Separate cis-acting DNA elements of the mouse pro-alpha 1(I) collagen promoter direct expression of reporter genes to different type I collagen-producing cells in transgenic mice.

The genes coding for the two type I collagen chains, which are active selectively in osteoblasts, odontoblasts, fibroblasts, and some mesenchymal cells, constitute good models for studying the mechanisms responsible for the cell-specific activity of genes which are expressed in a small number of discrete cell types. To test whether separate genetic elements could direct the activity of the mouse pro-alpha 1(I) collagen gene to different cell types in which it is expressed, transgenic mice were generated harboring various fragments of the proximal promoter of this gene cloned upstream of the Escherichia coli beta-galactosidase gene. During embryonic development, X-gal staining allows for the precise identification of the different cell types in which the beta-galactosidase gene is active. Transgenic mice harboring 900 bp of the pro-alpha 1(I) proximal promoter expressed the transgene at relatively low levels almost exclusively in skin. In mice containing 2.3 kb of this proximal promoter, the transgene was also expressed at high levels in osteoblasts and odontoblasts, but not in other type I collagen-producing cells. Transgenic mice harboring 3.2 kb of the proximal promoter showed an additional high level expression of the transgene in tendon and fascia fibroblasts. The pattern of expression of the lacZ transgene directed by the 0.9- and 2.3-kb pro-alpha 1(I) proximal promoters was confirmed by using the firefly luciferase gene as a reporter gene. The pattern of expression of this transgene, which can be detected even when it is active at very low levels, paralleled that of the beta-galactosidase gene. These data strongly suggest a modular arrangement of separate cell-specific cis-acting elements that can activate the mouse pro-alpha(I) collagen gene in different type I collagen-producing cells. At least three different types of cell-specific elements would be located in the first 3.2 kb of the promoter: (a) an element that confers low level expression in dermal fibroblasts; (b) a second that mediates high level expression in osteoblasts and odontoblasts; and (c) one responsible for high level expression in tendon and fascia fibroblasts. Our data also imply that other cis-acting cell-specific elements which direct activity of the gene to still other type I collagen-producing cells remain to be identified.

A potent far-upstream enhancer in the mouse pro alpha 2(I) collagen gene regulates expression of reporter genes in transgenic mice.

We have identified three DNase I-hypersensitive sites in chromatin between 15 and 17 kb upstream of the mouse pro alpha 2 (I) collagen gene. These sites were detected in cells that produce type I collagen but not in cells that do not express these genes. A construction containing the sequences from -17 kb to +54 bp of the mouse pro alpha 2 (I) collagen gene, cloned upstream of either the Escherichia coli beta-galactosidase or the firefly luciferase reporter gene, showed strong enhancer activity in transgenic mice when compared with the levels seen previously in animals harboring shorter promoter fragments. Especially high levels of expression of the reporter gene were seen in dermis, fascia, and the fibrous layers of many internal organs. High levels of expression could also be detected in some osteoblastic cells. When various fragments of the 5' flanking sequences were cloned upstream of the 350-bp proximal pro alpha 2(I) collagen promoter linked to the lacZ gene, the cis-acting elements responsible for enhancement were localized in the region between -13.5 and -19.5 kb, the same region that contains the three DNase I-hypersensitive sites. Moreover, the DNA segment from -13.5 to -19.5 kb was also able to drive the cell-specific expression of a 220-bp mouse pro alpha 1(I) collagen promoter, which is silent in transgenic mice. Hence, our data suggest that a far-upstream enhancer element plays a role in regulating high levels of expression of the mouse pro alpha 2(I) collagen gene.

An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes.

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood. We have used the gene for a chondrocyte marker, the proalpha1(II) collagen gene (Col2a1), as a model to delineate a minimal sequence needed for chondrocyte expression and identify chondrocyte-specific proteins binding to this sequence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2a1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondrocytes but not in 10T1/2 fibroblasts. They also directed cartilage specificity in transgenic mouse embryos. These 48 bp include two 11-bp inverted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS cells and chondrocytes but not in fibroblasts. Transgenic mice harboring 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues except skin and brain. In gel retardation assays, an RCS cell-specific protein and another closely related protein expressed only in RCS cells and primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mutations in these 10 bp abolished activity of the multimerized 18-bp enhancer, and deletion of these 10 bp abolished enhancer activity of 465- and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the chondrocyte proteins belong to this family. Together, our results indicate that an 18-bp sequence in Col2a1 intron 1 controls chondrocyte expression and suggest that RCS cells and chondrocytes contain specific POU domain proteins involved in enhancer activity.

The gene for the homeodomain-containing protein Cart-1 is expressed in cells that have a chondrogenic potential during embryonic development.

We have examined the expression of the gene for Cart-1, a new homeodomain-containing protein, during rat embryonic development. In early embryos, Cart-1 RNA was detected at high levels in head mesenchyme, lateral mesoderm, sclerotomes and limb bud mesenchyme. These tissues contain prechondrocytic mesenchymal cells responsible for the formation of the cartilaginous skeleton. In addition, Cart-1 RNA was also found in lung buds, tendons and mesonephros. Cells in these tissues have the potential of undergoing chondrogenesis either in explants for mesonephros and tendons, or in vivo for tendons and the precursors of bronchi cartilages. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver and muscle. Our results support the hypothesis that Cart-1 may play a role in the pathway of chondrogenesis. The gene for Cart-1 was mapped to a segment of mouse chromosome 10 between the genes for phenylalanine hydroxylase and interferon gamma.

Coordinate patterns of expression of type I and III collagens during mouse development.

The extracellular proteins types I and III collagen are abundantly expressed during development. Here, the patterns of the pro alpha 1(I), pro alpha 2(I), and pro alpha 1(III) collagen mRNAs are systematically examined from 7.5 to 17.5 days of development (E7.5 to E17.5) in the mouse using in situ hybridization with specific riboprobes. Coordinated expression of pro alpha 1(I) and pro alpha 2(I) collagen mRNA was found throughout development in all regions examined. Widespread type I collagen expression starting at E8.5 occurred in embryonic mesoderm, sclerotomes, dermatomes, and in the forming connective tissues. After E14.5, regions of ossification showed highest levels of type I collagen expression. Pro alpha 1(III) collagen expression was specific to and coordinated with patterns of type I collagen expression in many fibroblast-containing tissues. No expression of type III collagen occurred in osteoblasts. This comprehensive study of the transcripts of abundantly expressed structural proteins should provide a basis for comparison of other key extracellular matrix molecules and serve as a reference for studies on the patterns of activities of various promoter/enhancer-reporter gene constructions of type I and III collagen genes in transgenic mice.

Parallel expression of Sox9 and Col2a1 in cells undergoing chondrogenesis.

To assess the role of the transcription factor Sox9 in cartilage formation we have compared the expression pattern of Sox9 and Col2a1 at various stages of mouse embryonic development. Expression of Col2a1 colocalized with expression of Sox9 in all chondroprogenitor cells. In the sclerotomal compartment of somites the onset of Sox9 expression preceded that of Col2a1. A perfect correlation was also seen between high levels of Sox9 expression and high levels of Col2a1 expression in chondrocytic cells. However, no Sox9 expression was detected in hypertrophic chondrocytes; only low levels of Col2a1 RNA were found in the upper hypertrophic zone. Coexpression of Sox9 and Col2a1 was also seen in the notochord. At E11.5 Sox9 expression in the brain and spinal neural tube was more widespread than that of Col2a1 although at E14.5 Sox9 and Col2a1 transcripts were colocalized in discrete areas of the brain. Distinct differences between Sox9 and Col2a1 expression were observed in the otic vesicle at E11.5. At E8.5, expression of Sox9 but not of Col2a1 was seen in the dorsal tips of the neural folds and after neural tube closure also in presumptive crest cells emigrating from the dorsal pole of the neural tube. No Col2a1 expression was detected in gonadal ridges in which high levels of Sox9 expression were detected. Together with our previous results showing that the chondrocyte-specific enhancer element of the Col2a1 gene is a direct target for Sox9, these results suggest that Sox9 plays a major role in expression of Col2a1. The correlation between high expression levels of Sox9 and high expression levels of Col2a1 in chondrocytes suggests the hypothesis that high levels of Sox9 are needed for full expression of the chondrocyte phenotype; lower levels of Sox9 such as in neuronal tissues which are also associated with lower expression levels of Col2a1 would be compatible with other cell specifications.

Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.

We identified a rat cDNA that encodes cartilage homeoprotein 1 (Cart-1). The deduced amino acid sequence of Cart-1 contains a paired-type homeodomain. Northern blot hybridization and RNase protection assay revealed that Cart-1 RNA was present at high levels in a well-differentiated rat chondrosarcoma tumor and in a cell line derived from this tumor. Cart-1 RNA was detected in primary mouse and rat chondrocytes but not in various fibroblasts including mouse 10T1/2 cells, NIH 3T3 cells, BALB 3T3 cells, and rat skin fibroblasts. It was also undetectable in mouse C2 myoblasts, S194 myeloma cells, and embryonic stem cells. Cart-1 RNA was present at a very low level in tested but was not detected in other soft tissues of 8-week-old rats. In situ hybridization of rat embryos between 14.5 and 16.5 days post coitum revealed relatively high levels of Cart-1 RNA in condensed prechondrocytic mesenchymal cells and in early chondrocytes of cartilage primordia. The levels of Cart-1 RNA were lower in mature chondrocytes. No hybridization was observed in brain, spinal cord, heart, spleen, gastrointestinal tract, liver, and muscle. We speculate that Cart-1 has a role in chondrocyte differentiation.

A gene for a novel zinc-finger protein expressed in differentiated epithelial cells and transiently in certain mesenchymal cells.

We have identified a novel zinc-finger protein whose mRNA is expressed at high levels in the epidermal layer of the skin and in epithelial cells in the tongue, palate, esophagus, stomach, and colon of newborn mice. Expression in epithelial cells is first detected at the time of their differentiation during embryonic development. In addition, during early embryonic development there is expression in mesenchymal cells of the skeletal primordia and the metanephric kidney which is later down-regulated. The expression pattern suggests that the protein could be involved in terminal differentiation of several epithelial cell types and could also be involved in early differentiation of the skeleton and kidney. The carboxyl terminus of the protein contains three zinc fingers with a high degree of homology to erythroid krüppel-like factor and binds to DNA fragments containing CACCC motifs. The amino-terminal portion of the protein is proline and serine-rich and can function as a transcriptional activator. The chromosomal location of the gene was mapped using mouse interspecific backcrosses and was shown to localize to mouse chromosome 4 and to cosegregate with the thioredoxin gene.

rDlx, a novel distal-less-like homeoprotein is expressed in developing cartilages and discrete neuronal tissues.

From a rat chondrosarcoma we isolated a cDNA that encodes a novel homeoprotein rDlx. The homeodomain of rDlx shows a high degree of sequence identity with those of Drosophila Distal-less, mouse Dlx, and Xenopus Xdll proteins. Northern hybridization of rDlx revealed a 1.4- to 1.6-kb RNA species in a rat chondrosarcoma and a cell line derived from this tumor and in mouse C3H10T1/2 cells, but no rDlx RNA was detected in mouse NIH3T3 fibroblasts, rat skin fibroblasts, mouse C2 myoblasts, mouse myeloma S194 cells, human B-cell lymphoma Daudi cells, or human acute myelocytic leukemia cells. RNase protection assays showed that rDlx transcripts were present at high levels in 14-day-old rat embryos, 18-day-old rat embryo skeletal tissues, and adult rat brain. rDlx RNAs were present at lower levels in newborn rat rib cartilage, 18-day-old rat embryo soft tissues, newborn rat skin, and adult rat heart. rDlx transcripts were not detected in adult rat liver, spleen, lung, kidney, testis, or skeletal muscle. In situ hybridization of rat embryos at different stages revealed that rDlx transcripts were present in otic vesicle, branchial arches, apical ectodermal ridge of limb bud, developing cartilages, perichondria of mature cartilages, mesenchymal cells of developing membranous bones, developing teeth, ganglionic eminence of the telencephalon, diencephalon, olfactory epithelia, and epidermis of the skin. rDlx RNAs were also detected in the developing parasympathetic mesenteric ganglia of the gastrointestinal tract. Hence, rDlx RNAs are mainly expressed in several neuronal tissues and developing skeletal tissues.

Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo.

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-alpha1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity. We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1-3) that were essential for its chondrocyte-specific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bp Col2a1 intron 1 sequence in vivo. SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as larger Col2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocyte-specific complex between the 48-bp element, Sox9, and other protein(s). Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex. Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.

Identification of a minimal sequence of the mouse pro-alpha 1(I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts.

Based on our previous transgenic mice results, which strongly suggested that separate cell-specific cis-acting elements of the mouse pro-alpha 1(I) collagen promoter control the activity of the gene in different type I collagen-producing cells, we attempted to delineate a short segment in this promoter that could direct high-level expression selectively in osteoblasts. By generating transgenic mice harboring various fragments of the promoter, we identified a 117-bp segment (-1656 to -1540) that is a minimal sequence able to confer high-level expression of a lacZ reporter gene selectively in osteoblasts when cloned upstream of the proximal 220-bp pro-alpha 1(I) promoter. This 220-bp promoter by itself was inactive in transgenic mice and unable to direct osteoblast-specific expression. The 117-bp enhancer segment contained two sequences that appeared to have different functions. The A sequence (-1656 to -1628) was required to obtain expression of the lacZ gene in osteoblasts, whereas the C sequence (-1575 to -1540) was essential to obtain consistent and high-level expression of the lacZ gene in osteoblasts. Gel shift assays showed that the A sequence bound a nuclear protein present only in osteoblastic cells. A mutation in the A segment that abolished the binding of this osteoblast-specific protein also abolished lacZ expression in osteoblasts of transgenic mice.

Expression and localization of PG-Lb/epiphycan during mouse development.

We have examined the expression pattern of the PG-Lb/epiphycan gene that encodes a small leucine-rich repeat proteoglycan during mouse embryonic development. PG-Lb/epiphycan mRNA transcripts were first detected at E12.5 days postcoitus (dpc) at high levels in structures that were developing cartilage elements. The gene is expressed in a very specific temporal and spatial fashion in cartilaginous structures. To examine PG-Lb/epiphycan gene expression during cartilage development in more detail, we performed in situ hybridization on hindlimb sections at specific stages of mouse embryonic development. The expression of PG-Lb/epiphycan was compared to that of collagen type II and collagen type X, which are early and late markers for cartilage development, respectively. The expression of PG-Lb/epiphycan occurs later than collagen type II in cartilage development, but its expression appears in the growth plate before and is excluded from the zone of hypertrophic chondrocytic cells expressing collagen type X. An antibody against PG-Lb/epiphycan localized the protein within the entire growth plate of the E17.5 dpc embryonic hindlimb cartilage including the hypertrophic zone where PG-Lb/epiphycan gene expression is turned off. Our results show that PG-Lb/epiphycan gene expression is an intermediate marker for chondrogenesis, and that the protein can be localized to the extracellular matrix surrounding resting, proliferating, and hypertrophic chondrocytes by immunofluorescence histochemistry.

Molecular cloning and characterization of a novel rat activin receptor.

We report the isolation of a full-length rat cDNA for a new activin receptor. The deduced amino acid sequence of this receptor shows 67 percent overall identity with that of a previously identified mouse activin receptor. As predicted for the mouse activin receptor, the amino acid sequence of the rat receptor is consistent with a polypeptide containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and a serine/threonine kinase intracellular domain. In an expression assay, this new receptor was found to bind I125 radiolabeled activin.

A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice.

Type II collagen is a major chondrocyte-specific component of the cartilage extracellular matrix and it represents a typical differentiation marker of mature chondrocytes. In order to delineate cis-acting elements of the mouse pro alpha 1(II) collagen gene that control chondrocyte-specific expression in intact mouse embryos, we generated transgenic mice harboring chimeric constructions in which varying lengths of the promoter and intron 1 sequences were linked to a beta-galactosidase reporter gene. A construction containing a 3,000 bp promoter and a 3,020 bp intron 1 fragment directed high levels of beta-galactosidase expression specifically to chondrocytes. Expression of the transgene coincided with the temporal expression of the endogenous gene at all stages of embryonic development. Successive deletions of intron 1 delineated a 182 bp fragment which targeted beta-galactosidase expression to chondrocytes with the same specificity as the larger intron 1 fragment. Transgenic mice harboring a 309 bp Col2a1 promoter lacking intron 1 tester sequences showed no beta-galactosidase expression in chondrocytes. Reduction of the 182 bp fragment to a 73 bp subfragment surrounding a decamer sequence previously reported to be involved in chondrocyte specificity, resulted in loss of transgene expression in chondrocytes. When the Col2a1 promoter was replaced with a minimal beta-globin promoter, the 182 bp intron 1 sequence was still able to target expression of the transgene to chondrocytes. We conclude that a 182 bp intron 1 DNA segment of the mouse Col2a1 gene contains the necessary information to confer high-level, temporally correct, chondrocyte expression on a reporter gene in intact mouse embryos and that Col2a1 promoter sequences are dispensable for chondrocyte expression.

The organization and expression of the mdm2 gene.

The mdm2 gene encodes a zinc finger protein that negatively regulates p53 function by binding and masking the p53 transcriptional activation domain. Two different promoters control expression of mdm2, one of which is also transactivated by p53. We cloned and characterized the mdm2 gene from a murine 129 library. It contained at least 12 exons and spanned approximately 25 kb of DNA. Sequencing of the mdm2 gene revealed three nucleotide differences that resulted in amino acid substitutions in the previously published mdm2 sequence. Sequencing of normal BalbC/J DNA and the original cosmid clone isolated from the 3T3DM cell line revealed that they are identical, suggesting that the published sequence is in error at these three positions. In addition, we analyzed the expression pattern of mdm2 and found ubiquitous low-level expression throughout embryo development and in adult tissues. Analysis of mRNA from numerous tissues for several mdm2 spliced variants that had been identified in the transformed 3T3DM cell line revealed that these variants could not be detected in the developing embryo or in adult tissues.

Activation of a fibroblast-specific enhancer of the proalpha2(I) collagen gene in tight-skin mice.

OBJECTIVE: Reporter transgenes were introduced into the type 1 tight-skin (Tsk1/+) mouse model of scleroderma to test the hypothesis that fibroblast-specific genetic programs are activated in fibrosis. METHODS: Transgenes harboring upstream fragments of the 5' flanking region of the mouse proalpha2(I) collagen gene (Col1a2), linked to a 400-bp minimal Col1a2 promoter driving an Escherichia coli beta-galactosidase (LacZ) reporter gene, were introduced into Tsk1/+ mice by breeding. Expression of these transgenes, which function as lineage-specific markers of fibroblast differentiation, was compared between the Tsk-LacZ mice and non-Tsk littermates. Responsiveness of these constructs to the profibrotic cytokine, transforming growth factor beta1 (TGFbeta1), was investigated by transient transfection of reporter constructs in tissue-culture cells. RESULTS: There was significant activation of reporter genes harboring the upstream enhancer in Tsk1/+ mice starting from 1 week of age. This was maximal at 6 weeks old (mean +/- SD 237 +/- 24% of non-Tsk controls; P= 0.001). Recombinant TGFbeta1 significantly activated reporter genes regulated by the upstream enhancer in transient transfection, and Tsk-LacZ fibroblasts showed elevated LacZ expression in tissue culture. CONCLUSION: These data suggest that activating signals in Tsk1/+ mice may act via fibroblast-specific regulatory elements within the murine Col1a2 gene. Although TGFbeta has been implicated in the pathogenesis of fibrosis, and reporter genes regulated by the upstream enhancer appear to be TGFbeta responsive in vitro, our results suggest that fibroblast-specific pathways may also be involved.

Expression pattern and gene characterization of asporin. a newly discovered member of the leucine-rich repeat protein family.

We have discovered a new member of the class I small leucine-rich repeat proteoglycan (SLRP) family which is distinct from the other class I SLRPs since it possesses a unique stretch of aspartate residues at its N terminus. For this reason, we called the molecule asporin. The deduced amino acid sequence is about 50% identical (and 70% similar) to decorin and biglycan. However, asporin does not contain a serine/glycine dipeptide sequence required for the assembly of O-linked glycosaminoglycans and is probably not a proteoglycan. The tissue expression of asporin partially overlaps with the expression of decorin and biglycan. During mouse embryonic development, asporin mRNA expression was detected primarily in the skeleton and other specialized connective tissues; very little asporin message was detected in the major parenchymal organs. The mouse asporin gene structure is similar to that of biglycan and decorin with 8 exons. The asporin gene is localized to human chromosome 9q22-9q21.3 where asporin is part of a SLRP gene cluster that includes extracellular matrix protein 2, osteoadherin, and osteoglycin. Further analysis shows that, with the exception of biglycan, all known SLRP genes reside in three gene clusters.