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. 2017 Dec 12;114(50):E10717-E10725.
doi: 10.1073/pnas.1715772114. Epub 2017 Nov 27.

Smek1/2 Is a Nuclear Chaperone and Cofactor for Cleaved Wnt Receptor Ryk, Regulating Cortical Neurogenesis

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Free PMC article

Smek1/2 Is a Nuclear Chaperone and Cofactor for Cleaved Wnt Receptor Ryk, Regulating Cortical Neurogenesis

Wen-Hsuan Chang et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The receptor-like tyrosine kinase (Ryk), a Wnt receptor, is important for cell fate determination during corticogenesis. During neuronal differentiation, the Ryk intracellular domain (ICD) is cleaved. Cleavage of Ryk and nuclear translocation of Ryk-ICD are required for neuronal differentiation. However, the mechanism of translocation and how it regulates neuronal differentiation remain unclear. Here, we identified Smek1 and Smek2 as Ryk-ICD partners that regulate its nuclear localization and function together with Ryk-ICD in the nucleus through chromatin recruitment and gene transcription regulation. Smek1/2 double knockout mice displayed pronounced defects in the production of cortical neurons, especially interneurons, while the neural stem cell population increased. In addition, both Smek and Ryk-ICD bound to the Dlx1/2 intergenic regulator element and were involved in its transcriptional regulation. These findings demonstrate a mechanism of the Ryk signaling pathway in which Smek1/2 and Ryk-ICD work together to mediate neural cell fate during corticogenesis.

Keywords: Ryk signaling; neural stem cell; neurogenesis; noncanonical Wnt signaling.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Smek1 and Smek2 are identified as Ryk-interacting proteins, and Smek regulates Ryk nuclear translocation. (A) Ryk-ICD interacts with Smek1 and Smek2. Coimmunoprecipitation of overexpressed Ryk-ICD with Smek1 and Smek2 in HEK 293T cells. FUIGW-Flag serves as a negative control. IB, immunoblot. (B) Ryk-ICD binds Smek2 directly. (Left) Coomassie blue staining of purified GST, GST-tagged Ryk-ICD, and His-tagged Smek2. (Right) Immunoblot (IB) of the His-Smek2 and GST-tagged Ryk-ICD GST pull-down assay. The arrow indicates GST-tagged Ryk-ICD, and the asterisk indicates degraded GST-tagged Ryk-ICD. (C) Illustration of conserved domains of Smek protein. The Smek DUF domain mediates Ryk interaction. Immunoblot of the Myc-tagged Ryk and Flag-tagged Smek2 subdomains expressing HEK 293T cell lysate (Left), and immunoprecipitation assay with the FLAG antibody (Right). Arm, armadillo repeat region; DUF625, domain of unknown function 625; NLS, nuclear localization signal; RanBD, Ran-binding domain. (D) Mouse primary NSCs were infected with lentiviruses harboring mCherry only, Smek1-mCherry, Smek2-mCherry, Smek1ΔNLS-mCherry, or Ryk-ICD-GFP. White arrows indicate Smek1-mCherry–infected cells. Orange arrows indicate GFP-ICD–positive, Smek2-mCherry–negative cells. (Scale bars: 10 μm.) (E) Ryk-ICD nuclear localization is correlated with Smek1 expression. Doxycycline-inducible GFP-ICD-Flag lentiviruses were used to infect primary WT neural stem cells. Cells were stained against Smek1 antibody 2 d after doxycycline treatment under differentiation condition. Orange box Inset shows the location of the enlarged images on the right. (Scale bars: 20 μm.) (F) The average intensity of the cell nucleus and neurite was measured by ImageJ. The ratio of the nucleus area and neurite is shown here. [WT (n = 13), dKO (n = 9) ****P < 0.0001.]
Fig. 2.
Fig. 2.
Smek1/2 play important roles in neurogenesis during cortical development. (A) Smek1 and Smek2 expression in E14.5 WT mouse brains. (Scale bar: 200 μm.) (B) Smek1 and Smek2 are expressed in both differentiated neurons and neural stem cells. Map2 marked neurons at the cortical plate (CP). Nestin marked neural stem cells at the ventricular zone (VZ) and subventricular zone (SVZ) of the dorsal and ventral telencephalon. White boxes indicate location of Insets. (Scale bar: 200 μm.) WT and Smek1−/−; Smek2−/− mutant mouse E12.5 (C) and E14.5 (D) brain tissue was collected and stained for Tbr1 and Map2 expression. DAPI served as a nuclear stain. Quantification of C and D is shown in E and F, respectively. (E) The Map2 immunoreactivity in the whole-imaged area (0.36 mm2) was measured and normalized to the value obtained from the control group. (F) Tbr1+ cells in the whole-imaged area (0.36 mm2) were counted and normalized to the value obtained from the control group. LV, left ventricle; Ncx, neocortex. (Scale bars: 100 μm.) Values represent the mean ± SEM (n = 3, **P < 0.01, ***P < 0.001).
Fig. 3.
Fig. 3.
Smek deficiency restricts NSC differentiation and causes neurogenesis defect. E12.5 and E14.5 mouse brain coronal sections from control and Smek1−/−; Smek2−/− mice stained with Sox2 and Tuj1 (A), Phospho-Histone H3 (pH3) (C), BrdU (F), Ki67 (F) antibody, respectively. Smek1+/+; Smek2+/+, Smek1+/−; Smek2+/+, and Smek1+/+; Smek2+/− mice all served as control mice. Cell nuclei were stained with DAPI. (B) Quantification of Sox2+ cells by measuring the thickness of the Sox2+ layer relative to the whole cortex. Four measurements were taken on each image. Values represent mean ± SEM (n = 3; ****P < 0.0001; n.s., not statistically significant). (D) Quantification of Tuj1+ immunoreactivity area normalized to control (n = 3, **P < 0.01, Student’s t test). (E) Quantification of pH3+ cells per area (0.36 mm2) relative to control. VZ and SVZ/IZ were analyzed separately (n = 3; n.s., not statistically significant; *P < 0.1). (F) Cell cycle exit assay showed that Smek deficiency keeps NSCs from exiting the cell cycle. A BrdU pulse was administered 24 h before embryo collection at E12.5. BrdU+Ki67 cells (yellow arrowhead) marked that the cell population left the cell cycle in 24 h. (Scale bar: 100 μm.) (G) Quantification of (F) BrdU+Ki67 cells in each image field (0.72 mm2) were counted and normalized to BrdU+ cells (n = 3, ***P < 0.001). CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone.
Fig. 4.
Fig. 4.
Smek1/2 controls GABAergic neurogenesis by direct regulation of Dlx gene expression. (A) Coronal sections of E14.5 WT cortex stained with Smek1, Smek2, or Dlx2 antibody. Dlx2 marks migrating GABAergic neurons in the neocortex. The close-up Inset shows that all of the Dlx2 staining overlaps with Smek staining. White arrow indicates cells that show both Smek and Dlx2 positive staining. NCx, neocortex. (B) Dlx2 staining showed a decrease of GABAergic neurons in Smek-deficient mice. White arrowheads indicate tangentially migrating Dlx2+ interneurons. Quantification is shown in D. Cx, cortex; LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence. (C) Smek-deficient mice showed less calbindin+ cells at both E12.5 and E14.5 stage. Quantification is shown in E. (Scale bars: A, 100 μm, 50 μm; B, 50 μm, 100 μm, 100 μm; C, 100 μm.) Values represent mean ± SEM (n = 3, **P < 0.01, ***P < 0.001).
Fig. 5.
Fig. 5.
Genome-wide Smek binding sites and expression profiling of Smek-deficient NSCs. (A) Scatter plot of significantly changed genes from RNA sequencing results. Several important neuronal genes were handpicked and labeled. Three pairs of control and Smek1−/−; Smek2−/− NSCs were differentiated for 2 d before RNA extraction, followed by sequencing library preparation. (B) Top 10 IPA annotations of the disease and biofunction of the genes decreased more than twofold in Smek1−/−; Smek2−/− cells relative to controls (q ≤ 0.5). (C) Selected Smek1 binding tracks (ChIP-seq) on the University of California, Santa Cruz (UCSC) Genome Browser. (D) Selected enriched GO terms were generated from the list of Smek1 binding genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) Bioinformatics online tool. FPKM, fragments per kilobase of transcript per million mapped reads.
Fig. 6.
Fig. 6.
Ryk-ICD is recruited to the downstream target genes and Ryk functions with Smek to regulate neurogenesis. (A) Ryk-ICD and Smek1 bind to some similar genomic loci. ChIP-qPCR was performed with FLAG and Smek1 antibody using the FLAG knockin cell line. Primer sets were designed based on the Smek1 ChIP-seq result. (B) Real-time PCR of Dlx1 and Dlx2 transcripts in NSCs from WT, Smek1−/−, and Smek1−/−; Smek2−/− mice. Relative mRNA expression levels are normalized to Gapdh. (C) Smek1 and Ryk-ICD regulate Dlx1/2 transcription activity. The Dlx1/2 intergenic regulatory region was cloned into the pGL3.Basic luciferase reporter. Control vector (FUGW), Smek1, Ryk-ICD, β-galactosidase reporter, and the Dlx1/2 reporter were cotransfected into HEK293T cells. Luciferase activity was normalized to β-galactosidase activity in each condition and then normalized to the “Neg” condition. Neg, the experimental group, which was only transfected with β-galactosidase reporter and control vector. Values represent mean ± SEM (n = 3; *P < 0.1; **P < 0.01; n.s., not significant; unpaired t test). (D) Coexpression of ICD and Smek rescue neurogenesis defect in Ryk−/− NSCs. Ryk+/+ (WT) and Ryk−/− (KO) NSCs were transduced with lentiviruses expressing GFP (FUGW), ICD-GFP, mCherry, Smek1-mCherry, and Smek2-mCherry. Cells were allowed to differentiate for 2 d and were stained with Tuj1. Quantification of neurons is presented by the percentage of Tuj+ mCherry+ GFP+ cells among mCherry+ GFP+ cells. Values represent mean ± SEM in independent cell lines (n = 3, one-way ANOVA, P < 0.0001; post hoc t test results are labeled on the graph as *P < 0.05, ***P < 0.001,; n.s., not significant).

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