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Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo.

Mazur PK, Grüner BM, Nakhai H, Sipos B, Zimber-Strobl U, Strobl LJ, Radtke F, Schmid RM, Siveke JT - PLoS ONE (2010)

Bottom Line: Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function.Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.

View Article: PubMed Central - PubMed

Affiliation: 2nd Department of Internal Medicine, Klinikum Rechts der Isar University Hospital, Technical University of Munich, Munich, Germany.

ABSTRACT

Background: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D) mice with ablation of Notch1 and/or Notch2.

Methodology/principal findings: Surprisingly, mice with activated Kras(G12D) and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.

Conclusions/significance: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.

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Phenotype of K5;N1ko and K5;N2ko mice.A: Notch1 is expressed in all layers of the adult skin whereas Notch2 is expressed only in the suprabasal layer as assessed using immunohistochemical staining and Notch1-GFP and Notch2LacZ reporter mice. B: Gross phenotype of K5;N1ko, K5;N2ko and WT mice at 4 weeks of age (left). Spontaneous skin tumors (white arrows) and hyperplastic opaque corneas (black arrowhead) start to develop in 9 months old K5;N1ko mice (middle and right). C: Skin histopathologies of K5;N1ko mice include epidermal cyst (asterisk), hair follicle malformation (black arrowhead, left), skin tumors (middle), hyperplasia of the cornea (black arrows, right). D: HE stain shows morphology and thickness (indicated by scale lines) of WT, K5;N1ko, K5;N2ko epidermis (left panel). Immunohistochemical staining reveals ubiquitous expression of active β-catenin in K5;N1ko (black arrows) comparing to WT and K5;N2ko mice epidermis (right panel). E: The thickness of K5;N1ko epidermis is significantly reduced compared to K5;N2ko and WT. The scale bars represent 50 µm.
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pone-0013578-g005: Phenotype of K5;N1ko and K5;N2ko mice.A: Notch1 is expressed in all layers of the adult skin whereas Notch2 is expressed only in the suprabasal layer as assessed using immunohistochemical staining and Notch1-GFP and Notch2LacZ reporter mice. B: Gross phenotype of K5;N1ko, K5;N2ko and WT mice at 4 weeks of age (left). Spontaneous skin tumors (white arrows) and hyperplastic opaque corneas (black arrowhead) start to develop in 9 months old K5;N1ko mice (middle and right). C: Skin histopathologies of K5;N1ko mice include epidermal cyst (asterisk), hair follicle malformation (black arrowhead, left), skin tumors (middle), hyperplasia of the cornea (black arrows, right). D: HE stain shows morphology and thickness (indicated by scale lines) of WT, K5;N1ko, K5;N2ko epidermis (left panel). Immunohistochemical staining reveals ubiquitous expression of active β-catenin in K5;N1ko (black arrows) comparing to WT and K5;N2ko mice epidermis (right panel). E: The thickness of K5;N1ko epidermis is significantly reduced compared to K5;N2ko and WT. The scale bars represent 50 µm.

Mentions: Although the role of Notch receptors in the skin has already been intensively studied [12]–[17], we aimed to characterize epidermal Notch1 and Notch2 deficiency in our model. To do so, Notch1fl/fl [21] and Notch2fl/fl [22] mice were crossed with basal keratinocyte-specific Keratin5-Cre mice [35] (named K5;N1ko and K5;N2ko respectively). These mice were born at the expected Mendelian ratio (Fig. 5B) and successful recombination of the floxed loci was confirmed in isolated primary keratinocytes by immunoblot (Fig. 6A).


Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo.

Mazur PK, Grüner BM, Nakhai H, Sipos B, Zimber-Strobl U, Strobl LJ, Radtke F, Schmid RM, Siveke JT - PLoS ONE (2010)

Phenotype of K5;N1ko and K5;N2ko mice.A: Notch1 is expressed in all layers of the adult skin whereas Notch2 is expressed only in the suprabasal layer as assessed using immunohistochemical staining and Notch1-GFP and Notch2LacZ reporter mice. B: Gross phenotype of K5;N1ko, K5;N2ko and WT mice at 4 weeks of age (left). Spontaneous skin tumors (white arrows) and hyperplastic opaque corneas (black arrowhead) start to develop in 9 months old K5;N1ko mice (middle and right). C: Skin histopathologies of K5;N1ko mice include epidermal cyst (asterisk), hair follicle malformation (black arrowhead, left), skin tumors (middle), hyperplasia of the cornea (black arrows, right). D: HE stain shows morphology and thickness (indicated by scale lines) of WT, K5;N1ko, K5;N2ko epidermis (left panel). Immunohistochemical staining reveals ubiquitous expression of active β-catenin in K5;N1ko (black arrows) comparing to WT and K5;N2ko mice epidermis (right panel). E: The thickness of K5;N1ko epidermis is significantly reduced compared to K5;N2ko and WT. The scale bars represent 50 µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2962652&req=5

pone-0013578-g005: Phenotype of K5;N1ko and K5;N2ko mice.A: Notch1 is expressed in all layers of the adult skin whereas Notch2 is expressed only in the suprabasal layer as assessed using immunohistochemical staining and Notch1-GFP and Notch2LacZ reporter mice. B: Gross phenotype of K5;N1ko, K5;N2ko and WT mice at 4 weeks of age (left). Spontaneous skin tumors (white arrows) and hyperplastic opaque corneas (black arrowhead) start to develop in 9 months old K5;N1ko mice (middle and right). C: Skin histopathologies of K5;N1ko mice include epidermal cyst (asterisk), hair follicle malformation (black arrowhead, left), skin tumors (middle), hyperplasia of the cornea (black arrows, right). D: HE stain shows morphology and thickness (indicated by scale lines) of WT, K5;N1ko, K5;N2ko epidermis (left panel). Immunohistochemical staining reveals ubiquitous expression of active β-catenin in K5;N1ko (black arrows) comparing to WT and K5;N2ko mice epidermis (right panel). E: The thickness of K5;N1ko epidermis is significantly reduced compared to K5;N2ko and WT. The scale bars represent 50 µm.
Mentions: Although the role of Notch receptors in the skin has already been intensively studied [12]–[17], we aimed to characterize epidermal Notch1 and Notch2 deficiency in our model. To do so, Notch1fl/fl [21] and Notch2fl/fl [22] mice were crossed with basal keratinocyte-specific Keratin5-Cre mice [35] (named K5;N1ko and K5;N2ko respectively). These mice were born at the expected Mendelian ratio (Fig. 5B) and successful recombination of the floxed loci was confirmed in isolated primary keratinocytes by immunoblot (Fig. 6A).

Bottom Line: Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function.Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.

View Article: PubMed Central - PubMed

Affiliation: 2nd Department of Internal Medicine, Klinikum Rechts der Isar University Hospital, Technical University of Munich, Munich, Germany.

ABSTRACT

Background: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D) mice with ablation of Notch1 and/or Notch2.

Methodology/principal findings: Surprisingly, mice with activated Kras(G12D) and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and β-catenin signaling inhibition capabilities.

Conclusions/significance: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.

Show MeSH
Related in: MedlinePlus