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Sox4 Links Tumor Suppression to Accelerated Aging in Mice by Modulating Stem Cell Activation M. Foronado, P. Martinez, S. Schoeftner, G. Gomez-Lopez, R. Schneider, J.M. Flores, D.G. Pisano, and M.A. Blasco Cell Reports 8: 1-14 (2014) Intro: What was known before the work described in the paper? -adult organs maintained through balance of proliferation, differentiation, and self-renewal of stem cells -alterations in the balance between these in cancer and aging Epidermis: Interfollicular Epidermis (IFE) Sebaceous Glands (SG) Hair Follicles (HF) Dividing Cells: Hair Follicle Stem Cells (HFSC) in Hair Bulge and HG Dermis secretes signaling molecules that regulate HFSC proliferation, differentiation, and self-renewal through Wnt/β catenin pathway and Sox4 transcription factor Fig. 7.1 Sox4 transcription factor -belongs to family of SRY box-containing transcription factors that bind and induce bending in specific DNA sequences -expressed mainly during embryogenesis in mesenchymal cells -in adults, expression & function is restricted to progenitor cell populations in neuronal, cardiac, immune and blood cell lineages -overexpressed in all major cancer types, correlates with metastatic potential -Sox4 null mutant mice die in utero at day 14.5 New Question? What phenotype results from Sox4-knockout in a specific tissue only (conditional Knock Out or cKO)? Materials & Methods: Were any new materials or methods developed specifically for this study? Why were they needed? Mouse conditional knock-out technology through Cre/loxP System Sox4lox/lox Skin HFSC or HG cell Sox4 Sox4cKO in skin progenitor cell Cre is a recombination enzyme that acts specifically at loxP sites. Mouse cell-type specific promoters used to drive expression in a specific tissue where gene deletion is desired. Other tissues remain non-mutant, avoiding whole animal lethality. How were the Sox4lox/lox and Cre Transgenic Mice Made? Add DNA w/ selectable marker (inserts into genome in 1/104 ES cells) Blastocysts w/ transfected ES cells develop into adults, some carrying transfected DNA in GERMLINE Fig. 18.49 Remove ES cells from Blastocyst and culture in vitro Inject transfected ES cells into Blastocyst and implant in mouse Results: What question is being addressed in each figure, what experiment was used to answer that question, and how were the results interpreted? Fig. 1 Sox4lox/lox Mice Display Cancer Resistance & Accelerated Aging Question: Phenotype in Sox4lox/lox mice (knock-down intermediate)? A) B) C) D) E) F) G) H) I) J) YES Strategy for making Sox4lox/lox PCR confirmation that it was made Sox4lox/lox smaller in size Sox4lox/lox smaller in weight Sox4lox/lox has lower survival in wks and Humane End Point Sox4lox/lox has lower Bone Density Sox4lox/lox has higher Hernia Rate Sox4lox/lox has lower mean telomere length in blood cells Sox4lox/lox has lower spontaneous cancer rate Reduced Sox4 expression in all tissues of Sox4lox/lox (lox sites flanking gene must interfere with its transcription) Fig. 2 Normal Skin Stratification in Sox4cKO, but Bulge Cells Have Reduced Replicative History A) B) C) D) E) Normal hair coat appearance Sox4 expression absent in SoxcKO mice Expression of skin-specific differentiation genes normal Telomeres longer in HFSCs in Bulge of Sox4cKO mice (reduced replicative history) 5% HFSC telomeres < 50 a.u.f.; 95% > 800 a.u.f. Fig. 3 Sox4 is Induced after Hair Plucking and Required for Normal Hair Regeneration A) Induced genes following hair plucking include Sox 9 B) and Sox 4 C) Hair regeneration reduced in Sox4cKO relative to Sox4wt Fig. 4 Sox4 is Required for Normal HFSC Activation during Hair Regeneration and Wound Healing A-H) Decrease in proliferating cells as evidenced by decreased Ki67 (proliferation marker-rRNA txn) & increased γH2AX, p53 (cell cycle arrest markers) in Sox4cKO I) Reduced number of large, differentiated colonies in cultured keratinocytes from Sox4lox/lox or Sox4cKO mice J) Reduced wound healing in SoxcKO mice Fig. 5 Sox 4 is Required for Induction of Proliferative and Differentiation Pathways during Post-Plucking Telogen > Anagen B) Microarray mRNA expression profiling of Sox4wt vs Sox4cKO after plucking . Differentially Expressed Genes (DEGs) include known Sox4 targets) C) PCR confirmation of microarray E) Most DEGs specific to Telogen vs. Anagen difference (stimulated by plucking) Gene Set Enrichment Analysis compares results from other gene expression studies, revealed enrichment of genes for cell cycle regulation, DNA repair, and Wnt/β catenin signaling F) Heat maps of top 20 DEGs in these pathways in Soxwt vs SoxcKO G) FDR(False Discovery Rate): Likelihood that similarity between sets is random Induced Proliferation Fig. 6 Sox4 is Required for Oncogenic Transformation A) Sox4lox/lox MEFs are resistant to oncogenic transformation by activated Ras (RasG12V) B-D) DMBA/TPA mutagenesis: Sox4cKO resistant to skin tumor formation relative to wt (and size of tumors formed smaller) E) Sox4cKO/- and Sox4lox/lox (partial loss of function) also resistant to skin tumor formation relative to wt (and size of tumors formed smaller) Cells recovered from each genotype at time prior to tumor formation had less proliferation markers (Ki67 & P-H3) in SoxcKO relative to wt Discussion: What did we learn from the study? But also needed for oncogenic transformation Stress Proliferation Sox4 needed to maintain stem cell populations for skin tissue homeostasis & regeneration Delicate Balance Required Are there any caveats to the conclusions? What new or remaining questions are there?