Journal: bioRxiv

Article Title: ID1 and CEBPA Coordinate Epidermal Progenitor Cell Differentiation

doi: 10.1101/2022.03.09.483653

Figure Lengend Snippet: (A) Schematic representation of in utero lentiviral injections. Virus is injected into the amniotic cavity at E9.5 and embryos are collected between E14.5 and E18.5. (B) E14.5 ID1 immunoreactivity is reduced in H2BGFP reporter positive cells upon injection of lentiviral shId1, but not control shScr. (C) Id1 mRNA expression is reduced in epidermal progenitors targeted in vitro with shId1 compared to shScr . (D) The percentage of transduced H2BGFP-positive epidermal progenitors is reduced in shId1 targeted epidermis compared to shScr at E14.5. (E) Quantification of percentage of H2BGFP positive targeted K14 expressing basal progenitors and H2BGFP positive, K14 negative suprabasal keratinocytes indicate selective loss of shId1 targeted progenitor cells. (F) Total number of epidermal nuclei per 150um is significantly reduced in shId1 compared to shScr targeted epidermis at E16.5. (G) Quantification of K14-positive basal progenitor layer thickness in shId1 and shScr epidermis at E16.5. (H) Representative images of K14 thickness at E16.5 in shId1 and shScr epidermis. (I) K10-positive spinous layer thickens with epidermal development. Silencing of Id1 impairs spinous layer development. (J) Representative images of K10 distribution at E16.5 in shId1 and shScr epidermis. (K and L) Terminal differentiation is temporally and spatially normal in shId1 compared to shScr as judged by expression of differentiation marker TGM1 and INVOLUCRIN at E15.5. (M) Differentiation markers expression in shScr and shId1 cells. Statistical analysis is performed between shScr and shId1 within 0 or 96 hrs, respectively. Data are represented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 using multiple unpaired t test. Scale bars 50 μm.

Article Snippet: ID1 (Biocheck, #BCH-1, 1:500), Cleaved Caspase-3 (Cell Signaling Technology, #9661, 1:400), K10 (Biolegend, #905404, 1:1000), K5 (Biolegend, #905901, 1:1000), K14 (OriGene, #BP5009, 1:1000), Involucrin (Biolegend, #924401, 1:1000), TGM1 (Abcam, #ab103814, 1:1000), Flag (Sigma, F1804, 1:500), Cebpa (Cell Signaling Technology, #2295, 1:500).

Techniques: In Utero, Injection, Expressing, In Vitro, Marker

Journal: bioRxiv

Article Title: ID1 and CEBPA Coordinate Epidermal Progenitor Cell Differentiation

doi: 10.1101/2022.03.09.483653

Figure Lengend Snippet: (A) ID1 is induced in a doxycycline dependent manner and progenitor cells are differentiated and transcriptionally profiled. (B) Downregulated genes after ID1 overexpression and 24 hours of differentiation are linked to keratinocyte differentiation and regulation of signaling. (C) Spinous gene expression is reduced in epidermal progenitors upon ID1 overexpression. (D) Identification of ID1 gene signatures by merging expression data from shId1 and ID1 overexpression profiling of epidermal progenitors reveal a cohort of statistically significant genes (marked in purple) (LogFC >1 and FDR >0.05). Q1 and Q3 represent genes whose expression correlates to modulation of ID1. Q2 and Q4 represent genes that are only induced (Q2) or repressed (Q4) when ID1 levels are altered. (E) ID1 signature genes are functionally linked to adhesion and extracellular matrix modulation, differentiation or cell cycle regulation. (F) HOMER transcription factor binding motif analysis show distinct enrichment binding motifs in promoters (+400 bp) of genes found in Q1+Q3 compared to promoters in the expressed transcriptome. (G) bHLH motif enriched in Q1+Q3 gene promoters. (H) Immunoreactivity of CEBPA and K5 in E14.5 and E16.5 localizes CEBPA to suprabasal keratinocytes and scattered basal K5-positive progenitors. Arrows indicate CEBPA-positive basal cells. (I and J) Overexpression of CEBPA significantly reduces EdU incorporation in vitro . Quantification done after 3 days of doxycycline treatment. Scale bar 100 μm. (K and L) Proliferating K5-positive progenitors are largely CEBPA negative. Data are presented as mean ± SD. (M and N) K14/CEBPA double-positive progenitors are significantly enriched in shId1 targeted epidermis when compared to shScr . Arrows highlight K14/CEBPA positive cells. Data are represented as mean ± SEM. **p < 0.01 ***p < 0.001 using multiple unpaired t-test. Scale bars 50 μm except for J (100 μm).

Article Snippet: ID1 (Biocheck, #BCH-1, 1:500), Cleaved Caspase-3 (Cell Signaling Technology, #9661, 1:400), K10 (Biolegend, #905404, 1:1000), K5 (Biolegend, #905901, 1:1000), K14 (OriGene, #BP5009, 1:1000), Involucrin (Biolegend, #924401, 1:1000), TGM1 (Abcam, #ab103814, 1:1000), Flag (Sigma, F1804, 1:500), Cebpa (Cell Signaling Technology, #2295, 1:500).

Techniques: Over Expression, Expressing, Binding Assay, In Vitro