Transcriptomic Analysis of Mouse Embryonic Skin Cells Reveals Previously Unreported Genes Expressed in Melanoblasts Sophie Colombo, Delphine Champeval,

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Presentation transcript:

Transcriptomic Analysis of Mouse Embryonic Skin Cells Reveals Previously Unreported Genes Expressed in Melanoblasts Sophie Colombo, Delphine Champeval, Florian Rambow, Lionel Larue Journal of Investigative Dermatology Volume 132, Issue 1, Pages 170-178 (January 2012) DOI: 10.1038/jid.2011.252 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Dissociation and isolation of melanoblasts. Dissociated cells from Tyr::Cre/°; °/° (a–c) and Tyr::Cre/°; ZEG/° (d–f) epidermis observed under an inverted fluorescent microscope (Leica, Wetzlar, Germany). Green fluorescent protein (GFP) is observed only in defloxed cells from Tyr::Cre; ZEG/° embryos (d–f) and no GFP is observed in non-defloxed cells from Tyr::Cre/°; °/° embryos (a–c). FACS dot plots of cells isolated from Tyr::Cre/°; °/° (g) and Tyr::Cre/°; ZEG/° (h) epidermis. Fluorescence observed on the FITC-A channel shows GFP+ cells only in Tyr::Cre/°; ZEG/° cell population. SSC-A, side scatter area. a,b,d,e bar=30μm. Journal of Investigative Dermatology 2012 132, 170-178DOI: (10.1038/jid.2011.252) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Quality analysis of RNA and amplified cDNA. (a, b) The quality of the RNA was analyzed with the 2100 Bioanalyzer (Agilent) using an RNA 6000 Pico chip. An example of RNA obtained from green fluorescent protein (GFP)+ (a) and GFP- (b) cells from one representative experiment at E16.5 is illustrated. The RIN (RNA integrity number) was generally between 7.7 and 10. (c, d) The quality of the cDNA obtained after reverse transcription and amplification was also analyzed with the 2100 Bioanalyzer and an RNA 6000 Nano chip. The profiles of the amplified cDNA were, in all cases, similar, with most fragments being 200bp to 2kb long. Journal of Investigative Dermatology 2012 132, 170-178DOI: (10.1038/jid.2011.252) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Quality analysis of cell-sorting specificity. (a) The purity of the green fluorescent protein (GFP)+ and GFP- cell populations sorted by FACS was tested by PCR using amplified cDNA to detect GFP transcripts (499bp). One typical experiment at each E15.5 and E16.5 stages is presented. The GFP mRNA is only present in the GFP+ cell population. The positive and negative controls were cDNA from total skin of Tyr::Cre/°; ZEG/°; and Tyr::Cre/°; °/° mice, respectively. (b) The specificity of sorting was also verified by semiquantitative real-time PCR with amplified cDNA. One representative experiment at E16.5 is presented. Mitf-M was expressed only in GFP+ cells, all of which were melanoblasts, whereas LacZ was only expressed in GFP cells, which were the non-defloxed cells. Journal of Investigative Dermatology 2012 132, 170-178DOI: (10.1038/jid.2011.252) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Comparative transcriptomic analysis of E15.5 GFP+ and GFP– cells. The cDNAs were fragmented, labeled, and hybridized on whole-mouse genome arrays (Affymetrix, Santa Clara, CA). (a) The 50 probe sets (each corresponding to one gene) displaying the highest green fluorescent protein (GFP)+/GFP- ratio of fluorescence intensity are presented. The genes Tyr, Ptgds, Ednrb, and Ap1s2 are present several times because several probe sets corresponding to these genes displayed a high ratio. Several independent experiments were performed with E15.5 GFP+ and GFP- cells and analyzed. (b, c) Scatter plots showing the intensity of fluorescence of GFP+ cells (from 200 to 800 for b, and 400 to 2,900 for c) versus the intensity of fluorescence of GFP- cells are presented (from 200 to 800 for b, and 400 to 2,900 for c). Note the inset in panel c presenting data for the Dct gene, with an intensity of fluorescence of 134 for GFP- cells and of 4,326 for GFP+ cells. Journal of Investigative Dermatology 2012 132, 170-178DOI: (10.1038/jid.2011.252) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Quantitative real-time RT-PCR analysis of E15.5 GFP+ and GFP- cells. Semiquantitative real-time PCR was used to assay mRNA in E15.5 GFP+ cells (melanoblasts from three independent experiments) and E15.5 GFP- cells (mostly keratinocytes (KCs) from three independent experiments). Melb-a cells, melan-a cells, B16F10 cells, and adult murine intestine cells were used as controls. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) was used as an internal control. (a) PLP1 (b) Fabp7, (c) Zeb2, (d) Shc4, and (e) Sox11. Journal of Investigative Dermatology 2012 132, 170-178DOI: (10.1038/jid.2011.252) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions