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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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Pdx1 is physiologically expressed in the adult mouse epidermis.A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows). Pdx1-Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1. Inclusion (iii) shows positive staining of pancreatic islet cells. Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii). B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin. Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion). Double immunofluorescence (iii) demonstrates that the majority of PDX1+ cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1+ cells can be found in the basal layer of the epidermis (arrow). Asterisks indicate unspecific staining of stratum corneum. C: Pdx1 expression in cultured keratinocytes is increased during Ca++-induced differentiation. Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro. D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14. The scale bars represent 50 µm.
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pone-0013578-g002: Pdx1 is physiologically expressed in the adult mouse epidermis.A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows). Pdx1-Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1. Inclusion (iii) shows positive staining of pancreatic islet cells. Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii). B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin. Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion). Double immunofluorescence (iii) demonstrates that the majority of PDX1+ cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1+ cells can be found in the basal layer of the epidermis (arrow). Asterisks indicate unspecific staining of stratum corneum. C: Pdx1 expression in cultured keratinocytes is increased during Ca++-induced differentiation. Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro. D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14. The scale bars represent 50 µm.

Mentions: The observation that Pdx1-Cre;Kras;N1ko mice develop skin neoplastic lesions with high penetrance and undergo Cre-mediated recombination are evidence of Cre expression in the epidermis possibly due to Pdx1-Cre transgene misexpression or physiological PDX1 expression in the skin. To test both hypotheses, immunohistochemical expression analysis was performed in the skin of wildtype and Pdx1-Cre mice, which showed a small subset of PDX1+ cells (Fig. 2A). Thus, the observed phenotype is due to physiological PDX1 expression in the skin rather than transgenic misexpression of Cre recombinase.


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)

Pdx1 is physiologically expressed in the adult mouse epidermis.A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows). Pdx1-Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1. Inclusion (iii) shows positive staining of pancreatic islet cells. Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii). B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin. Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion). Double immunofluorescence (iii) demonstrates that the majority of PDX1+ cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1+ cells can be found in the basal layer of the epidermis (arrow). Asterisks indicate unspecific staining of stratum corneum. C: Pdx1 expression in cultured keratinocytes is increased during Ca++-induced differentiation. Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro. D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14. The scale bars represent 50 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2962652&req=5

pone-0013578-g002: Pdx1 is physiologically expressed in the adult mouse epidermis.A: Immunohistochemical PDX1 staining of normal wildtype epidermis (i, ii) reveals that PDX1 is expressed in suprabasal keratinocytes (black arrowheads) and only rarely in basal cells (black arrows). Pdx1-Cre;Kras;N1ko papilloma (iii) is strongly positive for PDX1. Inclusion (iii) shows positive staining of pancreatic islet cells. Nuclei were contrastained with methyl green (i, ii) or hematoxilin (iii). B: Immunofluorescent PDX1 staining (i) indicates positive keratinocytes in the suprabasal (white arrowheads) and the basal (arrow) layer of the skin. Signal strength is comparable to that in duodenum cells (ii, arrowheads) and weaker than in pancreatic islet cells (ii, inclusion). Double immunofluorescence (iii) demonstrates that the majority of PDX1+ cells co-localize with a suprabasal marker Keratin10 (arrowheads) however, a small subset of PDX1+ cells can be found in the basal layer of the epidermis (arrow). Asterisks indicate unspecific staining of stratum corneum. C: Pdx1 expression in cultured keratinocytes is increased during Ca++-induced differentiation. Quantitative RT-PCR of Pdx1, Keratin10, Loricrin and p63 transcripts in induced primary keratinocytes in vitro. D: Schematic representation of PDX1 expression in the epidermal layers: (SC) Stratum Corneum, (GL) Granular Layer, (SL) Spinous Layer, (BL) Basal Layer, (BM) Basement Membrane, (D) Dermis and their markers: Loricrin, K1/10, K5/14. The scale bars represent 50 µm.
Mentions: The observation that Pdx1-Cre;Kras;N1ko mice develop skin neoplastic lesions with high penetrance and undergo Cre-mediated recombination are evidence of Cre expression in the epidermis possibly due to Pdx1-Cre transgene misexpression or physiological PDX1 expression in the skin. To test both hypotheses, immunohistochemical expression analysis was performed in the skin of wildtype and Pdx1-Cre mice, which showed a small subset of PDX1+ cells (Fig. 2A). Thus, the observed phenotype is due to physiological PDX1 expression in the skin rather than transgenic misexpression of Cre recombinase.

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