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Epsins Regulate Mouse Embryonic Stem Cell Exit from Pluripotency and Neural Commitment by Controlling Notch Activation - PubMed

  • ️Tue Jan 01 2019

Epsins Regulate Mouse Embryonic Stem Cell Exit from Pluripotency and Neural Commitment by Controlling Notch Activation

Marina Cardano et al. Stem Cells Int. 2019.

Abstract

Epsins are part of the internalization machinery pivotal to control clathrin-mediated endocytosis. Here, we report that epsin family members are expressed in mouse embryonic stem cells (mESCs) and that epsin1/2 knockdown alters both mESC exits from pluripotency and their differentiation. Furthermore, we show that epsin1/2 knockdown compromises the correct polarization and division of mESC-derived neural progenitors and their conversion into expandable radial glia-like neural stem cells. Finally, we provide evidence that Notch signaling is impaired following epsin1/2 knockdown and that experimental restoration of Notch signaling rescues the epsin-mediated phenotypes. We conclude that epsins contribute to control mESC exit from pluripotency and allow their neural differentiation by appropriate modulation of Notch signaling.

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Figures

Figure 1
Figure 1

EPN1 and EPN2 are expressed in mESCs. (a) mESCs express EPN1 and EPN2. Western blot analysis shows EPN1 and EPN2 expression in mESCs. β-Actin was used as the loading control. (b, c) Representative immunofluorescence images of (b) EPN1 and EPN2 and (c) clathrin (heavy chain) localization in mESC: both EPNs and clathrin are localized as punctuated dots at the plasma membrane and in the cytoplasm, a pattern typical of endocytic proteins. Nuclei are counterstained with Hoechst 33258. For (a, b, and c), n = 3 biologically independent experiments.

Figure 2
Figure 2

EPN1 and EPN2 silencing in mESCs impairs Notch pathway activation. (a) EPN1 and EPN2 KD does not affect clathrin levels. Western blot assay shows comparable clathrin levels in shCTRL and shEPN cells. β-Actin was used as the loading control. (b) EPN KD does not affect clathrin-mediated endocytosis. Representative images of clathrin-mediated uptake of Alexa488-transferrin in shCTRL and EPN1 and EPN2 KD cultures. Nuclei are counterstained with Hoechst 33258. (c) EPN KD impairs Notch activation. EPN KD abolishes uptake of N1FC and reduces NICD levels. Nuclei are counterstained with Hoechst 33258. β-Actin was used as the loading control. (d) EPN1 and EPN2 KD result in reduced expression levels of Notch-regulated target genes. qRT-PCR analysis of HES1 and p21 in shEPN1 and shEPN2 KD mESCs. Levels are normalized over the control line (shCTRL), using β-actin as the housekeeping gene. All data are expressed as the means ± STDV (n = 3 biologically independent experiments). Statistical significance (unpaired t-test): ∗ p < 0.05 and ∗∗ p < 0.001.

Figure 3
Figure 3

EPN1 and EPN2 KD affects pluripotency and stimulates mESC differentiation. (a) EPN1 and EPN2 silencing in mESCs impairs colony formation capability. Colony formation assays show that EPN1 and EPN2 silencing results in an increase of colonies with low alkaline phosphatase activity and a flat, differentiated morphology. Values are normalized over the control line (shCTRL). (b) EPN1 and EPN2 silencing in mESCs induces nestin upregulation upon LIF withdrawal. Representative immunofluorescence analysis for nestin expression on self-renewing or LIF-deprived EPN1 and EPN2 KD mESCs. Nuclei are counterstained with Hoechst 33258. (c) EPN1 and EPN2 KD results in a significant reduction of OCT4+ve cells after 72 hours of LIF withdrawal. Values are normalized over the control line (shCTRL). (d) qRT-PCR analysis showing relative expression of pluripotency (Nanog and OCT4), neuroectodermal (nestin), mesodermal (Brachyury), and endodermal (FOXA-2) markers in EPNs silenced 46C mESCs. Data are normalized over the control line (shCTRL), using β-actin as the housekeeping gene. (e) Representative immunofluorescence and western blot analyses show the decrease of E-cadherin in EPN1 and EPN2 KD mESCs. α-Tubulin is used as the loading control, and nuclei are counterstained with Hoechst 33258. All data are expressed as the means ± STDV (n = 3 biologically independent experiments). Statistical significance (unpaired t-test): ∗ p < 0.05 and ∗∗ p < 0.001.

Figure 4
Figure 4

EPN1 and EPN2 silencing affects neuroepithelial cells polarization and neural rosette lumen size. (a) Nestin immunofluorescence analysis reveals impaired neuroectoderm polarization and neural rosette lumen size in 7 day-neuralized EPN1 and EPN2 KD 46C mESC cultures. (b, c) Immunofluorescence analysis and relative size quantification of N-cadherin+ve lumens show that EPN1 KD and EPN2 KD induce a decrease in the percentage of large-lumen rosettes. (d) After 11 days of exposure to neuralizing conditions, EPN1 and EPN2 KD mESC cultures show an increase in βIII-tubulin+ve and MAP 2+ve neurons as scored by immunofluorescence analysis. Nuclei are counterstained with Hoechst 33258. All data are expressed as the means ± STDV ((a, d): n = 3 biologically independent experiments; (b, c): n = 5 biologically independent experiments). Statistical significance (unpaired t-test): ∗ p < 0.05.

Figure 5
Figure 5

NICD overexpression rescues EPN1 and EPN2 KD phenotype. (a) NICD overexpression in EPN1 and EPN2 KD ESCs can rescue the regular and well-polarized rosette morphology during neuralization. eGFP signal indicates either mock or NICD-transfected cells. (b) Immunofluorescence analyses on mESC-derived NS cells show that NICD overexpression allows the obtainment of expandable undifferentiated EPN1 and EPN2 KD cultures (no or very few GFAP, βIII-tubulin, and MAP 2-positive cells), homogeneously immunopositive for SOX-2, nestin, and PAX6 and with similar proliferation rate (pH 3 expression). For (a, b), n = 3 biologically independent experiments.

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