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. 2019 Jan 1;30(1):56-68.
doi: 10.1091/mbc.E18-05-0286. Epub 2018 Oct 31.

Ventricular-subventricular zone fractones are speckled basement membranes that function as a neural stem cell niche

Yuya Sato  1 , Daiji Kiyozumi  1 , Sugiko Futaki  1 , Itsuko Nakano  1 , Chisei Shimono  1 , Naoko Kaneko  2 , Masahito Ikawa  3 , Masaru Okabe  3 , Kazunobu Sawamoto  2   4 , Kiyotoshi Sekiguchi  1   5
Affiliations

Ventricular-subventricular zone fractones are speckled basement membranes that function as a neural stem cell niche

Yuya Sato et al. Mol Biol Cell. .

Abstract

Neural stem cells (NSCs) are retained in the adult ventricular-subventricular zone (V-SVZ), a specialized neurogenic niche with a unique cellular architecture. It currently remains unclear whether or how NSCs utilize basement membranes (BMs) in this niche. Here, we examine the molecular compositions and functions of BMs in the adult mouse V-SVZ. Whole-mount V-SVZ immunostaining revealed that fractones, which are fingerlike processes of extravascular BMs, are speckled BMs unconnected to the vasculature, and differ in their molecular composition from vascular BMs. Glial fibrillary acidic protein (GFAP)-positive astrocytes and NSCs produce and adhere to speckled BMs. Furthermore, Gfap-Cre-mediated Lamc1flox(E1605Q) knockin mice, in which integrin-binding activities of laminins are specifically nullified in GFAP-positive cells, exhibit a decreased number and size of speckled BMs and reduced in vitro neurosphere-forming activity. Our results reveal niche activities of fractones/speckled BMs for NSCs and provide molecular insights into how laminin-integrin interactions regulate NSCs in vivo.

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Figures

FIGURE 1:
FIGURE 1:
Molecular profiling of BM proteins in the adult mouse V-SVZ. (A) Schematic diagram depicting the position (arrow) of the images shown in the figure panels. (B) Morphological differences between vascular BMs and fractones. Left, vascular BMs are immunoreactive for both anti-panLM (green) and anti-PECAM (red) antibodies, while fractones are negative for the anti-PECAM antibody. Nuclei were stained with TO-PRO-3 (blue). Asterisks, vascular BMs; closed arrowheads, fractones; open arrowheads, fractones negative for the anti-PECAM antibody. Scale bar, 10 μm. Right, box-and-whisker plot showing the distributions of diameters for vascular BMs and fractones. (C, D) Immunohistochemical localization of individual LM subunits (C, red) and representative BM proteins (D, red) in the adult mouse V-SVZ. Anti-panLM or anti-perlecan antibodies (green) were used as BM markers. Nuclei were stained with Hoechst 33342 (blue). Asterisks, vascular BMs; arrowheads, fractones. Scale bars, 10 μm. (E) Summary of the BM protein profiles in the V-SVZ. See also Supplemental Figure S1.
FIGURE 2:
FIGURE 2:
Fractones are visible as BM speckles in whole-mount V-SVZ immunostaining. (A) Whole-mount images of a mouse V-SVZ stained with an anti-panLM antibody. Left, image from the ventricle. Right, three-dimensional reconstruction of the left panel. See also Supplemental Movie S1. (B) Whole-mount V-SVZs were labeled with antibodies against individual BM proteins (red) together with anti-panLM or anti-LMγ1 antibodies (green). Each panel shows a merged image (top) and higher-magnification images (bottom) of the two channels in the boxed area. Asterisks, blood vessel BMs; arrowheads, speckled BMs. Scale bars, 10 μm.
FIGURE 3:
FIGURE 3:
Speckled BMs are produced postnatally and localized between ependymal cells. (A) Time series observations of speckled BMs at the V-SVZ labeled with anti-panLM (green), anti-β-catenin (red), and anti-GFAP (cyan) antibodies. Images show superficial optical slices. Scale bar, 20 µm. (B–D) Representative images of V-SVZs colabeled with an anti-panLM antibody (green) and antibodies against (B) β-catenin (ependymal cell-cell junctions), (C) Dcx (neuroblasts), or (D) GFAP (NSCs/astrocytes). Orthogonal images at the yellow and/or magenta lines are shown in the right and bottom boxes, respectively (B, C). Speckled BMs (arrowheads) are located between ependymal cells and colocalized with GFAP-positive cells. See also Supplemental Figure S3. (E) Quantification of ependymal cells (n = 1204), neuroblasts (n = 1997), and apical type B cells (n = 639) adhering to speckled BMs. Randomly obtained images (n = 8–10) from three mice were analyzed. Error bars represent SD. (F) EdU was administered to P21 mice once a day for 1 wk and chased for 2 wk, followed by whole-mount V-SVZ staining to visualize label-retaining cells. Note that GFAP+/EdU+ cells adhere to blood vessels (open arrowhead) and speckled BMs (closed arrowhead) and have an apical process that makes contact with the ventricle (arrow). Scale bars, 10 μm.
FIGURE 4:
FIGURE 4:
GFAP-positive NSCs produce speckled BMs. (A) Schematic diagrams of the wild-type Lama5 allele, targeting vector, targeted floxed(neo) allele, and floxed allele. Cre-mediated recombination removes exon 3, resulting in a frame-shift error. Open boxes, exons; closed triangles, loxP sites; gray ovals, FRT sites. Whole-mount V-SVZs from Tie2-Cre;Lama5fl/fl (B) and Gfap-Cre;Lama5fl/fl (C) mice were labeled with anti-panLM (green) and anti-LMα5 (red) antibodies. Each panel shows a merged image (top) and higher-magnification images (bottom) of the two channels in the boxed area. Asterisks, vascular BMs; closed arrowheads, speckled BMs; open arrowheads, speckled BMs without LMα5 deposition. (D) Quantification of the speckled BMs positive for panLM or LMα5. Randomly obtained images from three control littermates (n = 6 images in total) and three Gfap-Cre;Lama5fl/fl mice (n = 7 images in total) were analyzed. Data represent means ± SEM. ***p < 0.001. (E, F) Whole-mount V-SVZs from Gfap-Cre;Lama5fl/fl mice were labeled with anti-panLM (green) and anti-LMα3 (red) antibodies. Each panel shows a merged image (top) and higher-magnification images (bottom) of the two channels in the boxed area. Note that immunoreactivity for LMα3 is up-regulated two- to threefold in Gfap-Cre;Lama5fl/fl mice compared with control mice (F). ***p < 0.001. Scale bars, 10 μm.
FIGURE 5:
FIGURE 5:
Integrin-binding activity of speckled BMs. (A) Cryosections of adult mouse brains were incubated with recombinant integrins (green) in the presence of 1 mM MnCl2 (left ) or 10 mM EDTA (right). An anti-LMα5 (red) antibody was used as a marker for speckled and vascular BMs. Nuclei were stained with Hoechst 33342 (blue). Each panel shows a merged image (top) and higher magnification images (bottom) of the two channels in the boxed area. Arrowheads, speckled BMs. See also Supplemental Figure S4. (B) Cryosections of adult mouse brains were labeled with anti-α6 integrin (green) and anti-panLM (red) antibodies. Closed and open arrowheads indicate speckled BMs positive and negative for α6 integrin, respectively. Scale bars, 10 μm.
FIGURE 6:
FIGURE 6:
Disruption of LM integrin-binding activity causes impaired formation of speckled BMs. (A) Schematic model of a γ1-containing LM. The Glu (E) residue at the third position from the C-terminus of LMγ1 (highlighted by a yellow sphere in the inset) is a prerequisite for interactions with integrins. (B) Schematic diagrams of the wild-type Lamc1 allele, targeting vector, targeted floxedcEQ(neo) allele, and floxedcEQ allele. Cre-mediated recombination removes the wild-type exon 28, resulting in transcription of exon 28 (EQ). Open boxes, exons; closed triangles, loxP sites; gray ovals, FRT sites. (C) Cryosections of adult brains from Gfap-Cre;Lamc1cEQ/cEQ mice were probed with α3β1 integrin (green) in the presence of 1 mM MnCl2. An anti-LMα5 (red) antibody was used as a marker for speckled BMs. Nuclei were stained with Hoechst 33342 (blue). Open and closed arrowheads indicate speckled BMs with and without bound α3β1 integrin, respectively. Scale bars, 10 μm. (D) Whole-mount V-SVZs from Gfap-Cre;Lamc1cEQ/cEQ mice were labeled with an anti-panLM antibody. Scale bars, 20 µm. Quantification of speckled BMs (E) and histogram of speckled BM areas (F). Data represent means ± SEM in 20 and 15 randomly obtained images from four control (CTL) and three Gfap-Cre;Lamc1cEQ/cEQ mice, respectively. *p < 0.05; **p < 0.01; ***p < 0.001.
FIGURE 7:
FIGURE 7:
NSCs from conditional Lamc1 mutant mice exhibit reduced proliferation in vitro. (A) Whole-mount V-SVZs from Gfap-Cre;Lamc1cEQ/cEQ mice were labeled with anti-panLM (green) and anti-GFAP (red) antibodies. Data show superficial optical slices at the ependymal cell layer. Scale bars, 10 μm. Numbers of apical B cells making contact with speckled BMs (B), total numbers of apical B cells (C), and numbers of GFAP-high apical B cells exhibiting saturated GFAP signals (D). Data represent means ± SEM of cell numbers in 20 and 15 randomly obtained images from four control littermates (CTL) and three Gfap-Cre;Lamc1cEQ/cEQ mice, respectively. (E) Representative images of primary neurospheres generated from Gfap-Cre;Lamc1cEQ/cEQ and control Lamc1cEQ/+ mice. Arrowheads indicate neurospheres. (F) Quantification of cells plated at a density of 4000 cells/cm2 with EGF and basic FGF under nonadherent conditions and cultured for 7 d. Data represent means ± SD of three independent examinations. *p < 0.05; **p < 0.01.
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