title: Cellular heterogeneity during embryonic stem cell differentiation to epiblast stem cells is revealed by the ShcD/RaLP adaptor protein.
abstract: The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naïve (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity.
authors.affiliation: Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.
authors.forename: Margherita Y
authors.initials: MY
authors.lastname: Turco
authors.forename: Laura
authors.initials: L
authors.lastname: Furia
authors.forename: Anja
authors.initials: A
authors.lastname: Dietze
authors.forename: Luis
authors.initials: L
authors.lastname: Fernandez Diaz
authors.forename: Simona
authors.initials: S
authors.lastname: Ronzoni
authors.forename: Anna
authors.initials: A
authors.lastname: Sciullo
authors.forename: Antonio
authors.initials: A
authors.lastname: Simeone
authors.forename: Daniel
authors.initials: D
authors.lastname: Constam
authors.forename: Mario
authors.initials: M
authors.lastname: Faretta
authors.forename: Luisa
authors.initials: L
authors.lastname: Lanfrancone
mesh_terms: D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis; D000071616:CDX2 Transcription Factor; D053148:Caspase 3 / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D018920:Coculture Techniques; D047108:Embryonic Development; D053595:Embryonic Stem Cells / Q000378:metabolism / Q000502:physiology*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015870:Gene Expression; D018507:Gene Expression Regulation, Developmental; D005855:Germ Layers / Q000166:cytology*; D018398:Homeodomain Proteins; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism*; D059008:Time-Lapse Imaging; D014157:Transcription Factors
chemical_list: D000943:Antigens, Differentiation; D000071616:CDX2 Transcription Factor; C487514:Cdx2 protein, mouse; D018398:Homeodomain Proteins; D050814:Octamer Transcription Factor-3; D010750:Phosphoproteins; C494174:Pou5f1 protein, mouse; D055765:Shc Signaling Adaptor Proteins; C521729:Shc4 protein, mouse; D014157:Transcription Factors; D048049:Extracellular Signal-Regulated MAP Kinases; C505409:Casp3 protein, mouse; D053148:Caspase 3