beta1 integrin maintains integrity of the embryonic neocortical stem cell niche

Abstract

During embryogenesis, the neural stem cells (NSC) of the developing cerebral cortex are located in the ventricular zone (VZ) lining the cerebral ventricles. They exhibit apical and basal processes that contact the ventricular surface and the pial basement membrane, respectively. This unique architecture is important for VZ physical integrity and fate determination of NSC daughter cells. In addition, the shorter apical process is critical for interkinetic nuclear migration (INM), which enables VZ cell mitoses at the ventricular surface. Despite their importance, the mechanisms required for NSC adhesion to the ventricle are poorly understood. We have shown previously that one class of candidate adhesion molecules, laminins, are present in the ventricular region and that their integrin receptors are expressed by NSC. However, prior studies only demonstrate a role for their interaction in the attachment of the basal process to the overlying pial basement membrane. Here we use antibody-blocking and genetic experiments to reveal an additional and novel requirement for laminin/integrin interactions in apical process adhesion and NSC regulation. Transient abrogation of integrin binding and signalling using blocking antibodies to specifically target the ventricular region in utero results in abnormal INM and alterations in the orientation of NSC divisions. We found that these defects were also observed in laminin alpha2 deficient mice. More detailed analyses using a multidisciplinary approach to analyse stem cell behaviour by expression of fluorescent transgenes and multiphoton time-lapse imaging revealed that the transient embryonic disruption of laminin/integrin signalling at the VZ surface resulted in apical process detachment from the ventricular surface, dystrophic radial glia fibers, and substantial layering defects in the postnatal neocortex. Collectively, these data reveal novel roles for the laminin/integrin interaction in anchoring embryonic NSCs to the ventricular surface and maintaining the physical integrity of the neocortical niche, with even transient perturbations resulting in long-lasting cortical defects.

Figure 1. β1 integrin is expressed by radial glia and proliferating cells at the ventricular surface during neurogenesis.

(A–J) Fluorescent micrographs of E13 coronal (A, B, E–J) or E16 sagittal (C, D) sections immunostained as indicated. Both at the rostral (A–C, E–J) and medial (D) levels, β1 integrin is expressed in PH3+ proliferating cells (A, high magnification B) and radial glia RC2+ cells (E–J) at the apical surface but not in Tuj1+ neurons (C, D). All scale bars represent 100 µm.

Figure 2. Cell proliferation is disrupted in the embryonic telencephalon after intraventricular injection of a β1 integrin blocking antibody.

(A, B) Fluorescence micrographs of PH3 expression (green, dapi-counterstained nuclei in blue) after antibody injection at E12.5. Note the increase in PH3+ cells away from the ventricular surface in the β1 integrin blocking antibody-injected embryos (B), white arrows, compared to injected controls (A). (C, D) Quantification of PH3+ cells at the ventricular surface (VS) and nonventricular surface (nVS) after antibody injection at E12.5 (**, p<0.01 as assessed by a paired two-tailed t-test, C) and E15.5 (***, p<0.001, unpaired two-tailed t-test, D). (E, F) Fluorescence micrographs of BrdU expression (green, dapi-counterstained nuclei in blue) after antibody injection at E12.5, pulsed 1 h prior to sacrifice. Note the increase in the amount of BrdU+ cells in the telencephalon. (G, H) BrdU labelling index in E12.5- (G) and E15.5- (H) injected embryos pulsed with BrdU 1 h (G) or 6 h (H) prior to sacrifice. Note the increase in labelling index at 80–110 µm in the β1 integrin blocking antibody embryos, **, p<0.01 for bin 10 and 11, and *, p<0.05 for bin 12 calculated using Boneferroni post hoc tests. All scale bars represent 50 µm.

Figure 3. Intraventricular injection of β1 integrin blocking antibody prevents VZ horizontal mitotic cleavages throughout neurogenesis.

(A, B) Micrographs of cells stained with propidium iodide in E14 telencephalon 18 h post ITC (A) or β1 integrin blocking antibody (B) injection. (C–E) Distribution of mitotic rostral (C), medial (D), and caudal (E) progenitors according to their angle of cleavage in ITC (black symbols) or β1 integrin blocking antibody (white symbols)-injected forebrains. Boxed regions highlight the paucity of horizontal cleavages (lower than 60 degrees) in Ha2/5-injected embryos. Scale bar represents 15 µm.

Figure 4. Inhibition of β1 integrin signalling results in NSC detachment.

(A–F) Confocal fluorescence micrographs showing the VZ/SVZ of embryos injected at E15.5 with either an ITC (C, D) or β1 integrin blocking antibody (A, B, E, F) and simultaneously electroporated with CAG-RFP (red) and stained for phalloidin (green) 18 h later. (A) Note the bipolar morphology of the cell marked with an arrowhead and the dystrophic basal processes of detached cells (white arrows in A and B). (D, F) White and yellow dots represent soma and apical processes, respectively. (G, H) Quantification of the ratio of soma to apical processes in the co-injected/electroporated mouse brains at E13.5 (G) or E15.5 (H) and analyzed 18 h later. (I, J) Quantification of the percentage of apical processes still attached at the ventricular surface in the injected/electroporated brains at E13.5 (I) or E15.5 (J) and analyzed 18 h later. *, p<0.05; unpaired two-tailed t-test. All scale bars represent 50 µm.

Figure 5. Time lapse analysis of NSC morphology and detachment after β1 integrin signalling blockade at the VZ surface.

(A) Experimental paradigm of the multiphoton time lapse experiments. Organotypic brain slices were prepared 24 h after in utero electroporation with eGFP-F DNA into a wild-type E14.5 embryo. A drop of growth factor–reduced matrigel containing either ITC control or β1 blocking antibody was placed into the lateral ventricle. Automated multipoint scanning using a multiphoton laser (850 nm) was used to simultaneously monitor the behavior of two electroporated slices containing the drop of matrigel with either β1 blocking or the ITC control antibody. (B, C) Time lapse images of 10h recording of VZ neuroepithelial integrity in the presence of ITC control (B) or β1 blocking (C) antibody. (B) Slices in the presence of ITC antibody retain bipolar eGFP-F+ cells with straight processes (green arrows) and end-feet attached to the ventricular surface (green arrowheads) throughout the 10 h of recording. (C) In presence of β1 blocking antibody, the radial morphology of the eGFP-F cells is progressively disrupted; both basal and apical processes appear convoluted (red arrows) and the end-feet are detached from the ventricular surface (red arrowheads). Scale bar represents 25 µm.

Figure 6. VZ cell mitotic parameters and apical process attachment are altered in Lnα2^−/− deficient brains.

(A, B) Micrographs of dapi (blue) and BrdU (green) in E16 telencephalon of wild-type (A) and Lnα2^−/− (B) littermates after a 1 h BrdU pulse, scale bar, 100 µm. (C) Quantification of BrdU+ cells in the intermediate zone, outside the VZ/SVZ as marked by white dashed line in micrographs. Statistical analysis using an unpaired two-tailed t-test revealed a statistically significant difference ***, p<0.001. (D) Distribution of mitotic rostral and medial progenitors according to their cleavage angle. Boxed regions highlight the paucity of horizontal cleavages (lower than 60 degrees) in Lnα2^−/− embryos. n = 3 wild-type and four Lnα2^−/− embryos from one litter, ±SEM. (E, F) Confocal fluorescence micrographs showing VZ/SVZ of Lnα2^−/− mutant (F) or wild-type littermates (E) embryos electroporated at E15.5 with CAG-RFP (red) and stained for phalloidin (green) 18 h later. (G) Quantification of the ratio of soma to apical processes (n = 2 wild-type and 2 Lnα2^−/− embryos from one litter, ±SEM). (H) Quantification of the percentage of apical processes still attached at the ventricular surface (n = 2 wild-type and 2 Lnα2^−/− embryos from one litter, ±SEM). *, p<0.05; unpaired two-tailed t-test. Scale bars represents 100 µm (A) and 50 µm (E, F).

Figure 7. Inhibition of β1 integrin signalling at E15.5 alters cortical cell layering at P4.

Fluorescence micrographs of the P4 telencephalon stained with Topro-3 after injection of the ITC (A) or β1 (B) integrin blocking antibody at E15. (C) Quantification of cortical layer thickness shows a significant reduction in the thickness of layers I–V after injection of the β1 integrin blocking antibody (layer I: ITC = 71 µm±8, β1 block = 43 µm±2, *, p<0.02; layers II, III, IV: ITC = 214 µm±15, β1 block = 142 µm±13, *, p<0.004; layer V: ITC = 160 µm±10, β1 block = 127 µm±12, *, p<0.04; layer VI: ITC = 190 µm±19, β1 block = 142 µm±21, p<0.1). Fluorescence micrographs of the P4 telencephalon illustrating CAG-RFP+ cells after injection of the ITC (D) or β1 (E) integrin blocking antibody at E15.5. (F) Quantification of the thickness of CAG-RFP+ cell layers in primary motor (PM) and somato-sensory (SS) cortices shows a reduction at the level of the somato-sensory cortex after injection of the β1 integrin blocking antibody (PM: ITC = 104 µm±18, β1 block = 95 µm±16, p<0.7; SS: ITC = 143 µm±35, β1 block = 93 µm±14, p<0.3). n = 2 ITC and 3 β1 integrin blocking antibody-injected embryonic brains, ±SEM. Statistical analysis was done using an unpaired two-tailed t-test, *, p<0.05. All scale bars represent 50 µm.

Figure 8. Model depicting the role of β1 integrin in the VZ.

Schematic (left) showing the normal VZ with NSC attached at the ventricular surface undergoing mitosis in a variety of orientations. After β1 integrin blocking antibody injection (right), NSC detach from the ventricular surface and horizontal cell divisions are no longer present at the ventricular surface.