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The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development.

Bebee TW, Park JW, Sheridan KI, Warzecha CC, Cieply BW, Rohacek AM, Xing Y, Carstens RP - Elife (2015)

Bottom Line: Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development.Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease.Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

ABSTRACT
Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo.

No MeSH data available.


Related in: MedlinePlus

Generation of Esrp1 and Esrp2 KO alleles.(A) Schematic of the knock-in strategy used for generation of the Esrp1 floxed allele for conditional and ubiquitous KO. The floxed neomycin cassette targeted exons 7–9. Restriction site for SacI (S) and HincII (H) are indicated. The RNA Recognition Motifs (RRMs) 1–3 are indicated by brackets and loxP sites are red triangles. (B) Southern blot validation of V6.5, hybrid C57BL6/129Sv, mouse ES cells used for blastocyst injection. Clone 1D1 was verified as heterozygous, a representative southern is shown. (C) Schematic and genotyping for Esrp1 CKO (floxed), KO, and WT alleles are shown. Primers are indicated by arrows and representative genotyping gels are presented. (D) Sequencing histogram of the KO PCR product confirms Cre-mediated recombination. VS: variable sequence, as part of the targeting construct. (E) Schematic of full gene replacement of Esrp2 by LacZ:PGK-Neo cassette generated by Knockout Mouse Project and purchased from Velocigene. A representative genotyping gel is presented.DOI:http://dx.doi.org/10.7554/eLife.08954.005
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fig1s2: Generation of Esrp1 and Esrp2 KO alleles.(A) Schematic of the knock-in strategy used for generation of the Esrp1 floxed allele for conditional and ubiquitous KO. The floxed neomycin cassette targeted exons 7–9. Restriction site for SacI (S) and HincII (H) are indicated. The RNA Recognition Motifs (RRMs) 1–3 are indicated by brackets and loxP sites are red triangles. (B) Southern blot validation of V6.5, hybrid C57BL6/129Sv, mouse ES cells used for blastocyst injection. Clone 1D1 was verified as heterozygous, a representative southern is shown. (C) Schematic and genotyping for Esrp1 CKO (floxed), KO, and WT alleles are shown. Primers are indicated by arrows and representative genotyping gels are presented. (D) Sequencing histogram of the KO PCR product confirms Cre-mediated recombination. VS: variable sequence, as part of the targeting construct. (E) Schematic of full gene replacement of Esrp2 by LacZ:PGK-Neo cassette generated by Knockout Mouse Project and purchased from Velocigene. A representative genotyping gel is presented.DOI:http://dx.doi.org/10.7554/eLife.08954.005

Mentions: To characterize the functions of Esrp1 and Esrp2 in vivo during mammalian development we generated mice with conditional and/or germline Esrp KO alleles. Our previous cell culture based systems indicated that Esrp1 was the primary driver of ESRP-regulated splicing, and thus we hypothesized Esrp1 would be required during development. We generated a conditional Esrp1 KO allele wherein exons 7–9 were floxed. Mice with germline Esrp1 KO were generating in crosses with mice carrying Zp3-Cre transgenes. Excision of exons 7–9 following Cre-mediated recombination removes the first RNA Recognition Motif (RRM), and places the remaining transcript out of frame, generating a allele of Esrp1 (Figure 1—figure supplement 2A–D). Since we did not anticipate that Esrp2 KO alone would yield a phenotype we obtained an Esrp2 KO mouse ES cell line from the Knockout Mouse Project (KOMP) (Figure 1—figure supplement 2E).


The splicing regulators Esrp1 and Esrp2 direct an epithelial splicing program essential for mammalian development.

Bebee TW, Park JW, Sheridan KI, Warzecha CC, Cieply BW, Rohacek AM, Xing Y, Carstens RP - Elife (2015)

Generation of Esrp1 and Esrp2 KO alleles.(A) Schematic of the knock-in strategy used for generation of the Esrp1 floxed allele for conditional and ubiquitous KO. The floxed neomycin cassette targeted exons 7–9. Restriction site for SacI (S) and HincII (H) are indicated. The RNA Recognition Motifs (RRMs) 1–3 are indicated by brackets and loxP sites are red triangles. (B) Southern blot validation of V6.5, hybrid C57BL6/129Sv, mouse ES cells used for blastocyst injection. Clone 1D1 was verified as heterozygous, a representative southern is shown. (C) Schematic and genotyping for Esrp1 CKO (floxed), KO, and WT alleles are shown. Primers are indicated by arrows and representative genotyping gels are presented. (D) Sequencing histogram of the KO PCR product confirms Cre-mediated recombination. VS: variable sequence, as part of the targeting construct. (E) Schematic of full gene replacement of Esrp2 by LacZ:PGK-Neo cassette generated by Knockout Mouse Project and purchased from Velocigene. A representative genotyping gel is presented.DOI:http://dx.doi.org/10.7554/eLife.08954.005
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4566030&req=5

fig1s2: Generation of Esrp1 and Esrp2 KO alleles.(A) Schematic of the knock-in strategy used for generation of the Esrp1 floxed allele for conditional and ubiquitous KO. The floxed neomycin cassette targeted exons 7–9. Restriction site for SacI (S) and HincII (H) are indicated. The RNA Recognition Motifs (RRMs) 1–3 are indicated by brackets and loxP sites are red triangles. (B) Southern blot validation of V6.5, hybrid C57BL6/129Sv, mouse ES cells used for blastocyst injection. Clone 1D1 was verified as heterozygous, a representative southern is shown. (C) Schematic and genotyping for Esrp1 CKO (floxed), KO, and WT alleles are shown. Primers are indicated by arrows and representative genotyping gels are presented. (D) Sequencing histogram of the KO PCR product confirms Cre-mediated recombination. VS: variable sequence, as part of the targeting construct. (E) Schematic of full gene replacement of Esrp2 by LacZ:PGK-Neo cassette generated by Knockout Mouse Project and purchased from Velocigene. A representative genotyping gel is presented.DOI:http://dx.doi.org/10.7554/eLife.08954.005
Mentions: To characterize the functions of Esrp1 and Esrp2 in vivo during mammalian development we generated mice with conditional and/or germline Esrp KO alleles. Our previous cell culture based systems indicated that Esrp1 was the primary driver of ESRP-regulated splicing, and thus we hypothesized Esrp1 would be required during development. We generated a conditional Esrp1 KO allele wherein exons 7–9 were floxed. Mice with germline Esrp1 KO were generating in crosses with mice carrying Zp3-Cre transgenes. Excision of exons 7–9 following Cre-mediated recombination removes the first RNA Recognition Motif (RRM), and places the remaining transcript out of frame, generating a allele of Esrp1 (Figure 1—figure supplement 2A–D). Since we did not anticipate that Esrp2 KO alone would yield a phenotype we obtained an Esrp2 KO mouse ES cell line from the Knockout Mouse Project (KOMP) (Figure 1—figure supplement 2E).

Bottom Line: Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development.Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease.Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, United States.

ABSTRACT
Tissue- and cell-type-specific regulators of alternative splicing (AS) are essential components of posttranscriptional gene regulation, necessary for normal cellular function, patterning, and development. Mice with ablation of Epithelial splicing regulatory protein (Esrp1) develop cleft lip and palate. Loss of both Esrp1 and its paralog Esrp2 results in widespread developmental defects with broad implications to human disease. Deletion of the Esrps in the epidermis revealed their requirement for establishing a proper skin barrier, a primary function of epithelial cells comprising the epidermis. We profiled the global Esrp-mediated splicing regulatory program in epidermis, which revealed large-scale programs of epithelial cell-type-specific splicing required for epithelial cell functions. These mice represent a valuable model for evaluating the essential role for AS in development and function of epithelial cells, which play essential roles in tissue homeostasis in numerous organs, and provide a genetic tool to evaluate important functional properties of epithelial-specific splice variants in vivo.

No MeSH data available.


Related in: MedlinePlus