Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues

Alternative splicing of fibronectin pre-mRNA at two distinct regions, termed ED-A and IIICS, was investigated with human adult and fetal tissues by the nuclease S1 protection assay. A clear tissue specificity was observed in the splicing pattern at the ED-A region. More ED-A+ than ED-A- mRNAs were identified in lung, whereas ED-A- mRNAs were predominantly expressed in liver. Endometrium contained nearly equal amounts of ED-A+ and ED-A- mRNAs. The splicing pattern at the ED-A region was also different between adult and fetal liver but not between adult and fetal lung. Tissue type specific splicing was also observed at the IIICS region. Although the mRNA species containing the complete IIICS sequence comprised 40-65% of the total fibronectin mRNAs irrespective of tissue types, expression of the mRNA species lacking a part or all of the IIICS sequence was more pronounced in adult liver than in other tissues including fetal liver. These results strongly suggest that the alternative splicing of fibronectin pre-mRNA in vivo is regulated in a tissue type specific manner at both the ED-A and IIICS regions and that it is developmentally regulated in liver but not in lung. On the basis of these and other observations reported previously, a possibility that a part of the fibronectins synthesized and secreted by hepatocytes is deposited in the tissue matrix is discussed.     

Genetic analysis of the cell binding domain region of the chicken fibronectin gene

We have determined the nucleotide sequence of the cell binding domain region of the chicken fibronectin gene and analyzed it evolutionaly. We present here the complete nucleotide sequence of 4.3 kb HindIII/EcoRI segment from the clone lambda FC23 of the chicken fibronectin gene. There were five exons in this segment. When we lined up the amino acid of exons 28, 29 and 31, three alignments, known as the Type III repeat, appeared. Tetrapeptide, -RGDS-, called the cell binding domain, existed in the second repeat, coding exon 30. It was presumed that the Type III repeats were composed of two exons in the chicken gene, the same as in the rat and humans. We found repeatedly appearing amino-acid sequences such as -TIT- (three arrays in these Type III repeats) but also found one of the amino acids substituted in the tripeptide in these Type III repeats (seven arrays). We analyzed these repeats from the point of view of evolution. We used three of the nucleotide sequences (12-18 bp) coding such -TIT- repeats as a unit length for comparing the various homologies after dividing the coding region into 56 segments. The mutual homology of the divided segments to each one of three showed 53% on average. On the other hand, the mutual nucleotide homology of the Type III repeat was 44%. This suggested that the Type III repeat may have been developed by frequent duplication of small gene units.     

Ventricular myosin light chain-2 gene expression in developing heart of chicken embryos

Recent gene knock-out studies in mice have suggested that ventricular myosin light chain-2 (vMLC2) has a role in the regulation of cardiogenic development and that perturbation in expression of vMLC2 is linked to the onset of dilated cardiomyopathy. In an attempt to develop an avian model for such studies, we examined the expression pattern of vMLC2 in chicken embryos at various stages and analyzed the effect of antisense oligonucleotide-mediated interference of vMLC2 function in cultures of whole embryos. Our results showed vMLC2 to be a specific marker for ventricular chamber throughout chicken embryonic development and antisense vMLC2 treatment of primitive streak stage (stage 4) embryos to produce pronounced dilation of heart tube with severe deficiency in formation of striated myofibrils. Further studies with antisense mRNA techniques of whole embryo cultures should, therefore, be useful to evaluate the role of vMLC2 and other putative regulatory factors in cardiac myofibrillogenesis.     

Production of transgenic fish: introduction and expression of chicken delta-crystallin gene in medaka embryos

To produce a model of transgenic fish, recombinant plasmids containing chicken delta-crystallin gene were microinjected into the oocyte nucleus of a small teleost, medaka (Oryzias latipes). About 50% of the microinjected oocytes developed to 7-day-old embryos. By Southern blotting delta-crystallin gene was detected in 4 of 8 embryos, and, by Western blotting, delta-crystallin polypeptides in 5 of 16. In 1 of 6 examined histologically, delta-crystallin DNA was detected in all the tissues, and delta-crystallin polypeptides, in many of the tissues including the lens. Thus, the exogenous gene and/or its products were detected in 10 of 30 embryos examined. This is the first report of successful production of transgenic fish.     

Precardiac cell migration: fibronectin localization at mesoderm-endoderm interface during directional movement

The pathway of directional movement of chick precardiac mesoderm cells was studied by indirect immunofluorescence and by scanning electron microscopy. Directional movement of the precardiac cells begins at stage 6 from the lateral sides of the embryo at the level of Hensen's node. The cells move anteriorly in an arc to the embryo's midline. By stage 8 the cells arrive at the lateral sides of the anterior intestinal portal and movement ceases. The interval of this directional movement is approximately 10 hr. During migration the precardiac cells are in close association with the underlying endoderm. As migration proceeds, the cells encounter increasing amounts of fibrils in the substratum at the mesoderm-endoderm interface. Concomitant with increasing fibril formation there is an increase in fibronectin (FN) in the heart-forming region. During stage 5 FN first appears in the lateral heart-forming regions and increases in amount during the period of cell migration. By stage 7 a concentration difference of FN is apparent in the lateral regions with more FN cephalad and decreasing amounts caudad. At stages 7 and 8 large amounts of extracellular FN-associated fibrils are observed at the lateral sides of the anterior intestinal portal where the cells stop moving. The precardiac cells moving into this region are oriented perpendicular to the anterior intestinal portal and in close association with these fibrils. There is no evidence that the fibrillar meshwork forming the substratum of the precardiac mesoderm cells is physically oriented as a guide for directional movement. The correlations between FN distribution at the mesoderm-endoderm interface and directional cell movement suggest that the precardiac cells may migrate by haptotaxis, i.e., by moving along the substratum toward areas of greater adhesiveness.     

Alternative splicing during chondrogenesis: cis and trans factors involved in splicing of fibronectin exon EIIIA

Primary chicken mesenchymal cells from limb buds and vertebral chondrocytes have been used to study the changes that occur in alternative mRNA splicing of fibronectin exon EIIIA during chondrogenesis. The mesenchymal cell phenotype (exon EIIIA included) and chondrocyte phenotype (exon EIIIA excluded) were preserved in culture. Both primary cell types were transfected with an EIIIA minigene and alternative splicing was monitored by S1 protection assay. Differential cell-specific splicing of the reporter was observed. The roles of two regulatory elements, an exon splicing enhancer (ESE) and an exon splicing silencer (ESS) were examined. Both elements were required for EIIIA inclusion into mRNA in mesenchymal cells. Gel mobility shift assays revealed that both chondrocyte- and mesenchymal cell-derived nuclear extracts contained exon EIIIA binding factors, but the RNA binding factors present in the two cell types appeared to be distinct. The ESE and ESS appeared to cooperate in the formation of both cell type-specific complexes. These results suggest a model in which inhibitory factors enriched in chondrocytes compete with positive factors enriched in mesenchymal cells for binding to exon EIIIA, determining whether the exon is included.     

New tools for gene manipulation in chicken embryos

Genomics has changed the pace by which genes are analyzed. Rather than looking at genes one by one, gene expression today is studied at the genome level. Unfortunately, the data we get from microarray analysis do not give us any clues about the function of these genes. Functional analyses are still refractory to large-scale, high-throughput studies, particularly in vertebrates. With the development of in ovo RNAi as a tool for specific gene silencing, the chicken embryo has become an efficient in vivo system to study gene function during development. A major advantage of in ovo RNAi is the fact that the knowledge of a cDNA fragment of the gene of interest is sufficient to get loss-of-function phenotypes. Thus, this new approach is a valuable tool for functional genomics.     

Transforming growth factor-beta1 regulates fibronectin isoform expression and splicing factor SRp40 expression during ATDC5 chondrogenic maturation

Fibronectin (FN) isoform expression is altered during chondrocyte commitment and maturation, with cartilage favoring expression of FN isoforms that includes the type II repeat extra domain B (EDB) but excludes extra domain A (EDA). We and others have hypothesized that the regulated splicing of FN mRNAs is necessary for the progression of chondrogenesis. To test this, we treated the pre-chondrogenic cell line ATDC5 with transforming growth factor-beta1, which has been shown to modulate expression of the EDA and EDB exons, as well as the late markers of chondrocyte maturation; it also slightly accelerates the early acquisition of a sulfated proteoglycan matrix without affecting cell proliferation. When chondrocytes are treated with TGF-beta1, the EDA exon is preferentially excluded at all times whereas the EDB exon is relatively depleted at early times. This regulated alternative splicing of FN correlates with the regulation of alternative splicing of SRp40, a splicing factor facilitating inclusion of the EDA exon. To determine if overexpression of the SRp40 isoforms altered FN and FN EDA organization, cDNAs encoding these isoforms were overexpressed in ATDC5 cells. Overexpression of the long-form of SRp40 yielded an FN organization similar to TGF-beta1 treatment; whereas overexpression of the short form of SRp40 (which facilitates EDA inclusion) increased formation of long-thick FN fibrils. Therefore, we conclude that the effects of TGF-beta1 on FN splicing during chondrogenesis may be largely dependent on its effect on SRp40 isoform expression.     

Global gene expression analysis identifies PDEF transcriptional networks regulating cell migration during cancer progression

Prostate derived ETS factor (PDEF) is an ETS (epithelial-specific E26 transforming sequence) family member that has been identified as a potential tumor suppressor. In multiple invasive breast cancer cells, PDEF expression inhibits cell migration by preventing the acquisition of directional morphological polarity conferred by changes in cytoskeleton organization. In this study, microarray analysis was used to identify >200 human genes that displayed a common differential expression pattern in three invasive breast cancer cell lines after expression of exogenous PDEF protein. Gene ontology associations and data mining analysis identified focal adhesion, adherens junctions, cell adhesion, and actin cytoskeleton regulation as cell migration-associated interaction pathways significantly impacted by PDEF expression. Validation experiments confirmed the differential expression of four cytoskeleton-associated genes with known functional associations with these pathways: uPA, uPAR, LASP1, and VASP. Significantly, chromatin immunoprecipitation studies identified PDEF as a direct negative regulator of the metastasis-associated gene uPA and phenotypic rescue experiments demonstrate that exogenous urokinase plasminogen activator (uPA) expression can restore the migratory ability of invasive breast cancer cells expressing PDEF. Furthermore, immunofluorescence studies identify the subcellular relocalization of urokinase plasminogen activator receptor (uPAR), LIM and SH3 protein (LASP1), and vasodilator-stimulated protein (VASP) as a possible mechanism accounting for the loss of morphological polarity observed upon PDEF expression.     

Syndecan-1 regulates cell migration and fibronectin fibril assembly

Corneal scarring is a major cause of blindness worldwide and can result from the deposition of abnormal amounts of collagen fibers lacking the correct size and spacing required to produce a clear cornea. Collagen fiber formation requires a preformed fibronectin (FN) matrix. We demonstrate that the loss of syndecan1 (sdc1) in corneal stromal cells (CSC) impacts cell migration rates, the sizes and composition of focal and fibrillar adhesions, the activation of integrins, and the assembly of fibronectin into fibrils. Integrin and fibronectin expression are not altered on sdc1-null CSCs. Cell adhesion, spreading, and migration studies using low compared to high concentrations of FN and collagen I (CNI) or vitronectin (VN) with and without activation of integrins by manganese chloride show that the impact of sdc1 depletion on integrin activation varies depending on the integrin-mediated activity evaluated. Differences in FN fibrillogenesis and migration in sdc1-null CSCs are reversed by addition of manganese chloride but cell spreading differences remain. To determine if our findings on sdc1 were specific to the cornea, we compared the phenotypes of sdc1-null dermal fibroblasts with those of CSCs. We found that without sdc1, both cell types migrate faster; however, cell-type-specific differences in FN expression and its assembly into fibrils exist between these two cell types. Together, our data demonstrate that sdc1 functions to regulate integrin activity in multiple cell types. Loss of sdc1-mediated integrin function results in cell-type specific differences in matrix assembly. A better understanding of how different cell types regulate FN fibril formation via syndecans and integrins will lead to better treatments for scarring and fibrosis.     

Multiple sites of alternative splicing of the rat fibronectin gene transcript

We describe analyses of the structure and expression of the rat fibronectin gene with particular attention to the 40-kb stretch from the center of the gene which encodes 17 type-III repeating units. Each repeat is precisely separated from its neighbors by introns and most are encoded by pairs of exons. Three repeats are encoded precisely by single exons and two of these (EIIIA and EIIIB) are alternatively spliced in a cell type-specific fashion. A third site of alternative splicing (EIIIB) reported here is similar in expression to the previously described EIIIA segment. Both are excluded from mRNA in liver cells and are, therefore, absent from plasma fibronectin. These two alternative splices, plus a third one (V) reported previously, can occur in all possible combinations giving 12 fibronectin mRNAs from a single gene. These splicing variations account for most but not all of the known fibronectin subunit variants. We report investigations designed to detect other regions of alternative splicing. We also show that the pattern of alternative splicing is somewhat altered on oncogenic transformation.     

Stable integration and conditional expression of electroporated transgenes in chicken embryos

The in ovo electroporation in chicken embryos has widely been used as a powerful tool to study roles of genes during embryogenesis. However, the conventional electroporation technique fails to retain the expression of transgenes for more than several days because transgenes are not integrated into the genome. To overcome this shortcoming, we have developed a transposon-mediated gene transfer, a novel technique in chicken manipulations. It was previously reported that the transposon Tol2, originally found in medaka fish, facilitates an integration of a transgene into the genome when co-acting with Tol2 transposase. In this study, we co-electroporated a plasmid containing a CAGGS-EGFP cassette cloned in the Tol2 construct along with a transposase-encoding plasmid into early presomitic mesoderm or optic vesicles of chicken embryos. This resulted in persistent expression of EGFP at least until embryonic day 8 (E8) and E12 in somite-derived tissues and developing retina, respectively. The integration of the transgene was confirmed by genomic Southern blotting using chicken cultured cells. We further combined this transposon-mediated gene transfer with the tetracycline-dependent conditional expression system that we also developed recently. With this combined method, expression of a stably integrated transgene could be experimentally induced upon tetracycline administration at relatively late stages such as E6, where a variety of organogenesis are underway. Thus, the techniques proposed in this study provide a novel approach to study the mechanisms of late organogenesis, for which chickens are most suitable model animals.     

Dynamic gene expression of Lin-28 during embryonic development in mouse and chicken

The Caenorhabditis elegans heterochronic gene lin-28 regulates developmental timing in the nematode trunk. We report the dynamic expression patterns of Lin-28 homologues in mouse and chick embryos. Whole mount in situ hybridization revealed specific and intriguing expression patterns of Lin-28 in the developing mouse and chick limb bud. Mouse Lin-28 expression was detected in both the forelimb and hindlimb at E9.5, but disappeared from the forelimb at E10.5, and finally from the forelimb and hindlimb at E11.5. Chicken Lin-28, which was first detected in the limb primordium at stage 15/16, was also downregulated as the stage proceeded. The amino acid sequences of mouse and chicken Lin-28 genes are highly conserved and the similar expression patterns of Lin-28 during limb development in mouse and chicken suggest that this heterochronic gene is also conserved during vertebrate limb development.