Gao J et al. (2023), β-Catenin and SOX2 Interaction Regulate Visual ...

XB-ART-60288

Int J Mol Sci 2023 Sep 02;2417:. doi: 10.3390/ijms241713593.

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β-Catenin and SOX2 Interaction Regulate Visual Experience-Dependent Cell Homeostasis in the Developing Xenopus Thalamus.

Gao J , Lu Y , Luo Y , Duan X , Chen P , Zhang X , Wu X , Qiu M , Shen W .

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In the vertebrate brain, sensory experience plays a crucial role in shaping thalamocortical connections for visual processing. However, it is still not clear how visual experience influences tissue homeostasis and neurogenesis in the developing thalamus. Here, we reported that the majority of SOX2-positive cells in the thalamus are differentiated neurons that receive visual inputs as early as stage 47 Xenopus. Visual deprivation (VD) for 2 days shifts the neurogenic balance toward proliferation at the expense of differentiation, which is accompanied by a reduction in nuclear-accumulated β-catenin in SOX2-positive neurons. The knockdown of β-catenin decreases the expression of SOX2 and increases the number of progenitor cells. Coimmunoprecipitation studies reveal the evolutionary conservation of strong interactions between β-catenin and SOX2. These findings indicate that β-catenin interacts with SOX2 to maintain homeostatic neurogenesis during thalamus development.

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Species referenced: Xenopus laevis
Genes referenced: ctrl nkx2-2 pcna pth sox2
GO keywords: tissue homeostasis [+]
???displayArticle.antibodies??? BrdU Ab11 Ctnnb1 Ab23 Ctnnb1 Ab24 Ctnnb1 Ab3 Elavl3 Ab1 Fabp7 Ab3 Gapdh Ab8 Nkx2-2 Ab1 Pcna Ab10 Sox2 Ab1 Sox2 Ab7 sox9 Ab6 Vim Ab5
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	Figure 1. SOX2+ cells are distributed in the optic tectum and thalamus. (A) Representative images of a tadpole (left) and whole-mount immunofluorescent staining with an anti-SOX2 antibody (right) at stage 49 Xenopus. The red square indicates the whole optic tectum and the thalamus. Scale bar: 100 μm. (B) Representative images showing the colabeling with SOX2 and Nkx2.2 in the brain. The dotted white line indicates the outline of the optic tectum (OT). The white line represents the boundary of the SOX2 and Nkx2.2 immunoreactive thalamus (Th). Scale bar: 50 μm. (C) Six representative coronal planes of the whole brain with SOX2 immunostaining are shown at stage 49 Xenopus (Ca–Cf). Scale bar: 20 μm. (D) One representative sagittal section was immunostained with an anti-SOX2 antibody. The white lines (a–e) depict the positions of coronal sections for (Ca–Ce). Scale bar: 100 μm. III: third ventricle; IV: fourth ventricle; A: anterior; ABB: alar basal boundary; BH: basal hypothalamus; c-Th: caudal thalamus; D: dorsal; Hb: habenula; M: middle ventricle; Mes: mesencephalon; oc: optic chiasm; P: posterior; p1–3: prosomere1–3; Pa: pallium; PTh: prethalamus; r1–r7: rhomeres1–7; Spa: subpallium; OT: optic tectum; R: rostral; r-Th: rostral thalamus; Th: thalamus; Zli: zona limitans intrathalamica.
	Figure 2. The majority of thalamic SOX2+ cells are HuC/D+ or tubulin+ neurons. (A) Colabeling of SOX2 and BrdU showing that only a few SOX2+ cells (Aa) are BrdU+ cells (Ab) in the thalamus. Arrowheads indicate the SOX2− and BrdU+ cells (Ac). Arrows indicate the SOX2+ and BrdU+ cells (Ac). Scale bar: 20 μm. (B) Coimmunostaining of BrdU and PCNA in the thalamus. Arrows indicate the BrdU+ and PCNA+ cells. Scale bar: 20 μm. (C) Coimmunostaining of SOX2 (Ca) and PCNA (Cb) in the thalamus. Arrowheads indicate the SOX2+ and PCNA− cells. Arrows indicate the SOX2+ and PCNA+ neurons (Cc,Cd). Scale bar: 20 μm. (D) Coimmunostaining of SOX2 (Da) and HuC/D (Db) in the thalamus. Arrows indicate the SOX2+ and HuC/D+ neurons (Dc,Dd). Scale bar: 20 μm. (E) Colabeling of SOX2 (Ea) and tubulin (Eb) in the thalamus. Arrows indicate the SOX2+ and tubulin+ neurons (Ec,Ed). Scale bar: 20 μm. (F) Thalamic cells were immunostained with an anti-SOX2 antibody (red, Fb) followed by transfection with pSOX2::GFP (green, Fa), showing that the SOX2+ cells exhibited neuronal morphology with predicted dendrites (arrowheads, Fc,Fd). Arrows indicate the GFP–expressing and SOX2–immunoreactive cells. Scale bar: 20 μm.
	Figure 3. Visual deprivation alters synaptic transmission and dendritic growth. (A) Representative electrophysiological recordings of visual stimuli–evoked excitatory compound synaptic currents (eCSCs) in Ctrl-OT, Ctrl-Th, and VD-Th neurons in response to full–field light ON and OFF visual stimuli at an intensity of 20 cd/cm−2. Arrows indicate the onset and offset of the delay. Scale bar: 30 pA, 1 s. (B,C) Statistical results show the delay (B) and charge transfer (C) of eCSCs in Ctrl-OT, Ctrl-Th, and VD-Th neurons. White circles indicate the individual data. N = 17, 28, 17 for Ctrl-OT, Ctrl-Th, and VD-Th groups. (D) Representative recordings of optic chiasm stimuli–evoked excitatory postsynaptic currents (EPSCs). Scale bar: 20 pA, 20 ms. (E,F) Statistical results show the delay (E) and amplitude (F) of EPSCs. N = 14, 15, 13 for Ctrl-OT, Ctrl-Th, and VD-Th groups. (G) Three representative recording traces show current injection–induced spikes in Ctrl-OT, Ctrl-Th, and VD-Th neurons. Scale bar: 30 mV, 40 ms. (H) Statistical results show that the number of action potentials was significantly decreased in VD-Th neurons compared to Ctrl-Th neurons. N = 21, 16, 19 for Ctrl-OT, Ctrl-Th, and VD-Th groups. (I) Three representative neurons (upper panel) and their reconstructed images (lower panel) show pSOX2::GFP–expressing neurons in Ctrl-OT, Ctrl-Th, and VD-Th groups. Arrowheads indicate axons. Scale bar: 10 μm. (J) Total dendritic branch length (TDBL) was significantly decreased over 48 h in VD-Th neurons compared to Ctrl-Th neurons. (K) Total branch tip number (TBTN) was significantly decreased in VD-Th neurons compared to Ctrl-Th neurons. N = 19, 13, 13 for Ctrl-OT, Ctrl-Th, and VD-Th groups. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 4. Visual deprivation changes the balance between proliferation and differentiation in the thalamus. (A,B) Representative fluorescent images showing BrdU– (Ab) and SOX2–labeled (Aa) cells in Ctrl (Aa–Ac) and VD (Ba–Bc) thalamus. The white square indicates the BrdU– and SOX2–labeled cells in the zoomed–in thalamus (Aa–Ac). White dotted lines indicate the boundary of the thalamus (Ac). Arrows indicate the SOX2+ and BrdU+ cells (Ad–Af,Bd–Bf). Scale bar: 50 μm. Zoom Scale bar: 20 μm. (C–E) Summary data show that VD increased BrdU+ cells (C), decreased SOX2+ cells (D), and increased SOX2+/BrdU+ cells (E). N = 6, 6 for Ctrl and VD. (F) A representative immunofluorescent image showing z–stack for SOX2– and BrdU–labeled cells. Scale bar: 20 μm. (G,H) Summary data showing that VD increased BrdU+ cells (G) and decreased SOX2+ cells (H) in VD–treated tadpoles. N = 4, 6 for Ctrl and VD. (I,J) Representative immunofluorescent images showing SOX2– and HuC/D–labeled cells in the thalamus of Ctrl (Ia–Id) and VD (Ja–Jd) tadpoles. The white square indicates the zoomed–in images. Arrows indicate the SOX2+ and HuC/D+ cells (Ie–Ih,Je–Jh). Scale bar: 50 μm. Zoom scale bar: 20 μm. (K–M) Summary of data showing that VD decreased HuC/D+, SOX2+, and HuC/D+/SOX2+ cells. N = 5, 8 for Ctrl and VD. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 5. Visual deprivation reduces nuclearized β-catenin and differentiated neurons in the thalamus. (A,B) Representative fluorescent images show β-catenin– and SOX2–labeled cells in the Ctrl (Aa–Ad) and VD (Ba–Bd) thalamus. The white square indicates the β-catenin– and SOX2–labeled cells in the zoom of the thalamus. Arrows indicate the expressions of β-catenin– and SOX2 in the nuclei. Arrowheads indicate that β-catenin was expressed in the cytoplasm (Ae–Ah,Be–Bh). Scale bar: 50 μm, zoom scale bar: 20 μm. (C,D) Summary data show that VD decreased β-catenin nuclear localization in labeled cells. N = 5, 6 for β-catenin and SOX2. (E) Western blot analysis of homogenates from Ctrl and VD–treated brains using the anti-SOX2, anti-β-catenin, or anti-Phospho-β-catenin (P-β-Cat) antibody. (F–H) Summary of data showing the relative intensities of SOX2 ((F), N = 7), β-catenin ((G), N = 10), and P-β-Cat ((H), N = 13) to GAPDH. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 6. SOX2-MO and β-Cat-MO knockdown decreased β-catenin and SOX2 expression. (A) Western blot analysis of homogenates from Ctrl-MO–, SOX2-MO– and β-Cat-MO–transfected brains using the anti-SOX2 or anti-β-catenin antibody. (B) Quantification results show that SOX2 expression was significantly decreased in SOX2-MO or β-Cat-MO expressing cells compared to Ctrl-MO expressing cells. N = 4. (C) Summary of data showing that the relative intensity of the β-catenin group was significantly decreased compared to that of the Ctrl-MO group. N = 12. (D) Representative fluorescent images showing the immunostaining of β-catenin and BrdU in the Ctrl-MO– (Da–Dc) or β-Cat-MO–transfected (Dd–Df) cells. Scale bar: 20 μm. (E) Summary data show that the knockdown of β-catenin increased the number of BrdU+ cells. N = 7, 7 for Ctrl-MO and β-Cat-MO. (F) Representative images (Fa–Ff) showing the immunostaining of β-catenin (Fa,Fd) and HuC/D (Fb,Fe). Scale bar: 20 μm. (G) Summary data show that the knockdown of β-catenin decreased the number of HuC/D+ cells. N = 8, 4 for Ctrl-MO and β-Cat-MO. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 7. The visual deprivation-induced decrease in SOX2+ and β-catenin+ cells was prevented by TDZD-8. (A,B) Western blot analysis showing that the relative intensity of β-catenin to GAPDH was significantly decreased by IWR-1-endo but increased by TDZD-8. N = 4. (C,D) Western blot analysis showing that VD–induced decrease in β-catenin expression was blocked by TDZD-8 treatment. N = 8. (E,F) VD–induced increase in P-β-Cat was prevented by TDZD-8 treatment. N = 5. (G) Representative immunofluorescent images showing SOX2– and β-catenin–labeled cells in the thalamus of Ctrl (Ga–Gd), VD (Ge–Gh), TDZD-8 (Gi–Gl), and VD + TDZD-8 (Gm–Gp) tadpoles. Arrows indicate the double–labeling cells in the thalamus. Scale bar: 20 μm. (H,I) Summary of data showing that VD decreased SOX2+ (H) and β-catenin+ (I) cells. TDZD-8 prevented the VD–induced decrease in SOX2+ and β-catenin+ cells in the thalamus. N = 7, 4, 5, 5 for Ctrl, VD, TDZD-8, VD + TDZD-8. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 8. SOX2 is selectively associated with β-catenin. (A) Coimmunoprecipitation of SOX2 with β-catenin. N = 3 experiments. (B) Coimmunoprecipitation of β-catenin with SOX2. N = 4 experiments. (C) Coimmunoprecipitation of SOX2 with β-catenin in Ctrl and VD–treated thalamus. (D) Summary of data showing that VD increases the interaction between SOX2 with β-catenin. N = 6. * p < 0.05. (E,F) Coimmunoprecipitation in homogenates from the mouse thalamus. Coimmunoprecipitation of β-catenin with SOX2 (E). Coimmunoprecipitation of SOX2 with β-catenin (F). N = 2 experiments.

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	Figure 1. SOX2+ cells are distributed in the optic tectum and thalamus. (A) Representative images of a tadpole (left) and whole-mount immunofluorescent staining with an anti-SOX2 antibody (right) at stage 49 Xenopus. The red square indicates the whole optic tectum and the thalamus. Scale bar: 100 μm. (B) Representative images showing the colabeling with SOX2 and Nkx2.2 in the brain. The dotted white line indicates the outline of the optic tectum (OT). The white line represents the boundary of the SOX2 and Nkx2.2 immunoreactive thalamus (Th). Scale bar: 50 μm. (C) Six representative coronal planes of the whole brain with SOX2 immunostaining are shown at stage 49 Xenopus (Ca–Cf). Scale bar: 20 μm. (D) One representative sagittal section was immunostained with an anti-SOX2 antibody. The white lines (a–e) depict the positions of coronal sections for (Ca–Ce). Scale bar: 100 μm. III: third ventricle; IV: fourth ventricle; A: anterior; ABB: alar basal boundary; BH: basal hypothalamus; c-Th: caudal thalamus; D: dorsal; Hb: habenula; M: middle ventricle; Mes: mesencephalon; oc: optic chiasm; P: posterior; p1–3: prosomere1–3; Pa: pallium; PTh: prethalamus; r1–r7: rhomeres1–7; Spa: subpallium; OT: optic tectum; R: rostral; r-Th: rostral thalamus; Th: thalamus; Zli: zona limitans intrathalamica.
	Figure 2. The majority of thalamic SOX2+ cells are HuC/D+ or tubulin+ neurons. (A) Colabeling of SOX2 and BrdU showing that only a few SOX2+ cells (Aa) are BrdU+ cells (Ab) in the thalamus. Arrowheads indicate the SOX2− and BrdU+ cells (Ac). Arrows indicate the SOX2+ and BrdU+ cells (Ac). Scale bar: 20 μm. (B) Coimmunostaining of BrdU and PCNA in the thalamus. Arrows indicate the BrdU+ and PCNA+ cells. Scale bar: 20 μm. (C) Coimmunostaining of SOX2 (Ca) and PCNA (Cb) in the thalamus. Arrowheads indicate the SOX2+ and PCNA− cells. Arrows indicate the SOX2+ and PCNA+ neurons (Cc,Cd). Scale bar: 20 μm. (D) Coimmunostaining of SOX2 (Da) and HuC/D (Db) in the thalamus. Arrows indicate the SOX2+ and HuC/D+ neurons (Dc,Dd). Scale bar: 20 μm. (E) Colabeling of SOX2 (Ea) and tubulin (Eb) in the thalamus. Arrows indicate the SOX2+ and tubulin+ neurons (Ec,Ed). Scale bar: 20 μm. (F) Thalamic cells were immunostained with an anti-SOX2 antibody (red, Fb) followed by transfection with pSOX2::GFP (green, Fa), showing that the SOX2+ cells exhibited neuronal morphology with predicted dendrites (arrowheads, Fc,Fd). Arrows indicate the GFP–expressing and SOX2–immunoreactive cells. Scale bar: 20 μm.
	Figure 3. Visual deprivation alters synaptic transmission and dendritic growth. (A) Representative electrophysiological recordings of visual stimuli–evoked excitatory compound synaptic currents (eCSCs) in Ctrl-OT, Ctrl-Th, and VD-Th neurons in response to full–field light ON and OFF visual stimuli at an intensity of 20 cd/cm−2. Arrows indicate the onset and offset of the delay. Scale bar: 30 pA, 1 s. (B,C) Statistical results show the delay (B) and charge transfer (C) of eCSCs in Ctrl-OT, Ctrl-Th, and VD-Th neurons. White circles indicate the individual data. N = 17, 28, 17 for Ctrl-OT, Ctrl-Th, and VD-Th groups. (D) Representative recordings of optic chiasm stimuli–evoked excitatory postsynaptic currents (EPSCs). Scale bar: 20 pA, 20 ms. (E,F) Statistical results show the delay (E) and amplitude (F) of EPSCs. N = 14, 15, 13 for Ctrl-OT, Ctrl-Th, and VD-Th groups. (G) Three representative recording traces show current injection–induced spikes in Ctrl-OT, Ctrl-Th, and VD-Th neurons. Scale bar: 30 mV, 40 ms. (H) Statistical results show that the number of action potentials was significantly decreased in VD-Th neurons compared to Ctrl-Th neurons. N = 21, 16, 19 for Ctrl-OT, Ctrl-Th, and VD-Th groups. (I) Three representative neurons (upper panel) and their reconstructed images (lower panel) show pSOX2::GFP–expressing neurons in Ctrl-OT, Ctrl-Th, and VD-Th groups. Arrowheads indicate axons. Scale bar: 10 μm. (J) Total dendritic branch length (TDBL) was significantly decreased over 48 h in VD-Th neurons compared to Ctrl-Th neurons. (K) Total branch tip number (TBTN) was significantly decreased in VD-Th neurons compared to Ctrl-Th neurons. N = 19, 13, 13 for Ctrl-OT, Ctrl-Th, and VD-Th groups. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 4. Visual deprivation changes the balance between proliferation and differentiation in the thalamus. (A,B) Representative fluorescent images showing BrdU– (Ab) and SOX2–labeled (Aa) cells in Ctrl (Aa–Ac) and VD (Ba–Bc) thalamus. The white square indicates the BrdU– and SOX2–labeled cells in the zoomed–in thalamus (Aa–Ac). White dotted lines indicate the boundary of the thalamus (Ac). Arrows indicate the SOX2+ and BrdU+ cells (Ad–Af,Bd–Bf). Scale bar: 50 μm. Zoom Scale bar: 20 μm. (C–E) Summary data show that VD increased BrdU+ cells (C), decreased SOX2+ cells (D), and increased SOX2+/BrdU+ cells (E). N = 6, 6 for Ctrl and VD. (F) A representative immunofluorescent image showing z–stack for SOX2– and BrdU–labeled cells. Scale bar: 20 μm. (G,H) Summary data showing that VD increased BrdU+ cells (G) and decreased SOX2+ cells (H) in VD–treated tadpoles. N = 4, 6 for Ctrl and VD. (I,J) Representative immunofluorescent images showing SOX2– and HuC/D–labeled cells in the thalamus of Ctrl (Ia–Id) and VD (Ja–Jd) tadpoles. The white square indicates the zoomed–in images. Arrows indicate the SOX2+ and HuC/D+ cells (Ie–Ih,Je–Jh). Scale bar: 50 μm. Zoom scale bar: 20 μm. (K–M) Summary of data showing that VD decreased HuC/D+, SOX2+, and HuC/D+/SOX2+ cells. N = 5, 8 for Ctrl and VD. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 5. Visual deprivation reduces nuclearized β-catenin and differentiated neurons in the thalamus. (A,B) Representative fluorescent images show β-catenin– and SOX2–labeled cells in the Ctrl (Aa–Ad) and VD (Ba–Bd) thalamus. The white square indicates the β-catenin– and SOX2–labeled cells in the zoom of the thalamus. Arrows indicate the expressions of β-catenin– and SOX2 in the nuclei. Arrowheads indicate that β-catenin was expressed in the cytoplasm (Ae–Ah,Be–Bh). Scale bar: 50 μm, zoom scale bar: 20 μm. (C,D) Summary data show that VD decreased β-catenin nuclear localization in labeled cells. N = 5, 6 for β-catenin and SOX2. (E) Western blot analysis of homogenates from Ctrl and VD–treated brains using the anti-SOX2, anti-β-catenin, or anti-Phospho-β-catenin (P-β-Cat) antibody. (F–H) Summary of data showing the relative intensities of SOX2 ((F), N = 7), β-catenin ((G), N = 10), and P-β-Cat ((H), N = 13) to GAPDH. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 6. SOX2-MO and β-Cat-MO knockdown decreased β-catenin and SOX2 expression. (A) Western blot analysis of homogenates from Ctrl-MO–, SOX2-MO– and β-Cat-MO–transfected brains using the anti-SOX2 or anti-β-catenin antibody. (B) Quantification results show that SOX2 expression was significantly decreased in SOX2-MO or β-Cat-MO expressing cells compared to Ctrl-MO expressing cells. N = 4. (C) Summary of data showing that the relative intensity of the β-catenin group was significantly decreased compared to that of the Ctrl-MO group. N = 12. (D) Representative fluorescent images showing the immunostaining of β-catenin and BrdU in the Ctrl-MO– (Da–Dc) or β-Cat-MO–transfected (Dd–Df) cells. Scale bar: 20 μm. (E) Summary data show that the knockdown of β-catenin increased the number of BrdU+ cells. N = 7, 7 for Ctrl-MO and β-Cat-MO. (F) Representative images (Fa–Ff) showing the immunostaining of β-catenin (Fa,Fd) and HuC/D (Fb,Fe). Scale bar: 20 μm. (G) Summary data show that the knockdown of β-catenin decreased the number of HuC/D+ cells. N = 8, 4 for Ctrl-MO and β-Cat-MO. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 7. The visual deprivation-induced decrease in SOX2+ and β-catenin+ cells was prevented by TDZD-8. (A,B) Western blot analysis showing that the relative intensity of β-catenin to GAPDH was significantly decreased by IWR-1-endo but increased by TDZD-8. N = 4. (C,D) Western blot analysis showing that VD–induced decrease in β-catenin expression was blocked by TDZD-8 treatment. N = 8. (E,F) VD–induced increase in P-β-Cat was prevented by TDZD-8 treatment. N = 5. (G) Representative immunofluorescent images showing SOX2– and β-catenin–labeled cells in the thalamus of Ctrl (Ga–Gd), VD (Ge–Gh), TDZD-8 (Gi–Gl), and VD + TDZD-8 (Gm–Gp) tadpoles. Arrows indicate the double–labeling cells in the thalamus. Scale bar: 20 μm. (H,I) Summary of data showing that VD decreased SOX2+ (H) and β-catenin+ (I) cells. TDZD-8 prevented the VD–induced decrease in SOX2+ and β-catenin+ cells in the thalamus. N = 7, 4, 5, 5 for Ctrl, VD, TDZD-8, VD + TDZD-8. * p < 0.05, p < 0.01, * p < 0.001.
	Figure 8. SOX2 is selectively associated with β-catenin. (A) Coimmunoprecipitation of SOX2 with β-catenin. N = 3 experiments. (B) Coimmunoprecipitation of β-catenin with SOX2. N = 4 experiments. (C) Coimmunoprecipitation of SOX2 with β-catenin in Ctrl and VD–treated thalamus. (D) Summary of data showing that VD increases the interaction between SOX2 with β-catenin. N = 6. * p < 0.05. (E,F) Coimmunoprecipitation in homogenates from the mouse thalamus. Coimmunoprecipitation of β-catenin with SOX2 (E). Coimmunoprecipitation of SOX2 with β-catenin (F). N = 2 experiments.