Fibrillar networks are intimately involved in several morphogenetic processes which underlie the harmonious development of the vertebrate embryo. Recent genetic evidence has demonstrated that the minor types V and XI collagen are key regulators of types I and XI fibrillogenesis in non-cartilaginous and cartilaginous matrices, respectively. A comprehensive understanding of the expression and regulation of the genes coding for the chains of the minor collagen types is therefore relevant to animal morphogenesis and development. The present study was undertaken to elucidate the embryonic pattern of expression of the gene coding for the mouse a1 chain of type XI colagen ( C o l l l a l ) using the technique of in situ hybridization. Transcripts of the C o l l l a l gene were detected as early as 11 days of gestation. The oll(X1) transcripts were found to accumulate mostly in cartilaginous tissues, such as the chondrocranium and the developing limbs. Like the major cartilage-specific collagen (type XI), Colllal expression was also noted in the neuro-epithelium of the brain. However, al(X1) transcripts accumulated in several other non-cartilaginous sites. They include odontoblasts, trabecular bones, atrioventricular valve of the heart, the tongue, the intestine, and the otic vesicle. Altogether, the data confirm that C o l l l a l has a broader spectrum of expression than previously thought. This finding raises the possibility that the (ul(X1) chain may participate in the formation of stage-and tissue-specific trimers with distinct functional properties.

In order to eventually elucidate the mechanisms regulating alpha 1(XI) collagen expression in cartilaginous and non-cartilaginous tissues, we performed an initial analysis of the structural-functional features of the promoter of the human gene (COL11A1). After cloning and sequencing the 5' portion of COL11A1, primer extension and nuclease protection assays identified several minor transcriptional start sites clustered around a major one located 318 base pairs from the ATG codon. Consistent with this finding, analysis of the upstream sequence revealed the absence of a TATA motif and the presence of several GC boxes. Transient transfection experiments delineated the smallest promoter sequence directing relatively high expression of a reporter gene in a cell type-specific manner. Nine nuclear protein-bound areas were located within this promoter sequence of the COL11A1 gene. Sequence homologies suggested that the majority of the footprints correspond to potential binding sites for ubiquitous nuclear proteins, such as AP2 and Sp1. Additional experimental evidence indicated that one of the protected areas may bind a transcriptional complex that is identical or closely related to the one that regulates tissue specificity in the coordinately expressed alpha 2(V) collagen gene.

Type XIX collagen is a poorly characterized member of the fibril-associated collagens with an interrupted triple helices (FACIT) class of collagen molecules. As a first step toward elucidating its function, we have isolated full size cDNA clones from the mouse ␣1(XIX) collagen gene (Col19a1) and established its pattern of expression in the developing embryo and adult organism. Col19a1 transcripts can be detected as early as 11 days of gestation and in all embryonic tissues, except the liver, of an 18-day postcoitum mouse. In contrast, only a few adult tissues, brain, eye, and testis, seem to accumulate Col19a1 mRNA. Col19a1 transcripts are at least 10 times more abundant in adult than fetal brain and significantly less in adult than fetal muscle and skin. Consistent with the RNA data, polyclonal antibodies for ␣1(XIX) collagen reacted with a 150-kDa protein in the neutral salt extraction of adult mouse brain tissues. We therefore propose that type XIX collagen plays a distinct role from the other FACIT molecules, particularly in the assembly of embryonic matrices and in the maintenance of specific adult tissues.

We have isolated cDNAs and completed for the first time the primary structure for a novel collagenous chain that was partially characterized earlier and named alpha 1(Y) chain [Yoshioka, H. et al. (1992) Genomics 13, 884-886]. The size of the coding region was unexpectedly small compared with the length of the mRNA (> 10 kb), owing to the presence of a long 3' untranslated region (> 5 kb). The predicted polypeptide contained 1,142 amino acid residues with a 23-residue signal peptide consisting of 5 collagenous domains of 70-224 residues in length, interspersed and flanked with 6 noncollagenous (NC) domains. The primary structure is distinct from those of the 32 known collagen alpha-chains of types I through XVIII. Therefore, we designate this newly discovered collagen chain the alpha 1 chain of type XIX collagen. Sequence analysis suggested that this chain belongs to the recently discovered group of collagens known as FACITs (fibril associated collagens with interrupted triple-helices). Northern blotting analysis demonstrated hybridization of the cDNA to a large mRNA species (> 10 kb) extracted from a rhabdomyosarcoma cell line (CCL 136). We also isolated numerous truncated cDNA clones of which the 3' parts were different from the "proto" type of the mRNA of > 10-kb size. Sequence comparison between cDNAs and corresponding genomic DNA fragments indicated that unusual splicing events occurred through insufficient recognition at acceptor sites. Expression of the gene was extremely infrequent in the rhabdomyosarcoma cell line; it could be restricted to certain animal tissues both temporally and spatially during early development.

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