Lin HH et al. (2010), Neuronatin promotes neural lineage in ESCs via ...

XB-ART-42078

Stem Cells 2010 Nov 01;2811:1950-60. doi: 10.1002/stem.530.

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Neuronatin promotes neural lineage in ESCs via Ca(2+) signaling.

Lin HH , Bell E , Uwanogho D , Perfect LW , Noristani H , Bates TJ , Snetkov V , Price J , Sun YM .

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Neural induction is the first step in the formation of the vertebrate central nervous system. The emerging consensus of the mechanisms underlying neural induction is the combined influences from inhibiting bone morphogenetic protein (BMP) signaling and activating fibroblast growth factor (FGF)/Erk signaling, which act extrinsically via either autocrine or paracrine fashions. However, do intrinsic forces (cues) exist and do they play decisive roles in neural induction? These questions remain to be answered. Here, we have identified a novel neural initiator, neuronatin (Nnat), which acts as an intrinsic factor to promote neural fate in mammals and Xenopus. ESCs lacking this intrinsic factor fail to undergo neural induction despite the inhibition of the BMP pathway. We show that Nnat initiates neural induction in ESCs through increasing intracellular Ca(2+) ([Ca(2+) ](i)) by antagonizing Ca(2+) -ATPase isoform 2 (sarco/endoplasmic reticulum Ca(2+) -ATPase isoform 2) in the endoplasmic reticulum, which in turn increases the phosphorylation of Erk1/2 and inhibits the BMP4 pathway and leads to neural induction in conjunction with FGF/Erk pathway.

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Species referenced: Xenopus
Genes referenced: adm atp2a2 bag3 bmp4 chrd fgf4 fgf5 fst hnf4a krt18.1 map2 mapk1 mespb msx1 msx2 ncam1 nog nrp1 odc1 otx2 pax6 pou5f3 rbfox3 six3 smad1 sox1 tbxt

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	Figure 1. The expression of Nnat during ESC-derived neural differentiation and in the generated Nnat ES mutants. (A–G): Immunocytochemical analysis of Nnat expression. Nnat is expressed in a subpopulation of ESCs (before differentiation) (A) and 1 day after differentiated is initiated (B). After 2-day differentiation, Nnat is abundantly expressed in Sox1-eGFP+ early NSCs (C) and at higher magnification (D). As differentiation proceeded, Nnat-positive cells are also costained with the late NSC markers, Nes, and Pax6 (E), radial-glial-like progenitor marker, RC2 (F), and neuronal marker NeuN (G). (H): The levels of Nnatα and β in 12-day differentiated cells derived from control and Nnat ESC mutants were measured using Western blot analysis. Nnat ESC mutants are Nnatα-OE, Nnatβ-OE, and Nnat-KD ESCs. (I): Confirmation of Nnat-KD throughout 18 days of differentiation by qRT-PCR. Data shown are the mean ± SD (n = 2). Scale bar = 50 μm, 20 μm ([D]; [E], top panel; and [G]). All nuclei were stained with DAPI (blue). Abbreviations: DAPI: 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; Nnat, neuronatin; Nnat-KD, Nnat-knockdown; Nnatα-OE, neuronatin αoverexpression; Nnatβ-OE, neuronatin βoverexpression; NSC, neural stem cell; qRT-PCR, quantitative real time-polymerase chain reaction.
	Figure 2. The competence of Nnat overexpressing and knockdown ESCs to give rise to the three primary germ cells. (A): Quantitative RT-PCR analysis for markers of the three primary germ cells using RNAs derived from control, Nnatα-OE, and Nnat-KD ESCs using an embryoid body (EB) formation assay. The total RNA from each ESC line was collected from 6-day differentiated EBs and the cDNAs were used to analyze the various cell markers: Fgf5 (primitive ectodermal cells), Hnf4 (endodermal cells), T and Mesp (mesodermal cells), Otx2 (ectodermal cells), and Six3 and Sox1 (neuroectodermal cells). Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnatα-OE group, two-tailed Student's t test. (B): ICC analysis of the mesodermal and epidermal cells derived from control, Nnatα-OE, and Nnat-KD ESCs identified by staining with T and Krt18, respectively. Scale bar = 50 μm. All nuclei were stained with DAPI (blue). Abbreviations: DAPI: 4′,6′-diamidino-2-phenylindole; ICC, immunocytochemical; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; RT-PCR: real time-polymerase chain reaction.
	Figure 3. Nnat promotes the neural lineage. The role of Nnat in neural development was examined using an embryonic stem cell (ESC)-derived neural differentiation system over a 14-day time period. (A–X): Control, Nnat-KD, and Nnatα-OE ESCs were driven along neural differentiation using monolayer culture in N2B27 medium. Samples were collected for immunocytochemical analysis at ESC stage (before differentiation), NSC stage (6-day differentiation), and neuronal stage (14-day differentiation). The samples were stained with cell-specific markers, Oct4 (red), Nes (red), and Map2 (red), which represent ESCs, NSCs, and neurons, respectively. Scale bar = 50 μm. All nuclei were stained with DAPI (blue). (Y): Quantification of Sox1-eGFP+/Nes+ neural stem cells derived from control, Nnat-KD, and Nnatα-OE ESC using fluorescence-activated cell sorting (FACS) analysis. (Z): Quantification of NeuN+ neurons derived from control, Nnat-KD, and Nnatα-OE ESC using FACS analysis. Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnatα-OE group, two-tailed Student's t test. Abbreviations: DAPI: 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; ES, embryonic stem; Nnat-KD, neuronatin-knockdown; Nnatα-OE, neuronatin α-overexpression; NSC, neural stem cell.
	Figure 4. The effects of mouse Nnatα and β on neural patterning in Xenopus laevis. (A–I): The representative images of Xenopus embryos, which were injected in one cell at the two-cell stage with 1 ng of either Nnatα or Nnatβ and the morphology was evaluated at late neurula or early tadpole stages. At neurula stage, (A) uninjected control embryo, (B) Nnatα injection results in an edema-like structure (see black arrows), and an enlarged cement gland (arrowhead), whereas (C) Nnatβ injection not only causes an enlarged cement gland (see arrow) but also an expanded forebrain (double-headed arrow). At early tadpole stages, (D) uninjected control embryo, (E) Nnatα-injected embryos exhibit an abnormal phenotype with ectopic pigmentations (see arrows) and (F) Nnatβ-injected embryos show a distorted and truncated phenotype with an enlarged cement gland (arrow) and a clear expansion of the neural plate. The abnormal neural phenotypes were confirmed by Ncam staining (a pan-neural marker): (G) control uninjected embryo, (H) Nnatα-injected embryo with noticeable neural plate expansion in the injected side (), and (I) Nnatβ-injected embryo with a profound neural plate expansion in the injected side (). (J): In animal caps assay, injecting either Nnatα or β at one- to two-cell stage leads to increase in the expression of neural markers, nrp1 and otx, and a mesodermal marker nkx2.5, as assayed by RT-PCR. ODC was used as a loading control. Abbreviations: cg, cement gland; fb, forebrain; hb, hindbrain; mb, midbrain; sc, spinal cord; Ncam, neural cell adhesion molecule; Nnatα, neuronatin α; Nnatβ, neuronatin β; ODC, ornithine decarboxylase; RT, no reverse transcriptase; RT-PCR, real time-polymerase chain reaction.
	Figure 5. Nnat action is via Ca2+ signaling through negatively regulating SERCA2. (A): Dose-dependent pull down SERCA2 (100 kDa) by Nnat antibody using co-immunoprecipitation assay, suggesting that Nnat physically interacts with SERCA2. (B): Ca2+ imaging (top panel) and histograms (bottom panel) generated from Nnat-OE and Nnat-KD ESCs, in which the former exhibits higher [Ca2+]i than that of the latter using Ca2+ green-1AM dye. (C): SERCA blockers, Tg and BHQ, rescue Nnat-KD phenotypes in the production of Sox1-GFP+/Nes+ (red) NSCs, and NeuN+ (red) neurons. Scale bar = 50 μm. (D): Quantification of Sox1-eGFP+/Nes+ NSC derived from control, Nnat-KD ESCs, and Nnat-KD ESCs treated with Tg (Nnat-KD+Tg) using fluorescence-activated cell sorting (FACS) analysis. Data shown are the mean ± SD (n = 3). (E): Quantification of NeuN+ neurons derived from control, Nnat-KD, and Nnat-KD+Tg ESCs using FACS analysis. Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnat-KD group, two-tailed Student's t test. (F): Changes in intracellular [Ca2+]i levels in Nnat-KD ESCs following Tg or BHQ treatment were measured using the R340/380 emission intensities ratio of Fura PE-3. (G): Inhibition of Tg-rescued neural induction in Nnat-KD ESCs by chelating Tg-increased [Ca2+]i using an intracellular calcium chelator BAPTA-AM. Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: BAPTA-AM, 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxy methyl ester); BHQ, 2,5-di-t-butyl-1,4-benzohydroquinone; DAPI: 4′,6′-diamidino-2-phenylindole; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; Nnat-KD, neuronatin-knockdown; Nnatα-OE, neuronatin α-overexpression; NSC, neural stem cell; SERCA2, sarco/endoplasmic reticulum Ca2+-ATPase isoform 2; Tg, thapsigargin.
	Figure 6. Nnat-mediated Ca2+ signaling increases p-Erk1/2 and cross talks with FGF/Erk pathway in neural induction. (A): The inhibitory effect of PD173074 (a FGF-R blocker) and PD184352 (a p-Erk1/2 blocker) on neural induction. Without the presence of blockers, Nnat-KD ESCs failed to initiate neural induction (fail to generate Sox1-eGFP+/Nes+ [red] NSCs), which was rescued by Tg treatment (Nnat-KD+Tg). In the presence of the blockers (+PD173074 or +PD184352), neural induction in control and Nnat-KD+Tg ESCs was abolished, whereas PD173074 partially and PD184352 completely inhibited neural induction in Nnatα-OE ESCs. (B): The rescue effects of FGF4 or FGF5 on Nnat-KD phenotype in the production of Sox1-eGFP+/Nes+ (red) NSCs and NeuN+ (red) neurons. (C): Quantification of NSC and neuron population generated from control, Nnat-KD, and Nnat-KD ESCs treated with FGF4 (Nnat-KD+FGF4) or FGF5 (Nnat-KD+FGF5) using fluorescence-activated cell sorting analysis. Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05, significantly different from the Nnat-KD group, two-tailed Student's t test. (D): The FGF4 and FGF5 rescue of neural induction in Nnat-KD ESCs were abrogated by the presence of PD173074 or PD184352. (E): Increase in the phosphorylation of Erk1/2 after 5 minutes treated with various concentrations of Tg (top panel), the time points of the increment with 25 nM Tg treatment (middle panel), and the effect of PD173074 on Tg-induced p-Erk1/2 (bottom panel). Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; FGF4, fibroblast growth factor 4; FGF5, Fibroblast growth factor 5; GFP, green fluorescent protein; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; NSC, neural stem cell; Tg, thapsigargin; w/o, without.
	Figure 7. Nnat-mediated Ca2+ signaling interacts with BMP4 pathway in neural induction by suppressing the transcription of BMP4 and its target genes. (A): The inhibitory effect of BMP4 on neural induction. Without the presence of BMP4, Nnat-KD ESCs failed to generate Sox1-eGFP+/Nes+ (red) neural stem cells, which was rescued by Tg treatment (Nnat-KD+Tg). In the presence of BMP4 (+BMP4), neural induction in control and Nnat-KD+Tg ESCs was abolished, whereas BMP4 only partially inhibited neural induction in Nnatα-OE ESCs. (B): Gene expression profiles of BMP4 and its target genes, Msx1 and Msx2 in ESCs show that the expression of those genes is suppressed in Nnat-mediated Ca2+ signaling. Data shown are the mean ± SD (n = 3). *, p < .05, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnat-KD group. (C): The effect of Tg on BMP4-mediated C-terminal phosphorylation of Smad1 at indicated duration of Tg treatment. (D): Inhibition of BMP pathway by antagonists, Nog, Chrd and Fst, does not induce neural induction in Nnat-KD ESCs. Control and Nnat-KD ESCs were driven along neural differentiation. At 4-day differentiation, control ESCs generate many Sox1-eGFP+ neuroectodermal cells, whereas Nnat-KD ESCs fail to produce neuroectodermal cells. The failure of neural induction in Nnat-KD ESCs is rescued by Tg treatment, but not by BMP antagonists. Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; BMP4, bone morphogenetic protein 4; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; Tg, thapsigargin; w/o, without.

References [+] :

Aubert, Functional gene screening in embryonic stem cells implicates Wnt antagonism in neural differentiation. 2002, Pubmed

	Figure 1. The expression of Nnat during ESC-derived neural differentiation and in the generated Nnat ES mutants. (A–G): Immunocytochemical analysis of Nnat expression. Nnat is expressed in a subpopulation of ESCs (before differentiation) (A) and 1 day after differentiated is initiated (B). After 2-day differentiation, Nnat is abundantly expressed in Sox1-eGFP+ early NSCs (C) and at higher magnification (D). As differentiation proceeded, Nnat-positive cells are also costained with the late NSC markers, Nes, and Pax6 (E), radial-glial-like progenitor marker, RC2 (F), and neuronal marker NeuN (G). (H): The levels of Nnatα and β in 12-day differentiated cells derived from control and Nnat ESC mutants were measured using Western blot analysis. Nnat ESC mutants are Nnatα-OE, Nnatβ-OE, and Nnat-KD ESCs. (I): Confirmation of Nnat-KD throughout 18 days of differentiation by qRT-PCR. Data shown are the mean ± SD (n = 2). Scale bar = 50 μm, 20 μm ([D]; [E], top panel; and [G]). All nuclei were stained with DAPI (blue). Abbreviations: DAPI: 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; Nnat, neuronatin; Nnat-KD, Nnat-knockdown; Nnatα-OE, neuronatin αoverexpression; Nnatβ-OE, neuronatin βoverexpression; NSC, neural stem cell; qRT-PCR, quantitative real time-polymerase chain reaction.
	Figure 2. The competence of Nnat overexpressing and knockdown ESCs to give rise to the three primary germ cells. (A): Quantitative RT-PCR analysis for markers of the three primary germ cells using RNAs derived from control, Nnatα-OE, and Nnat-KD ESCs using an embryoid body (EB) formation assay. The total RNA from each ESC line was collected from 6-day differentiated EBs and the cDNAs were used to analyze the various cell markers: Fgf5 (primitive ectodermal cells), Hnf4 (endodermal cells), T and Mesp (mesodermal cells), Otx2 (ectodermal cells), and Six3 and Sox1 (neuroectodermal cells). Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnatα-OE group, two-tailed Student's t test. (B): ICC analysis of the mesodermal and epidermal cells derived from control, Nnatα-OE, and Nnat-KD ESCs identified by staining with T and Krt18, respectively. Scale bar = 50 μm. All nuclei were stained with DAPI (blue). Abbreviations: DAPI: 4′,6′-diamidino-2-phenylindole; ICC, immunocytochemical; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; RT-PCR: real time-polymerase chain reaction.
	Figure 3. Nnat promotes the neural lineage. The role of Nnat in neural development was examined using an embryonic stem cell (ESC)-derived neural differentiation system over a 14-day time period. (A–X): Control, Nnat-KD, and Nnatα-OE ESCs were driven along neural differentiation using monolayer culture in N2B27 medium. Samples were collected for immunocytochemical analysis at ESC stage (before differentiation), NSC stage (6-day differentiation), and neuronal stage (14-day differentiation). The samples were stained with cell-specific markers, Oct4 (red), Nes (red), and Map2 (red), which represent ESCs, NSCs, and neurons, respectively. Scale bar = 50 μm. All nuclei were stained with DAPI (blue). (Y): Quantification of Sox1-eGFP+/Nes+ neural stem cells derived from control, Nnat-KD, and Nnatα-OE ESC using fluorescence-activated cell sorting (FACS) analysis. (Z): Quantification of NeuN+ neurons derived from control, Nnat-KD, and Nnatα-OE ESC using FACS analysis. Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnatα-OE group, two-tailed Student's t test. Abbreviations: DAPI: 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; ES, embryonic stem; Nnat-KD, neuronatin-knockdown; Nnatα-OE, neuronatin α-overexpression; NSC, neural stem cell.
	Figure 4. The effects of mouse Nnatα and β on neural patterning in Xenopus laevis. (A–I): The representative images of Xenopus embryos, which were injected in one cell at the two-cell stage with 1 ng of either Nnatα or Nnatβ and the morphology was evaluated at late neurula or early tadpole stages. At neurula stage, (A) uninjected control embryo, (B) Nnatα injection results in an edema-like structure (see black arrows), and an enlarged cement gland (arrowhead), whereas (C) Nnatβ injection not only causes an enlarged cement gland (see arrow) but also an expanded forebrain (double-headed arrow). At early tadpole stages, (D) uninjected control embryo, (E) Nnatα-injected embryos exhibit an abnormal phenotype with ectopic pigmentations (see arrows) and (F) Nnatβ-injected embryos show a distorted and truncated phenotype with an enlarged cement gland (arrow) and a clear expansion of the neural plate. The abnormal neural phenotypes were confirmed by Ncam staining (a pan-neural marker): (G) control uninjected embryo, (H) Nnatα-injected embryo with noticeable neural plate expansion in the injected side (), and (I) Nnatβ-injected embryo with a profound neural plate expansion in the injected side (). (J): In animal caps assay, injecting either Nnatα or β at one- to two-cell stage leads to increase in the expression of neural markers, nrp1 and otx, and a mesodermal marker nkx2.5, as assayed by RT-PCR. ODC was used as a loading control. Abbreviations: cg, cement gland; fb, forebrain; hb, hindbrain; mb, midbrain; sc, spinal cord; Ncam, neural cell adhesion molecule; Nnatα, neuronatin α; Nnatβ, neuronatin β; ODC, ornithine decarboxylase; RT, no reverse transcriptase; RT-PCR, real time-polymerase chain reaction.
	Figure 5. Nnat action is via Ca2+ signaling through negatively regulating SERCA2. (A): Dose-dependent pull down SERCA2 (100 kDa) by Nnat antibody using co-immunoprecipitation assay, suggesting that Nnat physically interacts with SERCA2. (B): Ca2+ imaging (top panel) and histograms (bottom panel) generated from Nnat-OE and Nnat-KD ESCs, in which the former exhibits higher [Ca2+]i than that of the latter using Ca2+ green-1AM dye. (C): SERCA blockers, Tg and BHQ, rescue Nnat-KD phenotypes in the production of Sox1-GFP+/Nes+ (red) NSCs, and NeuN+ (red) neurons. Scale bar = 50 μm. (D): Quantification of Sox1-eGFP+/Nes+ NSC derived from control, Nnat-KD ESCs, and Nnat-KD ESCs treated with Tg (Nnat-KD+Tg) using fluorescence-activated cell sorting (FACS) analysis. Data shown are the mean ± SD (n = 3). (E): Quantification of NeuN+ neurons derived from control, Nnat-KD, and Nnat-KD+Tg ESCs using FACS analysis. Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnat-KD group, two-tailed Student's t test. (F): Changes in intracellular [Ca2+]i levels in Nnat-KD ESCs following Tg or BHQ treatment were measured using the R340/380 emission intensities ratio of Fura PE-3. (G): Inhibition of Tg-rescued neural induction in Nnat-KD ESCs by chelating Tg-increased [Ca2+]i using an intracellular calcium chelator BAPTA-AM. Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: BAPTA-AM, 1,2-Bis (2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxy methyl ester); BHQ, 2,5-di-t-butyl-1,4-benzohydroquinone; DAPI: 4′,6′-diamidino-2-phenylindole; GFP, green fluorescent protein; eGFP, enhanced green fluorescent protein; Nnat-KD, neuronatin-knockdown; Nnatα-OE, neuronatin α-overexpression; NSC, neural stem cell; SERCA2, sarco/endoplasmic reticulum Ca2+-ATPase isoform 2; Tg, thapsigargin.
	Figure 6. Nnat-mediated Ca2+ signaling increases p-Erk1/2 and cross talks with FGF/Erk pathway in neural induction. (A): The inhibitory effect of PD173074 (a FGF-R blocker) and PD184352 (a p-Erk1/2 blocker) on neural induction. Without the presence of blockers, Nnat-KD ESCs failed to initiate neural induction (fail to generate Sox1-eGFP+/Nes+ [red] NSCs), which was rescued by Tg treatment (Nnat-KD+Tg). In the presence of the blockers (+PD173074 or +PD184352), neural induction in control and Nnat-KD+Tg ESCs was abolished, whereas PD173074 partially and PD184352 completely inhibited neural induction in Nnatα-OE ESCs. (B): The rescue effects of FGF4 or FGF5 on Nnat-KD phenotype in the production of Sox1-eGFP+/Nes+ (red) NSCs and NeuN+ (red) neurons. (C): Quantification of NSC and neuron population generated from control, Nnat-KD, and Nnat-KD ESCs treated with FGF4 (Nnat-KD+FGF4) or FGF5 (Nnat-KD+FGF5) using fluorescence-activated cell sorting analysis. Data shown are the mean ± SD (n = 3). , p < .05 and *, p < .01, significantly different from the control group; #, p < .05, significantly different from the Nnat-KD group, two-tailed Student's t test. (D): The FGF4 and FGF5 rescue of neural induction in Nnat-KD ESCs were abrogated by the presence of PD173074 or PD184352. (E): Increase in the phosphorylation of Erk1/2 after 5 minutes treated with various concentrations of Tg (top panel), the time points of the increment with 25 nM Tg treatment (middle panel), and the effect of PD173074 on Tg-induced p-Erk1/2 (bottom panel). Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; eGFP, enhanced green fluorescent protein; FGF4, fibroblast growth factor 4; FGF5, Fibroblast growth factor 5; GFP, green fluorescent protein; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; NSC, neural stem cell; Tg, thapsigargin; w/o, without.
	Figure 7. Nnat-mediated Ca2+ signaling interacts with BMP4 pathway in neural induction by suppressing the transcription of BMP4 and its target genes. (A): The inhibitory effect of BMP4 on neural induction. Without the presence of BMP4, Nnat-KD ESCs failed to generate Sox1-eGFP+/Nes+ (red) neural stem cells, which was rescued by Tg treatment (Nnat-KD+Tg). In the presence of BMP4 (+BMP4), neural induction in control and Nnat-KD+Tg ESCs was abolished, whereas BMP4 only partially inhibited neural induction in Nnatα-OE ESCs. (B): Gene expression profiles of BMP4 and its target genes, Msx1 and Msx2 in ESCs show that the expression of those genes is suppressed in Nnat-mediated Ca2+ signaling. Data shown are the mean ± SD (n = 3). *, p < .05, significantly different from the control group; #, p < .05 and ##, p < .01, significantly different from the Nnat-KD group. (C): The effect of Tg on BMP4-mediated C-terminal phosphorylation of Smad1 at indicated duration of Tg treatment. (D): Inhibition of BMP pathway by antagonists, Nog, Chrd and Fst, does not induce neural induction in Nnat-KD ESCs. Control and Nnat-KD ESCs were driven along neural differentiation. At 4-day differentiation, control ESCs generate many Sox1-eGFP+ neuroectodermal cells, whereas Nnat-KD ESCs fail to produce neuroectodermal cells. The failure of neural induction in Nnat-KD ESCs is rescued by Tg treatment, but not by BMP antagonists. Scale bar = 50 μm and all nuclei were stained with DAPI (blue). Abbreviations: DAPI, 4′,6′-diamidino-2-phenylindole; BMP4, bone morphogenetic protein 4; Nnatα-OE, neuronatin α-overexpression; Nnat-KD, neuronatin-knockdown; Tg, thapsigargin; w/o, without.