myc maintains embryonic stem cell pluripotency and self-renewal

Abstract

While endogenous Myc (c-myc) and Mycn (N-myc) have been reported to be separately dispensable for murine embryonic stem cell (mESC) function, myc greatly enhances induced pluripotent stem (iPS) cell formation and overexpressed c-myc confers LIF-independence upon mESC. To address the role of myc genes in ESC and in pluripotency generally, we conditionally knocked out both c- and N-myc using myc doubly homozygously floxed mESC lines (cDKO). Both lines of myc cDKO mESC exhibited severely disrupted self-renewal, pluripotency, and survival along with enhanced differentiation. Chimeric embryos injected with DKO mESC most often completely failed to develop or in rare cases survived but with severe defects. The essential nature of myc for self-renewal and pluripotency is at least in part mediated through orchestrating pluripotency-related cell cycle and metabolic programs. This study demonstrates that endogenous myc genes are essential for mESC pluripotency and self-renewal as well as providing the first evidence that myc genes are required for early embryogenesis, suggesting potential mechanisms of myc contribution to iPS cell formation.

Fig. 1

Loss of c- and N-myc disrupts mESC growth and triggers lineage commitment. (A) Schematic of the floxed c- and N-myc loci. (B) Detection of c- and N-myc gene copy number changes by real-time qPCR. Gene copy numbers for c- and N-myc were normalized using two reference genes, β-actin and Nat1. Error bars are standard deviations. Decreases in c- and N-myc copy numbers had p values of 8.3×10⁻⁷ and 7.2×10⁻⁷, respectively, in cDKO2, and p values of 1.6×10⁻⁷ and 2.4×10⁻⁵ in cDKO3, respectively. (C) Phase contrast image of mESC lines in the presence or absence of CRE expression. Arrow denotes differentiated colony. GFP and GFP-Cre denoted cell cultures transduced with MSCV ires-GFP and MSCV Cre-ires-GFP retroviruses, respectively. (D) Real-time qRT-PCR of c-myc, N-myc, and a series of different lineage-specific marker genes in WT and DKO mESC lines. Levels of specific mRNAs measured by qRT-PCR were normalized to the levels of the loading control Nat1. Error bars are standard deviations. Changes in relative expression of N-myc, c-myc, Ngn3, Gata6, Bmp4, and Fgf5 had p values of 7×10⁻⁴, 2×10⁻⁴, 1×10⁻³, 8.6×10⁻⁵, 9×10⁻⁴, and 9.6×10⁻⁴, respectively, in cDKO2 and p values of 8.7×10⁻⁵, 1.4×10⁻⁵, 2.7×10⁻⁴, 3.4×10⁻⁴, 5.1×10⁻⁴, and 1.3×10⁻³, respectively, in cDKO3. (E) RT-PCR of hematopoietic, neural and sensory organ differentiation markers in mESC of the indicated genotype.

Fig. 2

myc depletion disrupts mESC pluripotency and self-renewal. (A) Immunofluorescent staining for SSEA-1 (red) and DAPI (blue) in the cDKO-2 mESC line. Arrow marks a representative GFP+colony, negative for SSEA-1. (B) Flow cytometric analysis of SSEA-1 expression in DKO-2 mESC line. mESC were analyzed by FACS for SSEA-1 levels with levels gated and defined as follows: negative (equal to or below levels of expression of isotype specific negative control), low (bottom third), medium (middle third), and high (top third) of SSEA-1 levels. Error bars are standard deviations. N=3. Decreases in high and medium SSEA-1 staining and increases in low and negative SSEA-1 staining had p values of 0.002, 0.005, 0.002, and 0.01, respectively, in cDKO2 and p values of 0.01, 0.002, 0.004, and 0.006 in cDKO3, respectively. (C) Fluorescent and phase contrast images of WT and DKO mESC stained for AP. Arrows denote AP staining in representative GFP-Cre positive colonies. (D) Percentage of self-renewing colonies of WT and DKO mESC lines calculated after alkaline phosphatase assays. Percentage of AP positive colonies in GFP only transduced mESC lines was defined as 100%. The error bars are SD and the data is the mean from three biological replicates (n=3). (E) Percentage of GATA6 positive cells out of GFP positive mESC (GFP alone or GFP-CRE) quantified by immunofluorescent staining for GATA6. The error bars are SD and the data is the mean from three biological replicates (n=3). P values were calculated by two-tail t-test assuming equal variances throughout this figure.

Fig. 3

myc knockdown stimulates the expression of early differentiation markers. Immunofluorescent staining for the expression of Bmp4, GATA6, and Ngn3 in GFP control and GFP-CRE transduced DKO mESC colonies. Numbers in the bottom left corner indicate the percentage of cells expressing Bmp4, GATA6 or Ngn3 differentiation markers out of GFP positive mESC (GFP alone or GFP CRE). Images taken at 20× magnification show the expression of differentiation markers in mESC colonies, and images taken at 60× magnification show the expression of differentiation markers in individual cells within mESC colonies. White square defines region of 20× field shown in 60× panel.

Fig. 4

myc-deficient cells do not exhibit widespread expression of late differentiation markers. Immunofluorescent staining for the expression of TUJ1, SMA, and AFP in GFP alone and GFP-CRE transduced DKO mESC colonies. Numbers in the bottom left corner indicate the percentage of cells expressing TUJ1, SMA, or AFP differentiation markers out of GFP positive mESC (GFP alone or GFP CRE). Images taken at 20× magnification show the expression of differentiation markers in mESC colonies, and images taken at 60× magnification show the expression of differentiation markers in individual cells within mESC colonies. White square defines region of 20× field shown in 60× panel.

Fig. 5

Myc is required for cell cycle progression and cell survival of mESC. (A) Cell cycle profiles of WT and cDKO mESC lines were obtained by measuring bromodeoxyuridine (BrdU) incorporation coupled to the staining of DNA content with 7-amino-actinomycin D (7-AAD). Changes in G₀/G₁, S, and G₂/M phases had p values of 0.02, 0.003, and 0.08 for cDKO2 and p values of 0.04, 0.0003, and 0.007 in cDKO3, respectively. (B) Percentage of apoptotic cells in GFP positive populations in mESC lines was measured by Annexin-V staining assay. The data are the means of three biological replicates (n=3) and error bars are standard deviations. Increases in apoptosis had p values of 0.004 and 0.006 in cDKO2 and cDKO3, respectively.

Fig. 6

Myc is essential for early embryogenesis. (A) Mean percentage of midgestational chimeric embryos recovered after 8–9 days postinjection in two independent microinjection experiments. The graph represents the percentage of chimeric embryos which were recovered compared to the total number of injected embryos for each cell type (defined as 100%). (B) Representative phase contrast and fluorescence microscopy images of chimeric embryos produced with cDKO-3 mESC transduced with GFP or GFP-Cre virus.