that may be developmentally expressed. The regulation of aggrecan splicing or transcription has not been studied in detail. To determine the spatial and temporal patterns of expression of extracellular matrix in the development of cartilage, we have examined the expression of type II collagen and aggrecan during chondrogenesis in the vertebral column and during elongation of a newborn growth plate. Our results indicate that there is a developmental sequence of type II collagen splice form expression during chondrogenesis with type IIA expressed in prechondrocytes and type IIB expressed in chondrocytes. During elongation of the growth plate, mature chondrocytes express type IIB procollagen and then differentiate into hypertrophic chondrocytes and initiate expression of type X collagen. In all cases, aggrecan was coordinately expressed with type IIB procollagen. As cartilage-like proteins have been observed in more primitive structures such as notochord, the expression of type II collagen mRNAs was also examined in the notochordal remnants of the vertebral column. In the notochord, the predominant collagen expressed was the type IIA collagen prechondrocyte isoform. Notochordal cells also expressed mRNAs more characteristic of fibroblasts such as versican and decorin: low expression of type I collagen, type IIB collagen, and aggrecan were observed.

Alternative splice form of type II procollagen mRNA (IIA) is predominant in skeletal precursors and non-cartilaginous tissues during early mouse development.

Type II collagen, generally considered to be characteristic of cartilage, has been localized in specific non-cartilaginous structures during embryogenesis and development of the skeleton. Type II procollagen is synthesized in two different forms generated by alternative splicing of exon 2 in the precursor mRNA transcript. One form (type IIA procollagen) contains a large cysteine-rich domain in the NH2-terminal propeptide, while the second form (type IIB procollagen) does not. These two forms are spatially expressed during development and chondrogenesis with the type IIB procollagen mRNA primarily expressed by chondrocytes while the IIA form is expressed in chondroprogenitor cells (Sandell et al. [1991] J. Cell Biol. 114:1307-1319). The present study demonstrates that the early non-cartilage expression, by somites, mesenchymal and epithelial cells, is predominantly the alternate splice form, type IIA procollagen mRNA. Later in development, the type IIB mRNA splice form is expressed by chondrocytes. During the development of intramembranous bones, such as the mandible, type IIA procollagen mRNA is also expressed. In this tissue, the splice form does not switch to type IIB mRNA and no cartilage is formed. These results show that expression of type IIA mRNA, whether by epithelial or mesenchymal cells, precedes formation of overt skeletal structures.

Alternatively spliced type II procollagen mRNAs define distinct populations of cells during vertebral development: differential expression of the amino-propeptide.

Type II collagen is a major component of cartilage providing structural integrity to the tissue. Type II procollagen can be expressed in two forms by differential splicing of the primary gene transcript. The two mRNAs either include (type IIA) or exclude (type IIB) an exon (exon 2) encoding the major portion of the amino (NH2)-propeptide (Ryan, M. C., and L. J. Sandell. 1990. J. Biol. Chem. 265:10334-10339). The expression of the two procollagens was examined in order to establish a potential functional significance for the two type II procollagen mRNAs. First, to establish whether the two mRNAs are functional, we showed that both mRNAs can be translated and the proteins secreted into the extracellular environment. Both proteins were identified as type II procollagens. Secondly, to test the hypothesis that differential expression of type II procollagens may be a marker for a distinct population of cells, specific procollagen mRNAs were localized in tissue by in situ hybridization to oligonucleotides spanning the exon junctions. Embryonic vertebral column was chosen as a source of tissue undergoing rapid chondrogenesis, allowing the examination of a variety of cell types related to cartilage. In this issue, each procollagen mRNA had a distinct tissue distribution during chondrogenesis with type IIB expressed in chondrocytes and type IIA expressed in cells surrounding cartilage in prechondrocytes. The morphology of the cells expressing the two collagen types was distinct: the cells expressing type IIA are narrow, elongated, and "fibroblastic" in appearance while the cells expressing type IIB are large and round. The expression of type IIB appears to be correlated with abundant synthesis and accumulation of cartilagenous extracellular matrix. The expression of type IIB is spatially correlated with the high level expression of the cartilage proteoglycan, aggrecan, establishing type IIB procollagen and aggrecan as markers for the chondrocyte phenotype. Transcripts of type II collagen, primarily type IIA, are also expressed in embryonic spinal ganglion. While small amounts of type II collagen have been previously detected in noncartilagenous tissues, the detection of this new form of the collagen in relatively high abundance in embryonic nerve tissue is unique. Taken together, these findings imply a potential functional difference between type IIA and type IIB procollagens and indicate that the removal of exon 2 from the pre-mRNA, and consequently the NH2-propeptide from the collagen molecule, may be an important step in chondrogenesis. In addition, type II procollagen, specifically type IIA, may function in noncartilage tissues, particularly during development.