Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice.

To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence elements in a modular arrangement, a mechanism which confers high flexibility in the temporal and spatial pattern of expression during development.

Spatial and temporal changes of type VI collagen expression during mouse development.

The expression of type VI collagen has been studied in mouse tissues. By Northern blotting, the mRNA for the alpha 1 (VI) chain was detectable in whole embryos at 10.5 days postcoitum and steeply increased afterward. The messenger levels were high at birth, but decreased rapidly in the following days, reaching low levels in adult animals. In 2-month-old mice, lung, skin, adrenal gland, heart, skeletal muscle and tail and fat were among the most active producers of alpha 1 (VI) mRNA. In situ hybridization first identified mRNA for alpha 1 (VI) collagen in mesenchymal cells of 10.5-day embryos in various locations, including serosae, branchial arches, large blood vessels and the cephalic mesenchyme. Staining increased at later stages of development and most connective tissues were positive at 16.5 days and later. Strongly staining tissues were joints, intervertebral disks, perichondrium, periostium, dermis, skeletal muscle and heart valves, whereas cartilage and bone were very poorly labelled. Epithelia and the central nervous system were completely negative. In several organs, notably lung, salivary glands and the digestive tract, staining was concentrated underneath epithelia. This staining pattern was different from that for collagen type I, which was evenly distributed in the subepithelial mesenchyme. The pattern of distribution of the protein, revealed by immunocytochemistry, was coincident with that of the alpha 1 (VI) mRNA. In addition, the results confirmed that type VI collagen is preferentially deposited in the pericellular environment. This was particularly evident in skeletal muscle. The data show that type VI collagen is mainly produced by mesenchymal cells and suggest a role for the protein in delineating the boundary of distinct domains in connective tissue.

Murine alpha 1(VI) collagen chain. Complete amino acid sequence and identification of the gene promoter region.

The entire primary structure of the murine alpha 1(VI) collagen chain was deduced from cloned cDNA. The predicted polypeptide consists of 1025 amino acids and shows extensive homology with the corresponding human and chicken chains. A genomic clone isolated with a cDNA probe was found to contain about 13 kilobases of the 5'-flanking region and the first and second exon, coding for the 5'-untranslated sequence and signal peptide and part of the N-terminal portion of the mature protein, respectively. Polymerase chain reaction and primer extension analyses revealed two major and several minor transcription start sites distributed over 76 base pairs (bp). The region just upstream of the transcription initiation sites lacks canonical TATA and CAAT boxes and Sp1 binding sites, but contains putative binding sites for other transcription factors and a 90-bp polypyrimidine tract with elements of dyad symmetry. Chimeric constructs were derived from different fragments of the 5'-flanking genomic region and the chloramphenicol acetyltransferase (CAT) gene and expression of the reporter gene was assayed following transfection of various cell types. A construct containing sequences extending from -215 to +41 directed high levels of CAT expression. The data indicate that this region harbours a functional promoter.