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Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

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EDA−/−skin wounds at day 7 after wounding show abnormal healing and increased number of BrdU-positive cells. (A) Full thickness cutaneous wounds of control (EDAwt/wt, n = 8) and mutant mice (EDA+/+ and EDA−/−, n = 8 and 9, respectively) mice were analyzed at 7 d after wounding. Representative sections are shown. The black arrowheads indicate the wound edges. Abbreviations are as described in Fig. 5. The ulcerated epithelium observed in the EDA−/− wound is indicated as “ue.” The experiment was repeated three times with similar results. Wound sections of three independent experiments (eight mice per genotype in each experiments) were microscopically scored for the presence of ulcerative processes at day 7 after wounding. The fraction of ulcerated wounds of the EDA−/− mice was statistically significant different from that of EDAwt/wt and EDA+/+ mice (63 and 22% for EDA−/− and EDAwt/wt, respectively; P < 0.0002 for EDA−/− vs. EDAwt/wt by both the Fisher Exact test and the χ2 test). There were no differences between the EDAwt/wt and EDA+/+ scores). (B) BrdU labeling shows an increased number of replicating cells in the EDA−/− wounds. Serial sections of the same wounds seen in A were incubated with an anti-BrdU mAb. The count of several microscopical fields showed that there are at least 10 times more BrdU-positive nuclei in the area below the newly formed epidermis in the EDA−/− wounds when compared with EDAwt/wt or EDA+/+ wounds. A representative field is shown. More than 50 positive nuclei are observed in the EDA−/− wound (bottom right), whereas less than five positive nuclei are observed in the case of EDAwt/wt or EDA+/+ samples (bottom left and center, respectively). Illustrative BrdU-labeled nuclei are indicated by white triangles. The black arrows indicate the wound edges.
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fig6: EDA−/−skin wounds at day 7 after wounding show abnormal healing and increased number of BrdU-positive cells. (A) Full thickness cutaneous wounds of control (EDAwt/wt, n = 8) and mutant mice (EDA+/+ and EDA−/−, n = 8 and 9, respectively) mice were analyzed at 7 d after wounding. Representative sections are shown. The black arrowheads indicate the wound edges. Abbreviations are as described in Fig. 5. The ulcerated epithelium observed in the EDA−/− wound is indicated as “ue.” The experiment was repeated three times with similar results. Wound sections of three independent experiments (eight mice per genotype in each experiments) were microscopically scored for the presence of ulcerative processes at day 7 after wounding. The fraction of ulcerated wounds of the EDA−/− mice was statistically significant different from that of EDAwt/wt and EDA+/+ mice (63 and 22% for EDA−/− and EDAwt/wt, respectively; P < 0.0002 for EDA−/− vs. EDAwt/wt by both the Fisher Exact test and the χ2 test). There were no differences between the EDAwt/wt and EDA+/+ scores). (B) BrdU labeling shows an increased number of replicating cells in the EDA−/− wounds. Serial sections of the same wounds seen in A were incubated with an anti-BrdU mAb. The count of several microscopical fields showed that there are at least 10 times more BrdU-positive nuclei in the area below the newly formed epidermis in the EDA−/− wounds when compared with EDAwt/wt or EDA+/+ wounds. A representative field is shown. More than 50 positive nuclei are observed in the EDA−/− wound (bottom right), whereas less than five positive nuclei are observed in the case of EDAwt/wt or EDA+/+ samples (bottom left and center, respectively). Illustrative BrdU-labeled nuclei are indicated by white triangles. The black arrows indicate the wound edges.

Mentions: The FN present in the skin of wild-type animals does not contain the EDA exon. However, the FN-EDA+ form is thought to participate actively in the reepithelization process (Clark et al., 1983; ffrench-Constant et al., 1989). To explore the role of the EDA exon in the wound healing process, we performed full thickness excisional skin wounds in EDAwt/wt, EDA−/−, and EDA+/+ mice. The wound healing process was analyzed at 1, 3, 5, 7, 10, and 14 d after wounding. Hematoxylin-eosin staining of tissues from full thickness wounds during the early phase of reepithelization (days 1 and 3) did not show any differences in reepithelization and neovascularization (unpublished data). During late phase (days 5 and 7), the reepithelization and formation of granulation tissue were indistinguishable between EDA+/+ and EDAwt/wt mice (Figs. 5 and 6)Figure 5.


Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan.

Muro AF, Chauhan AK, Gajovic S, Iaconcig A, Porro F, Stanta G, Baralle FE - J. Cell Biol. (2003)

EDA−/−skin wounds at day 7 after wounding show abnormal healing and increased number of BrdU-positive cells. (A) Full thickness cutaneous wounds of control (EDAwt/wt, n = 8) and mutant mice (EDA+/+ and EDA−/−, n = 8 and 9, respectively) mice were analyzed at 7 d after wounding. Representative sections are shown. The black arrowheads indicate the wound edges. Abbreviations are as described in Fig. 5. The ulcerated epithelium observed in the EDA−/− wound is indicated as “ue.” The experiment was repeated three times with similar results. Wound sections of three independent experiments (eight mice per genotype in each experiments) were microscopically scored for the presence of ulcerative processes at day 7 after wounding. The fraction of ulcerated wounds of the EDA−/− mice was statistically significant different from that of EDAwt/wt and EDA+/+ mice (63 and 22% for EDA−/− and EDAwt/wt, respectively; P < 0.0002 for EDA−/− vs. EDAwt/wt by both the Fisher Exact test and the χ2 test). There were no differences between the EDAwt/wt and EDA+/+ scores). (B) BrdU labeling shows an increased number of replicating cells in the EDA−/− wounds. Serial sections of the same wounds seen in A were incubated with an anti-BrdU mAb. The count of several microscopical fields showed that there are at least 10 times more BrdU-positive nuclei in the area below the newly formed epidermis in the EDA−/− wounds when compared with EDAwt/wt or EDA+/+ wounds. A representative field is shown. More than 50 positive nuclei are observed in the EDA−/− wound (bottom right), whereas less than five positive nuclei are observed in the case of EDAwt/wt or EDA+/+ samples (bottom left and center, respectively). Illustrative BrdU-labeled nuclei are indicated by white triangles. The black arrows indicate the wound edges.
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fig6: EDA−/−skin wounds at day 7 after wounding show abnormal healing and increased number of BrdU-positive cells. (A) Full thickness cutaneous wounds of control (EDAwt/wt, n = 8) and mutant mice (EDA+/+ and EDA−/−, n = 8 and 9, respectively) mice were analyzed at 7 d after wounding. Representative sections are shown. The black arrowheads indicate the wound edges. Abbreviations are as described in Fig. 5. The ulcerated epithelium observed in the EDA−/− wound is indicated as “ue.” The experiment was repeated three times with similar results. Wound sections of three independent experiments (eight mice per genotype in each experiments) were microscopically scored for the presence of ulcerative processes at day 7 after wounding. The fraction of ulcerated wounds of the EDA−/− mice was statistically significant different from that of EDAwt/wt and EDA+/+ mice (63 and 22% for EDA−/− and EDAwt/wt, respectively; P < 0.0002 for EDA−/− vs. EDAwt/wt by both the Fisher Exact test and the χ2 test). There were no differences between the EDAwt/wt and EDA+/+ scores). (B) BrdU labeling shows an increased number of replicating cells in the EDA−/− wounds. Serial sections of the same wounds seen in A were incubated with an anti-BrdU mAb. The count of several microscopical fields showed that there are at least 10 times more BrdU-positive nuclei in the area below the newly formed epidermis in the EDA−/− wounds when compared with EDAwt/wt or EDA+/+ wounds. A representative field is shown. More than 50 positive nuclei are observed in the EDA−/− wound (bottom right), whereas less than five positive nuclei are observed in the case of EDAwt/wt or EDA+/+ samples (bottom left and center, respectively). Illustrative BrdU-labeled nuclei are indicated by white triangles. The black arrows indicate the wound edges.
Mentions: The FN present in the skin of wild-type animals does not contain the EDA exon. However, the FN-EDA+ form is thought to participate actively in the reepithelization process (Clark et al., 1983; ffrench-Constant et al., 1989). To explore the role of the EDA exon in the wound healing process, we performed full thickness excisional skin wounds in EDAwt/wt, EDA−/−, and EDA+/+ mice. The wound healing process was analyzed at 1, 3, 5, 7, 10, and 14 d after wounding. Hematoxylin-eosin staining of tissues from full thickness wounds during the early phase of reepithelization (days 1 and 3) did not show any differences in reepithelization and neovascularization (unpublished data). During late phase (days 5 and 7), the reepithelization and formation of granulation tissue were indistinguishable between EDA+/+ and EDAwt/wt mice (Figs. 5 and 6)Figure 5.

Bottom Line: However, the precise role of the FN isoforms is poorly understood.One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging.Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon.

View Article: PubMed Central - PubMed

Affiliation: International Center for Genetic Engineering and Biotechnology (ICGEB), Padriciano 99, 34012 Trieste, Italy.

ABSTRACT
Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP-mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions.

Show MeSH
Related in: MedlinePlus