The niche factor syndecan-1 regulates the maintenance and proliferation of neural progenitor cells during mammalian cortical development

Abstract

Neural progenitor cells (NPCs) divide and differentiate in a precisely regulated manner over time to achieve the remarkable expansion and assembly of the layered mammalian cerebral cortex. Both intrinsic signaling pathways and environmental factors control the behavior of NPCs during cortical development. Heparan sulphate proteoglycans (HSPG) are critical environmental regulators that help modulate and integrate environmental cues and downstream intracellular signals. Syndecan-1 (Sdc1), a major transmembrane HSPG, is highly enriched in the early neural germinal zone, but its function in modulating NPC behavior and cortical development has not been explored. In this study we investigate the expression pattern and function of Sdc1 in the developing mouse cerebral cortex. We found that Sdc1 is highly expressed by cortical NPCs. Knockdown of Sdc1 in vivo by in utero electroporation reduces NPC proliferation and causes their premature differentiation, corroborated in isolated cells in vitro. We found that Sdc1 knockdown leads to reduced levels of β-catenin, indicating reduced canonical Wnt signaling. Consistent with this, GSK3β inhibition helps rescue the Sdc1 knockdown phenotype, partially restoring NPC number and proliferation. Moreover, exogenous Wnt protein promotes cortical NPC proliferation, but this is prevented by Sdc1 knockdown. Thus, Sdc1 in the germinal niche is a key HSPG regulating the maintenance and proliferation of NPCs during cortical neurogenesis, in part by modulating the ability of NPCs to respond to Wnt ligands.

Figure 1. Sdc1 is highly expressed in the germinal zones of the developing mouse cerebral cortex.

(A) Schematic to indicate area of analysis. (B) Sdc1 expression in a coronal section of the E15 mouse cortex (area indicated by the box in Fig. 1A). Sdc1 is highly expressed in both the VZ and SVZ (scale bar = 200 um). (C) Higher magnification of the boxed area indicated in Fig. 1B (scale bar = 100 um). (D) Sdc1 expression in the neural germinal zone where Sox2+ RGCs are located (scale bar = 100 um). LV, Lateral ventricle; CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone.

Figure 2. Sdc1 knockdown reduces NPC maintenance and proliferation in vitro.

(A) The FUWG H1 lentiviral vector expresses a shRNA under the H1 promoter and eGFP under the ubiquitin promoter. Two different shRNAs (Sdc1 UTR and Sdc1 ORF) specifically targeting Sdc1 inhibit Sdc1 expression at mRNA (B) and protein (C) levels. (D) E11 cortical NPCs were treated with scrambled control, Sdc1 UTR and Sdc1 ORF lentiviruses and assessed at 4 DIV. Knocking down Sdc1 reduces the NPC population and their proliferation, indicated by Nestin and Ki67, respectively; knocking down Sdc1 also promotes neuronal differentiation, indicated by Tuj1 staining (scale bar = 50 um). Data are quantified in (E). (F) Knocking down Sdc1 does not increase cell apoptosis, assessed by active Caspase-3. (G) Knocking down Sdc1 reduces neurosphere generation, assessed at 7 DIV and quantified in (H) (N = 3) (scale bar = 200 um). Error bars represent S.E.M, *P<0.05, **P<0.01, ***p<0.001.

Figure 3. Knockdown constructs shows Sdc1 is necessary for NPC maintenance and proliferation in vivo.

In utero electroporation was performed at E13 and results assessed at E15. (A) Sdc1 UTR and ORF shRNAs reduced NPC proliferation and promoted neuronal differentiation, indicated by Ki67 and Tuj1, respectively (scale bar = 50 um), as quantified in (B) (scrambled, N = 4; Sdc1 UTR, N = 3; Sdc1 ORF, N = 5). (C) Sdc1 UTR and ORF shRNAs decreased both RGC and IPC populations, indicated by reduced Pax6 and Tbr2, respectively (scale bar = 50 um) and quantified in (D) (scrambled, N = 4; Sdc1 UTR, N = 3; Sdc1 ORF, N = 5). (E) The distribution of eGFP⁺ cells in the cortical layers (scrambled, N = 5; Sdc1 ORF, N = 4). CP, cortical plate; IZ, intermediate zone; SVZ, subventricular zone; VZ, ventricular zone. Error bars represent S.E.M, *P<0.05, **P<0.01, ***p<0.001.

Figure 4. Sdc1 modulates NPC to response to Wnt ligand.

(A) Knocking down Sdc1 reduced the total β-catenin protein level compared to scrambled control group, quantified in (B) after normalized to GAPDH. (C) Schematic depicting the GSK 3β inhibitor (Chir 99021) treatment experiment (D) Chir 99021 treatment partially rescued proliferation of Sdc1 UTR or Sdc1 ORF treated NPCs (scale bar = 50 um), assessed by Ki67 and quantified in (E). (F) Schematic diagram depicting the Wnt3a conditioned medium treatment experiment. (G) Wnt3a increased proliferation in scrambled control treated NPCs, but had no significant effect in Sdc1 UTR or Sdc1 ORF treated NPCs (scale bar = 50 um), assessed by Ki67 and quantified in (H). Error bars represent S.E.M, *P<0.05, **P<0.01, ***p<0.001, NS = not significant.