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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

Conditional KO of the Esrps in the epidermis result in skin barrier defects.(A) Images of P0.5 Krt14-CON and Krt14-DKO pups, which present with dry flaky skin or red shinny skin. (B) qRT-PCR validation of Keratin14-Cre mediated KO of the floxed Esrp1 targeted cassette in isolated Krt14-CON (n = 5) and Krt14-DKO (n = 6) epidermis. (C) Western validation of Esrp1 protein KO in Krt14-Cre mice harboring floxed Esrp1 alleles (n = 2). (D) Hot RT-PCR gel images of Esrp SE targets, confirming Krt14-DKO epidermis have defects in Esrp-regulated splicing targets (n = 2). (E) Water loss assay for barrier defect in P0.5 pups. Fraction of initial weight is graphed over 5 hr in 30 min intervals. Multiple t-tests analysis indicate significant differences between the Krt14-CON (n = 16) and Krt14-DKO (n = 7) pups. (F) qRT-PCR validation of nine EDC genes identified with increased expression in DKO samples, and down regulation of three genes including the AS-NMD target, Grhl1 (n = 4).DOI:http://dx.doi.org/10.7554/eLife.08954.024
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fig7: Conditional KO of the Esrps in the epidermis result in skin barrier defects.(A) Images of P0.5 Krt14-CON and Krt14-DKO pups, which present with dry flaky skin or red shinny skin. (B) qRT-PCR validation of Keratin14-Cre mediated KO of the floxed Esrp1 targeted cassette in isolated Krt14-CON (n = 5) and Krt14-DKO (n = 6) epidermis. (C) Western validation of Esrp1 protein KO in Krt14-Cre mice harboring floxed Esrp1 alleles (n = 2). (D) Hot RT-PCR gel images of Esrp SE targets, confirming Krt14-DKO epidermis have defects in Esrp-regulated splicing targets (n = 2). (E) Water loss assay for barrier defect in P0.5 pups. Fraction of initial weight is graphed over 5 hr in 30 min intervals. Multiple t-tests analysis indicate significant differences between the Krt14-CON (n = 16) and Krt14-DKO (n = 7) pups. (F) qRT-PCR validation of nine EDC genes identified with increased expression in DKO samples, and down regulation of three genes including the AS-NMD target, Grhl1 (n = 4).DOI:http://dx.doi.org/10.7554/eLife.08954.024

Mentions: The increase in EDC gene expression in the epidermis of DKO embryos suggested a possible defect in skin barrier function. However, as the Esrp1/Esrp2 DKO mice are not viable at birth we were unable to analyze postnatal barrier defects in DKO mice. Therefore, we utilized a conditional KO approach using transgenic Keratin14-Cre (Krt14-Cre) mice (Andl et al., 2004). This Krt14-Cre transgene is first expressed at E11.5 in surface epithelium and maintains expression in basal layer keratinocytes of stratified epidermis (Zhang et al., 2008). Because basal keratinocytes undergo differentiation to give rise to all epithelial cells of the interfollicular epidermis, Krt14-Cre effectively induces KO in all epithelial cell layers of stratified epidermis. We generated control mice harboring at least one WT Esrp1 allele (Esrp1flox/+, Esrp2−/−, Krt14-Cre Tg− or Tg+) and littermate Krt14-DKO (Esrp1flox/flox, Esrp2−/−, Krt14-Cre Tg+) mice. Krt14-DKO neonates were viable at birth (P0.5), however no Krt14-DKO pups with confirmed Esrp1 KO survived to the following day (P1.5). While the early neonatal lethality in the Krt14-DKO mice precluded long term evaluation of barrier function, we were able to evaluate skin barrier function by means of a standard water loss assay, which measures the ability of a neonatal mouse epidermis to retain fluid and prevent dehydration by loss of water through the skin (Gladden et al., 2010). Litters of conditional KO neonates were isolated from their mother at P0, and weighed every 30 min for 5 hr. During the time-course control littermates lost weight at expected levels, but never exceeded ∼1.5% of total body weight, indicative of an intact epidermal barrier. However, Krt14-DKO pups lost significantly more weight (∼2.5–3% of total body weight) compared to littermate controls during the same time period (Figure 7E). The Krt14-Cre DKO mice that exhibited weight loss during the water loss assay also displayed skin phenotypes observed in other mouse KO models associated with skin barrier defects: thin red shinny skin with areas of dry flaky skin (Figure 7A) (Koch et al., 2000; Gladden et al., 2010).10.7554/eLife.08954.024Figure 7.Conditional KO of the Esrps in the epidermis result in skin barrier defects.


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)

Conditional KO of the Esrps in the epidermis result in skin barrier defects.(A) Images of P0.5 Krt14-CON and Krt14-DKO pups, which present with dry flaky skin or red shinny skin. (B) qRT-PCR validation of Keratin14-Cre mediated KO of the floxed Esrp1 targeted cassette in isolated Krt14-CON (n = 5) and Krt14-DKO (n = 6) epidermis. (C) Western validation of Esrp1 protein KO in Krt14-Cre mice harboring floxed Esrp1 alleles (n = 2). (D) Hot RT-PCR gel images of Esrp SE targets, confirming Krt14-DKO epidermis have defects in Esrp-regulated splicing targets (n = 2). (E) Water loss assay for barrier defect in P0.5 pups. Fraction of initial weight is graphed over 5 hr in 30 min intervals. Multiple t-tests analysis indicate significant differences between the Krt14-CON (n = 16) and Krt14-DKO (n = 7) pups. (F) qRT-PCR validation of nine EDC genes identified with increased expression in DKO samples, and down regulation of three genes including the AS-NMD target, Grhl1 (n = 4).DOI:http://dx.doi.org/10.7554/eLife.08954.024
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fig7: Conditional KO of the Esrps in the epidermis result in skin barrier defects.(A) Images of P0.5 Krt14-CON and Krt14-DKO pups, which present with dry flaky skin or red shinny skin. (B) qRT-PCR validation of Keratin14-Cre mediated KO of the floxed Esrp1 targeted cassette in isolated Krt14-CON (n = 5) and Krt14-DKO (n = 6) epidermis. (C) Western validation of Esrp1 protein KO in Krt14-Cre mice harboring floxed Esrp1 alleles (n = 2). (D) Hot RT-PCR gel images of Esrp SE targets, confirming Krt14-DKO epidermis have defects in Esrp-regulated splicing targets (n = 2). (E) Water loss assay for barrier defect in P0.5 pups. Fraction of initial weight is graphed over 5 hr in 30 min intervals. Multiple t-tests analysis indicate significant differences between the Krt14-CON (n = 16) and Krt14-DKO (n = 7) pups. (F) qRT-PCR validation of nine EDC genes identified with increased expression in DKO samples, and down regulation of three genes including the AS-NMD target, Grhl1 (n = 4).DOI:http://dx.doi.org/10.7554/eLife.08954.024
Mentions: The increase in EDC gene expression in the epidermis of DKO embryos suggested a possible defect in skin barrier function. However, as the Esrp1/Esrp2 DKO mice are not viable at birth we were unable to analyze postnatal barrier defects in DKO mice. Therefore, we utilized a conditional KO approach using transgenic Keratin14-Cre (Krt14-Cre) mice (Andl et al., 2004). This Krt14-Cre transgene is first expressed at E11.5 in surface epithelium and maintains expression in basal layer keratinocytes of stratified epidermis (Zhang et al., 2008). Because basal keratinocytes undergo differentiation to give rise to all epithelial cells of the interfollicular epidermis, Krt14-Cre effectively induces KO in all epithelial cell layers of stratified epidermis. We generated control mice harboring at least one WT Esrp1 allele (Esrp1flox/+, Esrp2−/−, Krt14-Cre Tg− or Tg+) and littermate Krt14-DKO (Esrp1flox/flox, Esrp2−/−, Krt14-Cre Tg+) mice. Krt14-DKO neonates were viable at birth (P0.5), however no Krt14-DKO pups with confirmed Esrp1 KO survived to the following day (P1.5). While the early neonatal lethality in the Krt14-DKO mice precluded long term evaluation of barrier function, we were able to evaluate skin barrier function by means of a standard water loss assay, which measures the ability of a neonatal mouse epidermis to retain fluid and prevent dehydration by loss of water through the skin (Gladden et al., 2010). Litters of conditional KO neonates were isolated from their mother at P0, and weighed every 30 min for 5 hr. During the time-course control littermates lost weight at expected levels, but never exceeded ∼1.5% of total body weight, indicative of an intact epidermal barrier. However, Krt14-DKO pups lost significantly more weight (∼2.5–3% of total body weight) compared to littermate controls during the same time period (Figure 7E). The Krt14-Cre DKO mice that exhibited weight loss during the water loss assay also displayed skin phenotypes observed in other mouse KO models associated with skin barrier defects: thin red shinny skin with areas of dry flaky skin (Figure 7A) (Koch et al., 2000; Gladden et al., 2010).10.7554/eLife.08954.024Figure 7.Conditional KO of the Esrps in the epidermis result in skin barrier defects.

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