©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Primary Structure and Developmental Expression of Fbn-1, the Mouse Fibrillin Gene (*)

(Received for publication, July 25, 1994; and in revised form, October 3, 1994)

Wushan Yin (1) Elizabeth Smiley (1) John Germiller (2) Chiara Sanguineti (1) Theresa Lawton (1) Lygia Pereira (§) Francesco Ramirez (§) Jeffrey Bonadio (1)(¶)

From the  (1)Department of Pathology and (2)Orthopaedic Research Laboratories, Section of Orthopaedic Surgery, University of Michigan, Ann Arbor, Michigan 48109-0650 and the Brookdale Center for Molecular Biology, Mt. Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous studies have reported >10 kilobases of human fibrillin-1 cDNA sequence, but a consensus regarding the 5` end of the transcript remains to be worked out. One approach to developing a clear consensus would be to search for regions of evolutionary conservation in transcripts from a related species such as mouse. As reported here, the mouse fibrillin-1 transcript encodes a highly conserved polypeptide of 2,871 amino acids. The upstream sequence that flanks the ATG is considerably less well conserved, however. Indeed, the ATG codon (which occurs in the context of a Kozak consensus sequence and is located just upstream of a consensus signal peptide) signals the point where human and mouse fibrillin-1 sequences cease to be nearly identical. Together, these results are consistent with previous efforts by Pereira et al. (Pereira, L., D'Alessio, M., Ramirez, F., Lynch, J. R., Sykes, B., Pangilinan, T., and Bonadio, J.(1993) Human Mol. Genet. 2, 961-968) to identify the human fibrillin-1 translational start site. Sequences immediately upstream of the ATG are GC-rich and devoid of TATA and CCAAT boxes, which suggests that the mouse fibrillin-1 gene will be broadly expressed. A survey of expression in mouse embryo tissues is consistent with this hypothesis and suggests two novel functions for fibrillin-associated microfibrils in non-elastic connective tissues.


INTRODUCTION

Ten-nm microfibrils are found in a wide variety of extracellular matrices. In elastic tissues such as aorta and fetal bovine nuchal ligament, microfibrils have been shown to facilitate elastic fiber formation (Mecham and Davis, 1994). Microfibrils are also found in non-elastic tissues where they may anchor epithelial cells to the interstitial matrix and may also participate in wound healing (Vracko and Thorning, 1990). Microfibril-associated glycoprotein (Gibson et al., 1991; Chen et al., 1993) and fibrillin (Sakai et al., 1986) are considered to be integral microfibril constituents. Based on biochemical and immunochemical criteria, several other candidate proteins have been identified including: 32- and 250-kDa proteins isolated from bovine zonular fibrils (Streeten and Gibson, 1988), a 35-kDa protein isolated from bovine ligamentum nuchae (Serafini-Fracassini et al., 1981), proteins of 78, 70, and 25 kDa (Gibson et al., 1989), and a 58-kDa protein that is post-translationally modified into a 32-kDa fragment called the associated microfibril protein (Horrigan et al., 1992). Extracellular proteins that may be associated with microfibrils include emilin (Bressan et al., 1993), GP128/thrombospondin (Arabeille et al., 1991), 36-kDa microfibril-associated protein (Kobayashi et al., 1989), proteoglycan (Cleary and Gibson, 1983), fibronectin (Goldfischer et al., 1985), amyloid P component (Inoue and Leblond, 1986), vitronectin (Dahlback et al., 1989), and, perhaps, lysyl oxidase (Kagan et al., 1986; Baccarini-Contri et al., 1989).

Fibrillin is now recognized as a gene family with two well-characterized members. The human fibrillin-1 gene (FBN-1) (^1)has been mapped to chromosome band 15q21, while the human fibrillin-2 gene has been localized to chromosome band 5q23 (Lee et al., 1991). The corresponding mouse fibrillin genes have been assigned to mouse chromosome 2, band F (Fbn-1) and mouse chromosome 18, band D-E1 (Fbn-2) (Li et al., 1993). The primary structure of both molecules is similar, and the fibrillin-1 and fibrillin-2 polypeptides have been immunolocalized to 10-nm microfibrils in developing elastic tissues (Zhang et al., 1994). A unique fibrillin-like protein has recently been identified (Mecham and Davis, 1994), and it has been suggested that another fibrillin gene may exist on human chromosome 17 (Lee et al., 1991). The fact that naturally occurring mutations in FBN-1 cause Marfan syndrome and dominantly inherited ectopia lentis emphasizes the importance of this gene family (Dietz et al., 1991). Moreover, FBN-2 has been genetically linked to congenital contractural arachnodactyly, a rare disorder that shares some of the skeletal manifestations of Marfan syndrome (Lee et al., 1991).

Previous studies have reported >10 kb of human fibrillin-1 cDNA sequence, but a consensus regarding the structure of the 5` end of the transcript remains to be worked out. Pereira et al.(1993) used primer extension mapping and S1 nuclease analysis to show that the 5`-untranslated region of the FBN-1 transcript produced by cultured osteosarcoma cells (MG-63) was 134 nt in length. A limited analysis also suggested that sequences upstream of the cap site were GC-rich and lacked TATA and CCAAT boxes. This relatively simple structure predicted that the human fibrillin-1 promoter would have a broad temporal and spatial expression pattern. On the other hand, Corson et al.(1993) found evidence of a longer and more complicated 5`-untranslated region. DNA sequencing plus Northern analysis identified a 1.8-kb CpG island that contained three partial exons (designated B, A, and C). The CpG island also included a fourth exon (designated M) containing the initiator Met residue. In vitro and in vivo studies revealed a strong bias favoring the expression of transcripts that contained exon A. By analogy with other extracellular matrix genes, therefore, the CpG island may contain the transcription initiation site, while exons B, A, and C may be alternatively spliced first exons under the control of separate promoters. The formal possibility also exists that the CpG island does not contain the transcription start site and that there may be upstream exons (i.e. yet to be discovered) that may or may not be associated with CpG islands.

The study by Corson et al.(1993) has raised important questions about the structure of the FBN-1 transcript that bear on normal connective tissue function as well as the pathogenesis of Marfan syndrome and related disorders. One approach to answering these questions is to search for regions of evolutionary conservation, and thus functional significance, in the fibrillin-1 transcript of a related species such as the mouse. As reported here, the mouse embryo and human fibrillin-1 transcript share a remarkable >95% DNA and amino acid sequence identity, extending 8,613 nt downstream of the initiator Met codon identified by Pereira et al.(1993). More than 2.0 kb of 5`-flanking sequence upstream of the mouse initiator Met codon has also been obtained and shown to be considerably less well conserved, which argues against the possibility that upstream exons B, A and C code for additional fibrillin-1 amino acid sequence. The available human and mouse data therefore suggests that the initiator Met codon identified by Pereira et al.(1993) is the one and only fibrillin-1 translation start site. As predicted, the mouse fibrillin-1 gene is broadly expressed in developing mouse tissues. A survey of the expression pattern of the fibrillin-1 transcript suggests a novel role for fibrillin-associated microfibrils during cardiac morphogenesis and during connective tissue remodeling of uterine stroma after embryo implantation.


EXPERIMENTAL PROCEDURES

cDNA Cloning

Aliquots (typically 40,000-50,000 plaque-forming units) of phage particles from a cDNA library in the ZAP II vector made from 3T3 cell mRNA (Stratagene) and fresh overnight XL1-blue cells (grown in Luria broth supplemented with 0.4% maltose in 10 mM MgSO(4)) were mixed, incubated for 15 min at 37 °C, mixed again with 9 ml of liquid (50 °C) top layer agarose (NZY broth plus 0.75% agarose), and then spread evenly onto a freshly poured 150-mm NZY-agar plate. Standard methods were used for the preparation of plaque lifts and filter hybridization (42 °C, in buffer containing 50% formamide, 5 times SSPE, 1 times Denhardt's, 0.1% SDS, 100 mg/ml salmon sperm DNA, 100 mg/ml heparin). Human FBN-1 cDNA probes (Pereira et al., 1993) were radiolabeled by the nick translation method (Chen et al., 1993). Purified phage clones were then converted to pBluescript plasmid clones, which were sequenced using Sequenase (version 2.0).

Genomic Cloning

A genomic library in the Lambda DASH vector (Stratagene) was plated at 60,000 plaque-forming units/plate, and nitrocellulose replicas were screened at high stringency as described above. Filters were washed in 0.2 times SSPE at 42 °C and autoradiographed. Duplicate positive plaques were rescreened until purified, and -DNA was prepared as described in the Magic Lambda Prep kit protocol (Promega). Selected regions of the subcloned inserts were sequenced using Sequenase (version 2.0). Sequence alignment and identity was determined using sequence analysis programs from the Genetics Computer Group.

Tissue in Situ Hybridization

To prepare sense and antisense Fbn-1 probes, a 335-base pair fragment from the mouse 3`-untranslated region (+8771 to +9105, counting the first A of the initiator Met codon as the +1 nt) was subcloned into the pBluescript KS+ plasmid (Stratagene, Inc.). Template DNA was linearized with either EcoRI or BamHI, extracted, and precipitated with ethanol. Sense and antisense transcripts were generated from 1 µg of template with T3 and T7 polymerases in the presence of [S]UTP at >6 mCi/ml (Amersham Corp., >1,200 Ci/mmol) and 1.6 units/ml RNasin (Promega), with the remaining in vitro transcription reagents provided in a kit (SureSite, Novagen Inc.). After transcription at 37 °C for 1 h, DNA templates were removed by a 15-min digest at 37 °C with 0.5 unit/ml RNase-free DNase I, extracted, and precipitated with ethanol. Riboprobes were hydrolyzed to an average final length of 150 base pairs by incubating in 40 mM NaHCO(3), 60 mM Na(2)CO(3), 80 mM dithiothreitol for 40 min at 60 °C. Hydrolysis was terminated by addition of sodium acetate, pH 6.0, and glacial acetic acid to 0.09 M and 0.005% (v/v), respectively, and the probes were then ethanol precipitated, dissolved in 0.1 M dithiothreitol, counted, and stored at -20 °C until use. Day 8.5-9.0, day 13.5, and day 16.5 mouse embryo tissue sections and the in situ hybridization protocol were exactly as described (Chen et al., 1993).


RESULTS

Molecular Cloning of the Mouse Fibrillin-1 cDNA

Seven overlapping phage clones were isolated from a 3T3 cell cDNA library to obtain most of the mouse Fbn-1 coding sequence (Fig. 1). The polymerase chain reaction (PCR) was used to generate clone PCR-WS, which resolved a small gap in the sequence between clones 4 and 3.5 m. A potential initiator Met codon was identified in clone pTP#1. The murine codon occurred in the context of a Kozak consensus sequence GGCACCATGC (Kozak, 1991). As expected, an open reading frame of 8,613 nt was identified downstream of the initiator codon, followed by a 3`-untranslated region of 958 nt^2. Greater than 95% identity was found to exist between mouse and human fibrillin-1 at the nucleotide and deduced amino acid levels. (^2)


Figure 1: Mouse Fbn-1 cDNA clones. A partial representation of restriction sites is shown. N, NcoI; Ss, SstII; Ha, HaeII; S, SacI; E, EcoRI; B, BamHI; H, HindIII; Hp, HpaI; and M, MstII.



The conceptual mouse prefibrillin-1 molecule is a polypeptide of 2,871 amino acids, with a calculated pI of 4.60 and a molecular mass of 312 kDa (Fig. 2). The predicted domain structure of mouse and human fibrillin-1 is identical (see Pereira et al., 1993 for additional details). Thus, the mouse molecule consists of a signal peptide and five structurally distinct regions (A-E). The signal peptide is located at the extreme NH(2) terminus and consists of an estimated 17 amino acids. Region A, which is contiguous with the signal peptide, consists of 42 amino acids and has a net basic charge (estimated pI, 9.9). Region B consists of 324 amino acids that are organized into a total of 8 cysteine-rich repeats. Region C is a 57 amino acid, proline-rich domain (25/57 residues, or 43.9% proline). Region D, the largest domain, consists of 2,247 amino acids which are organized into 49 cysteine-rich repeats. Region E consists of 184 amino acids and represents the carboxyl terminus of the molecule. The position and type of cysteine-rich repeats, all potential sites of N-linked glycosylation, and a single RGD sequence were all conserved in the mouse coding sequence.


Figure 2: Sequence comparison of mouse and human fibrillin-1 amino acid sequences. A, presents a schematic diagram of the mouse fibrillin-1 polypeptide. Regions A-E are depicted below. Cysteine-rich repeats are numbered in groups of five at the top. Symbols are as follows: open rectangles, EGF-CB repeats; open ovals, TGF-bp repeats; open circle, Fib motif; patterned rectangles, cysteine-rich repeats of varying pattern (see Pereira et al., 1993); patterned oval, TGF-bp-like repeat; patterned circle, Fib-like module. The amino-terminal signal peptide has been deleted from the schematic for the sake of simplicity. B presents the amino acid sequence of mouse fibrillin-1. Amino acid numbers are shown at the right. Human amino acids that differ from mouse are shown below individual lines of mouse sequence (in bold).



As noted, cysteine-rich repeats were found in regions B and D of the mouse and human fibrillin-1 sequence. Thirty of these showed the calcium binding consensus sequence: D/N-I/V-D/N-E/D-C(1). This consensus was derived from an analysis of 154 EGF-CB repeats in 23 different proteins and from structural analyses of the EGF-CB repeat, both bound and unbound to calcium ion (Selander-Sunnerhagen et al., 1992). Variations on the consensus have been noted previously, and some of these are present in mouse fibrillin (repeat numbers correspond to the numbering system used for human fibrillin-1, see Pereira et al., 1993): D-L-N/D-E-C(1) (repeat 29, 30, 52, and 53), D-I-D-Q-C(1) (repeat 26), D-N-D/N-E-C(1) (repeat 54), and D-T-D/N-E-C(1) (repeat 46). The following potential calcium binding sequences, which have not previously been reported, were also found: D-E-N/D-E-C(1) (repeat 48, 49, and 56), D-M-N/D-E-C(1) (repeat 12 and 45), and D-R-D/N-E-C(1) (repeat 39). All EGF-CB repeats contained a second consensus sequence, C(3)-X-D/N-X-X-X-X-Y/F-X-C(4), which is a recognition sequence for an Asp/Asn hydroxylase that co- and post-translationally modifies D/N residues (Stenflo et al., 1987; Gronke et al., 1989). Hydroxyaspartic acid and hydroxyasparagine residues have been found in direct analyses of the EGF-CB repeats of coagulation factors, the anticoagulant, protein C, and the latent TGF-beta-binding protein (Ohlin et al., 1988; Persson et al., 1989; Handford et al., 1990; Colosetti et al., 1993).

5`-Region Flanking the Mouse Fibrillin-1 Gene: Isolation of Genomic Clones

To assess the degree of sequence conservation between the mouse and human 5` upstream fibrillin-1 sequences, independent mouse genomic clones were isolated, and >2.0 kb of upstream sequence was obtained. One clone, F-2.18, was isolated from a Lambda DASH library (Stratagene) using a human fibrillin-1 cDNA probe (Li et al., 1993). As demonstrated by oligonucleotide hybridization and selected DNA sequencing, the F-2.18 clone has an 18-kb FBN-1 genomic insert that includes the initiator Met codon (see Pereira et al., 1993). A second clone (WY-129 m) was isolated using cDNA clone pTP#1 as the probe (see Fig. 1). Southern hybridization and DNA sequence analysis demonstrated that the WY-129 m clone has a >10-kb insert that also includes exon 1 of the Fbn-1 gene. It should be emphasized that these genomic clones were independently isolated in terms of time, place, operator, library, and probe.

5`-Region Flanking the Mouse Fibrillin-1 Gene: Sequence Analysis

The Fbn-1 DNA sequence upstream of the initiator Met codon was obtained by the independent efforts of four individuals (W. Y., E. S., C. S., and T. L.). All discrepancies were resolved by direct comparison of sequencing gels and by selected resequencing of many regions. Identical sequence was obtained from the F-2.18 and WY-129 m clones without exception.

Initial sequence comparisons using GAP and BESTFIT algorithms quickly demonstrated that the 5` mouse and human fibrillin-1 upstream sequences shared few regions of identity. Partially characterized human exon B, A, and C sequences, as defined by Corson et al.(1993), were therefore aligned with the entire mouse 5` upstream sequence using both programs. The BESTFIT algorithm generally gave higher sequence identity values, and these values have been reported here. To maximize alignment quality, putative splice donor sequences were included at the 3` end of the exon B, A, and C sequence files, whereas putative splice acceptor sequences were included at the 5` end of the exon M sequence file.

As shown in Fig. 3, mouse and human exon B showed 74% sequence identity with 1 gap (gap weight, 5.0; length weight, 0.05). Three potential open reading frames were found in the mouse sequence, but only one potentially conserved fragment of four amino acids was identified. The splice donor at the 3` end of mouse exon B did not appear to be conserved. Mouse and human exon A showed 94% sequence identity with 7 gaps (gap weight, 2.0; length weight, 0.05). One potential open reading frame was found in the mouse sequence, but fragments of conserved amino acid sequence were identified in all three reading frames. The splice donor at the 3` end of mouse exon A appeared to be conserved. Mouse and human exon C showed 85% sequence identity with 12 gaps (gap weight, 2.0; length weight, 0.05). One potential open reading frame was identified that contained several fragments of conserved amino acid sequence. The splice donor at the 3` end of mouse exon C appeared to be conserved. Mouse and human exon M upstream of the initiator Met codon showed 82% sequence identity with nine gaps (gap weight, 2.0; length weight, 0.05). A coding sequence in frame with the initiator Met codon was found, and this sequence contained several fragments of conserved amino acid sequence. The splice acceptor sequence at the predicted 5` boundary of mouse exon M appeared to be conserved.



Figure 3: Sequence comparison of 5` mouse and human Fbn-1 upstream exon sequences. Mouse and human nucleotide sequence alignments are shown on the left. Nucleotide sequence alignment and identity are presented using the BESTFIT sequence analysis program from the Genetics Computer Group, but essentially the same alignments were obtained using the GAP algorithm. Human sequence is presented atop the mouse sequence, and nucleotide numbers are shown on both the right and the left. Predicted splice donor/splice acceptor sites (a total of 5 nt) are shown on a separate line and have been underlined. Deduced murine amino acid sequences are shown on the right. Stop codons are indicated by an *. Fragments of conserved amino acid sequence are underlined.



5`-Region Flanking the Mouse Fibrillin-1 Gene: Structural Analysis

The evaluation of Fbn-1 has thus far identified significant differences in the degree of sequence conservation upstream and downstream of the putative initiator Met codon. Given the degree of conservation of mouse 5` fibrillin-1 upstream sequences, it was important to ensure that the F-2.18 and WY-129 m genomic clones were free of ``gene jumping'' artifacts. Several observations make the possibility of a cloning artifact unlikely. First, a subclone of pTP#1 (see Fig. 1) consisting solely of coding sequence downstream of the proposed translation start site was prepared and used to rescreen a mouse genomic library. This effort yielded an additional 17 genomic clones that overlap F-2.18 and WY-129 m. (^3)All 17 clones hybridized to mouse exon B, A, and C PCR probes. Second, clone pTP#1 was found to consist of >430 nt of sequence upstream of the Fbn-1 Met codon and 1 kb of downstream coding sequence. Significantly, the upstream 5` sequence in this unusual cDNA clone is identical to the genomic sequence found just upstream of the Fbn-1 Met codon. This result suggests that pTP#1 may have been generated from an unspliced pre-mRNA fragment. Third, Southern analysis provided direct evidence linking the 5` upstream and coding sequence. Mouse genomic DNA was double-digested with NsiI and NcoI (which should yield a single 1.7-kb fragment that contains exons B, A, and C) and also double-digested with NsiI and XhoI, which should yield a 1.0-kb fragment that contains mouse exon B and A plus a 0.6-kb fragment that contains mouse exon C). Using exon probes obtained by PCR, the predicted result was obtained in both instances.^3

Fbn-1 Expression during Early Mouse Development

The Fbn-1 analysis corroborates previous efforts to localize the fibrillin-1 translational start site. Pereira et al.(1993) have suggested that the human FBN-1 promoter is GC-rich and devoid of TATA and CCAAT boxes. Promoters of this type have previously been associated with genes that show a broad patterns of expression. To test this idea, tissue in situ hybridization was used to survey the expression pattern of fibrillin-1 transcripts in mouse embryo tissues.

The temporal and spatial expression patterns of Fbn-1 transcripts were determined using a specific 3`-untranslated region probe (see ``Experimental Procedures''). As shown in Fig. 4, dramatic Fbn-1 expression was observed in uterine tissues surrounding day 8.5-9.0 embryos. Particularly striking was the fact that gene expression was limited to the uterine stromal tissue (decidua) beneath and lateral to the placental implantation site, i.e. the mesometrial side of the gravid uterus. In contrast, expression was not significant in embryo tissues, placenta, placental membranes, or the ectoplacental cone.


Figure 4: Overview of Fbn-1 gene expression during murine development as determined by tissue in situ hybridization. Day 8.5-9.0 sections contained embryos surrounded by intact membranes, uterine tissues, and the placental disk, cut in random planes. Day 13.5 and 16.5 sections contain isolated whole embryos sectioned in the sagittal plane near or about the mid-line. Identical conditions were maintained throughout autoradiography and photography, thereby allowing a comparison of the overall strength of hybridization in all tissue sections. The Fbn-1 transcript is intensely expressed in the mesometrial decidua at day 8.5-9.0 of development. The anti-mesometrial decidua, placenta, placental membranes, and the embryo do not show significant hybridization. At day 13.5 and day 16 of development, the Fbn-1 transcript appears to be the widespread product of connective tissue cells. Significant hybridization is not observed in the brain, spinal cord, heart, and liver of day 13.5 and day 16.5 tissue sections (arrowheads).



Histological examination of the day 8.5-9.0 gravid uterus confirmed that the Fbn-1 gene was highly expressed by mesometrial decidual cells. Expression was above background in the anti-mesometrial decidua, trophoblastic cells of the ectoplacental cone, placenta, and placental membranes. (^4)Fbn-1 gene expression could not be detected in non-gravid uterine tissues derived from age-matched CD-1 females. Fbn-1 gene expression in day 8.5-9.0 mouse embryo tissues was generally negative with a single exception, evaluation of 24 embryos demonstrated that the endocardium alone expressed the Fbn-1 gene at levels above background (Fig. 5).


Figure 5: Fbn-1 gene expression in the day 8.5-9.0 embryo. At day 8.5-9.0 of development, the hybridization signal appears to be concentrated within endocardial tissue. Heart muscle, the neural epithelium, and the mesenchyme/connective tissues did not show significant hybridization (not shown). Bar, 20 µm.



Fbn-1 Expression during Late Mouse Development

As is clear from Fig. 3, Fbn-1 transcripts appeared to be the widespread product of connective tissue cells at day 13.5 and day 16.5 of development. Those organs in which we failed to detect significant Fbn-1 expression in the interstitium included the liver, spinal cord, brain, and cardiac muscle (atria and ventricles). Consistent with previous findings (Mecham and Davis, 1994), widespread Fbn-1 expression clearly antedates expression of the mouse elastin gene, which was not be detected until day 16.5.^4 Elastin expression was restricted to the lung and aorta.


DISCUSSION

Primary Structure of the Fbn-1 Gene Product

As demonstrated here, striking conservation exists between human and mouse fibrillin-1 at the DNA and protein levels. Region A appears to be a unique sequence that likely has a globular conformation. In addition, region A is highly basic and may allow fibrillin to bind acidic molecules (e.g. acidic proteoglycans) in the extracellular matrix. Sequences rich in basic amino acids have also been shown to function as endoproteolytic processing signals for several peptide hormones (Steiner et al., 1992), and perhaps the NH(2) terminus of the fibrillin-1 molecule is proteolytically processed in a tissue-specific manner. We note, however, that the potential for tissue-specific endoproteolytic processing should be considered separately from previous studies which described a shortened form of the fibrillin-1 molecule in the medium of human and bovine cultured cells (e.g. Milewicz et al., 1992). This form of fibrillin-1 was estimated to be 300 kDa, which implies a loss of 30-50 kDa from the native molecule. It is unlikely that endoproteolytic cleavage within region A would shorten the fibrillin-1 polypeptide to this degree. Regions B and D consist of consecutive cysteine-rich repeats, the majority of which are of the EGF-CB type. Besides binding calcium (Corson et al., 1993), these repeats may provide fibrillin with regions of extended conformation capable of interacting with other matrix macromolecules (Engel, 1989). Region C is proline-rich and may be capable of bending (or functioning like a hinge) in three-dimensional space (MacArthur and Thornton, 1991). Region E also appears to be a unique sequence having a globular conformation, and it contains a pair of conserved cysteine residues that may be of potential significance for microfibril assembly (Zhang et al., 1994).

The remarkable degree of evolutionary conservation between mouse and human fibrillin-1 gene products implies that strict structural requirements govern the assembly of extracellular 10-nm microfibrils. To facilitate assembly, regions B and D (which consist of consecutive EGF-like repeats) may function as rigid arms capable of projecting unique globular domains that interact with other microfibril constituents and/or with extracellular matrix molecules near the microfibril surface. A similar model has been proposed for the structure of other extracellular matrix molecules that have multiple EGF-like repeats, including laminin, tenascin, and thrombospondin-1 (Engel, 1989).

Fbn-1 Upstream Sequences

Data presented in this paper corroborate the initiator Met codon originally identified by Pereira et al.(1993) as the most likely start site of translation. This result implies that the mouse and human fibrillin-1 transcripts both have open reading frames that code for polypeptides of 2,871 amino acids.

The fibrillin-1 transcriptional start site is more problematical. Corson et al.(1993) have used DNA sequencing plus Northern analysis to identify a 1.8-kb CpG island that contained three putative exons plus a fourth downstream exon containing a putative initiator Met residue. These findings suggested that the CpG island contained the transcription initiation site and exons B, A, and C may be alternatively spliced first exons under the control of separate promoters. Alternatively, the CpG island may not represent the transcription start site and there may be additional exons, as yet undiscovered, within the upstream flanking sequence. Our preliminary studies have yielded conflicting results regarding these possibilities. On one hand, none of the upstream human fibrillin-1 sequences described by Corson et al.(1993) were identified as candidate exons when analyzed by the GRAIL program (Oak Ridge National Laboratory), (^5)a result that argues against the possibility of more than one transcript initiation site. On the other hand, Northern analysis of mouse mRNA identified fibrillin-1 transcripts that hybridize with exon A and C probes and therefore appear to posses heterogeneous 5` ends.^5 As expected from previous work (Corson et al., 1993), transcripts with exon A sequences were most readily identified, although they were extremely low abundance relative to fibrillin-1 transcripts that hybridize with a 3`-untranslated region probe (10-100-fold less, as determined by scanning gel densitometry). Fibrillin-1 transcripts with heterogeneous 5` ends therefore appear to be rare or restricted to a subset of developing mouse tissues and cell lines.

While the fibrillin-1 translational start site appears well defined more information clearly is necessary before the existence and function of alternative upstream exons can be determined and the sites of transcription initiation are localized. Perhaps these issues will be resolved through the analysis of fibrillin-1 genes from species even more closely related to human than the mouse, e.g. the pig fibrillin-1 gene.

Fbn-1 Expression in Developing Mouse Tissues

Tissue in situ hybridization studies reported here demonstrate for the first time that the Fbn-1 gene is highly expressed by decidual cells on the mesometrial side of the day 8.5-9.0 gravid uterus, but is expressed at low to undetectable levels in the non-gravid uterus. The interaction of trophoblast and decidual cells is complex and highly regulated. Cell migration and proliferation is associated with the new expression of hormones and cytokines and the alternate expression (``switching'') of cell surface adhesion molecules and matrix receptors (for discussion and additional references, see Farrar and Carson, 1992; Zhou et al., 1993; Sutherland et al., 1993). Extensive remodeling of the endometrial stroma also occurs, as exemplified by the expression of metalloproteinase enzymes and their inhibitors (Librach et al., 1991), the loss of collagen types I and VI (Mulholland et al., 1992), the development of an electron dense external lamina at the periphery of decidual cells, and the new expression of a number of basement membrane components including laminin, type IV collagen, heparan sulfate proteoglycan, entactin, and a molecule variously referred to as osteonectin/SPARC/BM40 (Farrar and Carson, 1992). Tenascin (Castellucci et al., 1991) and an alternatively spliced form of fibronectin known as oncofetal fibronectin (Feinberg et al., 1991) have also been associated with invading cytotrophoblast cells. The fibrillin-1 tissue in situ hybridization results therefore suggest that remodeling of the endometrial stroma involves formation of an extracellular matrix that contains fibrillin-associated microfibrils.

The endocardium was the only tissue in our study of day 8.5-9.0 embryos that expresses the Fbn-1 gene at levels above the background of the experiment. At this time the heart beats regularly and powerfully, and it is plausible that fibrillin-1 molecules are assembled into a microfibril-rich, subendocardial connective tissue that organizes muscle cells and helps them resist the mechanical forces of cardiac contraction. Fibrillin may certainly be expressed at earlier time points, however. For example, Gallagher et al.(1993) have shown that fibrillin-1 polypeptides are expressed along the primary axis of the avian embryo, including Hensen's node. These results further emphasize that fibrillin-associated microfibrils may play a critical role during connective tissue assembly.

In sharp contrast to the pattern of Fbn-1 expression, Magp is widely expressed in the mesenchyme of day 8.5-9.0 embryos, but is not expressed by decidual cells in any region of the gravid uterus or in endocardial tissue (Chen et al., 1993). By day 13 of mouse development, the overall pattern of Fbn-1 expression was similar to that of Magp in that both genes appeared to be the widespread product of connective tissue cells in the interstitium of many organs. The results of these tissue in situ hybridization studies indicate that microfibril genes are not always coordinately regulated in the mouse, which, in turn, may be an indication of tissue-specific differences in microfibril composition, structure, and function.


FOOTNOTES

*
This work was supported in part by the National Institutes of Health and by a grant from the American Heart Association, New York City Affiliate. This is article 157 from the Brookdale Center for Molecular Biology. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L29454[GenBank].

§
To whom correspondence should be addressed: Dept. of Pathology, University of Michigan, MSRB I, Rm. 3510, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0650. Tel.: 313-747-4774; Fax: 313-936-9353.

(^1)
The abbreviations used are: FBN-1, the human fibrillin gene on chromosome 15 involved with Marfan syndrome and ectopia lentis; Fbn-1, the mouse homologue of FBN-1; fibrillin-1, the FBN-1 gene product; FBN-2, the human fibrillin gene on chromosome 5 that links to congenital contractural arachnodactyly; Magp, the mouse MAGP gene; nt, nucleotide(s); EGF-CB, epidermal growth factor-like repeats with the potential to bind calcium; TGF-bp, a cysteine-rich motif initially identified in the transforming growth factor-beta1-binding protein; PCR, polymerase chain reaction; kb, kilobase(s); nt, nucleotide(s).

(^2)
E. Smiley and J. Bonadio, unpublished data.

(^3)
W. Yin and J. Bonadio, unpublished data.

(^4)
J. Germiller and J. Bonadio, unpublished data.

(^5)
E. Smiley and J. Bonadio, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful to R. Mecham and H. Dietz for communication of data prior to publication.


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