Sp5 induces the expression of Nanog to maintain mouse embryonic stem cell self-renewal

<div><p>Activation of signal transducer and activator of transcription 3 (STAT3) by leukemia inhibitory factor (LIF) maintains mouse embryonic stem cell (mESC) self-renewal. Our previous study showed that trans-acting transcription factor 5 (Sp5), an LIF/STAT3 downstream target, supports mESC self-renewal. However, the mechanism by which Sp5 exerts these effects remains elusive. Here, we found that Nanog is a direct target of Sp5 and mediates the self-renewal-promoting effect of Sp5 in mESCs. Overexpression of <i>Sp5</i> induced <i>Nanog</i> expression, while knockdown or knockout of <i>Sp5</i> decreased the <i>Nanog</i> level. Moreover, chromatin immunoprecipitation (ChIP) assays showed that Sp5 directly bound to the Nanog promoter. Functional studies revealed that knockdown of <i>Nanog</i> eliminated the mESC self-renewal-promoting ability of Sp5. Finally, we demonstrated that the self-renewal-promoting function of Sp5 was largely dependent on its zinc finger domains. Taken together, our study provides unrecognized functions of Sp5 in mESCs and will expand our current understanding of the regulation of mESC pluripotency.</p></div

Screening the downstream pluripotency genes regulated by <i>Sp5</i>.

<p>(A) HA-tagged <i>Sp5</i> was introduced into 46C mESCs and the protein level of HA-tagged <i>Sp5</i> was determined by Western blot. α-Tubulin was used as a loading control. (B) Phase-contrast and alkaline phosphatase (AP) staining images of PB and PB-<i>Sp5</i> mESCs cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (C) Immunofluorescence of PB and PB-<i>Sp5</i> mESCs cultured under basal media conditions in the absence of LIF. Scale bar, 100 μm. (D) qRT–PCR analysis of the expression levels of mESC pluripotency markers (<i>Oct4</i>, <i>Sox2</i>, and <i>Nanog</i>) and differentiation-associated genes (<i>Gata4</i>, <i>Gata6</i>, and <i>T</i>) in PB and PB-<i>Sp5</i> 46C mESCs cultured in the absence of LIF. Data represent the mean±s.d of three biological replicates. **p < 0.01 vs PB. (E) qRT-PCR analysis of <i>Klf2/4/5</i>, <i>Nanog</i>, <i>Esrrb</i>, <i>Gbx2</i>, <i>Myc</i>, <i>Tfcp2l1</i>, <i>Tbx3</i> and <i>Pim1/3</i> expression in PB and PB-<i>Sp5</i> 46C mESCs cultured under LIF/serum-containing conditions. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB. (F) qRT–PCR analysis of <i>Sp5</i>, <i>Nanog</i> and <i>Klf2</i> transcripts in <i>scramble</i> and <i>Sp5</i> shRNA mESCs cultured under LIF/serum-containing conditions. Data represent the mean±s.d. of three biological replicates. *p < 0.05, **p < 0.01 vs <i>scramble</i> shRNA control.</p

Sp5 directly regulates the transcription of <i>Nanog</i>.

<p>(A) Independent validation of Nanog as an Sp5-bound target by ChIP-qPCR with fifteen primers set to scan different fragments of the Nanog promoter. Primers set at sites 1, 3, 5, 8, 9 and 15 represent significant enrichment. Data represent the mean±s.d. of three biological replicates. *p < 0.05, **p < 0.01 vs IgG. (B) Schematic illustration of luciferase reporter plasmids and <i>Sp5</i> expression activates the P_Nanog-luciferase reporter. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB.</p

Delineation of the zinc finger domains of Sp5 impairs the self-renewal-promoting effect of Sp5.

<p>(A) Schematic illustration of the <i>Sp5</i> deletion (Δ) mutants. Zinc finger domains are shown as green boxes. (B) Western blot analysis of Flag-tagged <i>Sp5</i> and <i>Sp5</i> deletion mutants with anti-FLAG antibody. (C) AP staining images of PB, PB-<i>Sp5</i> and <i>Sp5</i> deletion mutant mESCs cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (D) Quantification of AP-positive colonies in Fig. 4C. (E) qRT–PCR analysis of the expression of <i>Nanog</i> in PB, PB-<i>Sp5</i> and <i>Sp5</i> deletion mutant mESCs cultured in LIF/serum-containing media. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB.</p

Sp5 relies on Nanog to promote mESC self-renewal.

<p>(A) qRT–PCR analysis of <i>Nanog</i> expression in PB-<i>Sp5</i> mESCs infected with <i>Nanog</i> knockdown lentiviruses and cultured in LIF/serum-containing media. The transcript level was normalized to the <i>scramble</i> shRNA control. Data represent the mean±s.d of three biological replicates. **p < 0.01 vs <i>scramble</i> shRNA control. (B) Immunofluorescence of PB-<i>Sp5</i> cells infected with <i>scramble</i> control and <i>Nanog</i> shRNA lentiviruses cultured under serum-containing conditions in the absence of LIF for eight days. Scale bar, 100 μm. (C) AP staining images of <i>scramble</i> control and <i>Nanog</i> shRNA mESCs overexpressing PB-<i>Sp5</i> cultured under serum-containing conditions. Scale bar, 100 μm. (D) qRT–PCR analysis of the expression of mESC pluripotency markers (<i>Oct4</i>, <i>Sox2</i>, <i>Klf4</i>, <i>Esrrb</i> and <i>Tfcp2l1</i>) and differentiation-associated genes (<i>Gata4</i>, <i>Gata6</i>, <i>Mixl1</i>, <i>Sox1</i> and <i>Cdx2</i>) in <i>scramble</i> and <i>Nanog</i>-knockdown mESCs transfected with PB-<i>Sp5</i> cultured in the absence of LIF. Data represent the mean±s.d of three biological replicates. *p < 0.05, **p < 0.01 vs scramble control. (E) qRT–PCR analysis of the expression of <i>Gata4</i> and <i>Gata6</i> in PB and PB-<i>Sp5</i> 46C mESC-derived embryoid bodies harvested on day 6. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs PB EBs. (F) qRT–PCR analysis of <i>Gata4</i> and <i>Gata6</i> transcript levels in wild type and S<i>p5</i>KO mESC-derived embryoid bodies. Data represent the mean±s.d. of three biological replicates. **p < 0.01 vs 46C EBs.</p

NS Cells Die or Begin to Differentiate in the Absence of EGF

<p>Unlike proliferating cultures in FGF plus EGF (A,C), NS cells on gelatin die by caspase-3-mediated programmed cell death 20 h after removal of EGF (B,D). This death can be overcome if cells are cultured on a laminin substrate in FGF-2 only (F). Under these conditions, cells become slow-dividing and extend longer processes (G,H). Most cells retain RC2 immunoreactivity (H), but a minority begin neuronal differentiation marked by TuJ1 expression (J).</p

Human ES Cell or Foetal-Derived NS Cells

<div><p>(A) Derivation from human ES cells: human ES cell primary culture (a), differentiation of human ES cells into neural-rosette structures (b), passage 9 in NS expansion medium (c), individual cells exhibit radial glial morphology (d), and immunostaining for NS cell/radial glia markers (e–h).</p> <p>(B) Derivation from human foetal forebrain: floating clusters (i) generated from cortex, attachment and outgrowth (j), passage 5 in NS expansion medium (k), radial glia morphology (l), and NS cell/radial glial markers (m–p).</p> <p>(C) Differentiation of human foetal NS cells: TuJ1 positive neuronal cells generated by sequential growth factor withdrawal (q), and GFAP positive astrocytes induced by exposure to serum (r).</p></div

Clonal NS Cells Generated through <i>Sox1</i> Neural Lineage Selection

<div><p>(A) Phase image of neural precursors at passage 1 (a) and 5 (c), with (b) and (d) showing corresponding <i>Sox1</i>-GFP fluorescence. Image (e) shows a single cell, 1 h after plating in Terasaki well, and (f) shows a phase-contrast image of clonal cell line at passage 20.</p> <p>(B) Differentiation of NS-5 cells into astrocytes (g,h) and neurons (j,k) with loss of nestin immunoreactivity (i,l).</p> <p>(C) These NS-5 cells are immunoreactive for neural precursor cell/radial glia markers (m–o,q,r) and negative for GFAP (p).</p> <p>(D) Clones of NS-5 cells exhibit homogenous expression of BLBP with no immunoreactivity for GFAP in the presence of EGF/FGF (s), and generate neurons upon growth factor withdrawal (t).</p> <p>(E) Metaphase spread of NS-5 (passage 31).</p></div

ES Cell–Derived or Forebrain-Derived NS Cells are Similar to Radial Glia

<div><p>NS cells were derived from independent ES cell lines (CGR8, E14Tg2a) or primary cortical (Cor-1) and striatal (Str-1) tissue.</p> <p>(A) RT-PCR of stem cell/radial glia markers.</p> <p>(B) RT-PCR for pan-neural and region-specific transcriptional regulators.</p> <p>(C) Double immunostaining for Pax6 and Pax6/RC2 (a,b), Olig2 and Olig2/RC2 (c,d) and Olig2/Pax6 (f). DAPI only for Olig2/Pax6 (e).</p> <p>(D) The ES cell–derived line (CGR8-NS) and foetal cell–derived line (Cor-1) are indistinguishable from LC1 by morphology and NS cell/radial glial marker immunoreactivity (g,h,k,l), and can each differentiate into neurons (i,m) and astrocytes (j,n).</p> <p>(E) The ability of Cor-1 to generate neurons (TuJ1+) is retained after 16 passages, more than 30 generations (p,o).</p></div

Pathological lymphangiogenesis models using Prox1-tdTomato mouse.

<p>(A-E) Bladder lymphangiogenesis model: Adult Prox1-tdTomato mice were i.p. injected with phosphate-buffered saline (A,C) or cyclophosphamide (B,D) at days 1 and 4. At day 9, lymphatic vessels in their bladders were visualized. Morphometric analyses revealed a significantly increase in the number of branch (marked with arrowhead), loop (arrow) and blind ends (double arrowhead) of lymphatic vessels in the CYP-treated bladder compared to the PBS-treated control (CTR) bladder (E). (F-J) Tumor implantation model: GFP-labeled tumor cells were implanted into the skin of immunodeficient athymic Prox1-tdTomato mouse, generated by crossing athymic nude mice and Prox1-tdTomato mice. Images of the GFP-labeled tumor mass (F), dermal lymphatic in the surrounding tissues (G) and their merged image (H) are shown. (I) and (J) are enlarged images of the boxed areas in panel (H) and (I), respectively. Arrows mark luminal valves.</p