Fig 1: Lzts1 is expressed at high levels at the AJs of delaminating cells. a Neuronal delamination and oRG generation by oblique aRG division are two different ways for departure from the apical surface. IP, intermediate progenitor cell. b Single-cell transcriptome profiles24 of E14 cells show increased expression of the lzts1 mRNA during neuronal differentiation. (Affymetrix ID: 1433988_s_at, annotated as C230098O21Rik, a transcript variant of lzts1). c Anti-Lzts1 immunohistochemistry (IHC) of the E14 mouse brain (see also Supplementary Figure 1). d Magnified view of the E13 brain section stained with the anti-Lzts1 antibody showing both dot-like and ring-like expression of Lzts1 at high levels in some apical endfeet. Z-projection images of 8-µm thick slices. e and f Lzts1 IHC of the E14 dorsal forebrain of Tbr2::EGFP Tg mice, with magnified view of Tbr2+Lzts1+ cells in the VZ e and Gadd45g::d4Venus Tg mice f showing that GFP+, Tbr2+, or Venus+ differentiating cells are Lzts1+. g and h Lzts1 is expressed at high levels at the AJs of the apical endfeet of differentiating cells. En face observations of anti-Lzts1 and anti-ZO1 IHC of the E13 dorsal forebrain g or anti-Lzts1 and anti-GFP IHC of E13 Gadd45g::d4Venus Tg mouse dorsal forebrain h from the apical surface. In the magnified view g, dot-like signals may represent the apical endfeet of cells that had almost completed the delamination from the apical surface. i Anti-Lzts1 signal intensities along the cellular junctions were negatively correlated with the apex (apical) area^(1/2), which is proportional to the planar circumferential length of the AJ ring. j and k Ultrastructural localization of Lzts1 in the E14 dorsolateral cerebrum. Immunoelectron microscopy using an anti-Lzts1 antibody shows that intracellular Lzts1 gold particles were closely located to the electron-dense zone of AJs (j, arrowheads). Particles with an intracellular distribution or located adjacent to the plasma membrane were also observed in a subset of the cells (k, arrows, ~ 100 µm from the apical surface). Bars, 100 µm in c, 10 µm in d, magnified view e, 30 µm in e, f, 5 µm in g, h, and 1 µm in magnified view g, j, k
Fig 2: Reduction of neuroprogenitors in Mcph1-?e8 embryonic cortex. (A) Immunofluorescence staining of sagittal sections of the E15.5 brain with Sox2 and Tbr2 antibodies. (B) Quantification of Sox2+ and Tbr2+ cells. N: the number of mice analyzed. The total numbers of cells of control (Con) and mutant (Mcph1-?e8) scored are summarized under the respective graphs. Unpaired Student’s t-test was used for statistical analysis. *, p < 0.05; n.s, not significant.
Fig 3: Prenatal neurogenesis is altered in FA experimental groups at peak neurogenesis. Analysis at E14.5 uncovers shifts in neural proliferation of the FA dietary groups at the extremes that mirror each other. (A–D) EdU/Ki67 analysis reveals a decrease in neuron generation in FA deficiency (0 mg/kg) and excess (20 mg/kg) as demonstrated by a reduced quit (Q-) fraction in both groups, when compared with controls (2 mg/kg) (E). (F–I) Immunofluorescence analysis of Pax6+ cells indicates a significant increase in RGCs of the two FA test groups (J). (K–N) The Tbr2+ IP population is significantly decreased in both FA experimental groups compared with controls (O). SVZ, subventricular zone; VZ, ventricular zone. Significant changes are indicated by asterisks. Scale bar in (A) is 200 µm and in (C) 20 µm.
Fig 4: Loss-of-function of Lzts1 retards radial migration through differentiation. a–d Lzts1-siRNA#1 or a negative control siRNA (siRNA-NC) was in vivo electroporated at E13, and sections of the E14 c or E15 a, b, d brain were examined using IHC with anti-Pax6 a, anti-BrdU (30-min BrdU pulse labeling) b, anti-Tbr2 c, anti-Ki67 and anti-BrdU d antibodies. The mitotic index (MI) d indicates the percentage of Ki67+ cells among BrdU+ GFP+ cells that received BrdU 20 h before fixation. Means ± s.d., a N = 9, 9; b N = 10, 9; d N = 8, 6; d N = 9, nine sections from three embryos per experiment, Wilcoxon rank sum test. e and f CRISPR/Cas9-induced disruption (KO) of lzts1 successfully reduces Lzts1 expression. The hCas9 and guide RNA for lzts1 were co-expressed with EGFP by performing in vivo electroporation at E13, and brains were examined at E15. The majority of EGFP+ cells were negative or exhibit weak Lzts1 immunoreactivity. f Magnified view. Bars, 30 µm. g Lzts1 KO retards the overall radial migration of cells from the apical surface. In vivo electroporation was performed at E13, and the distribution of EGFP+ cells in the cerebral wall was examined at E15 using 10 bins. Means ± s.d., Brunner–Munzel test, *p < 0.05, **p < 0.01, N = 8 (control) and 9 (KO) sections from three embryos per experiment (see also Supplementary Figure 4). h Scheme for Tbr2 expression in differentiating cells. i and j Both Lzts1 KD and KO significantly slowed the migration of Tbr2+ cells from the apical surface. This KD effect was rescued by the siRNA-resistant Lzts1, but not by wild-type Lzts1. In vivo electroporation was performed at E13, and the migration of EGFP+ Tbr2+ cells from the apical surface was examined at E14 in KD experiments or at E15 in KO experiments (Steel–Dwass test for multiple comparisons among the four conditions in KD experiments; Brunner–Munzel test for KO experiments; N = 547, 568, 351, 257, 306, and 344 cells from 8, 8, 11, 9, 7, and 8 sections of 4, 4, 4, 3, 3, and 3 embryos, respectively; Medians with Q1 and Q3 values). Bar, 50 µm. Source data are provided as a Source Data file
Fig 5: Classification of Fezf2-GFP progenitor cells in SGZ.Based on the expression of the cell-fate markers, neural progenitor cells in the sub-granular zone (SGZ) can be categorized into several developmental stages. Among all the Fezf2-GFP-positive cells (A–F), Sox2-positive type 1 cells (A1–F1) are radial glia-like cells (A–A4, C–C4) and can give rise to Sox2+/Tbr2+ type 2a transit-amplifying progenitor cells (B–B4, D–D4). Type 2a cells further differentiate into type 2b neuroblasts that are positive for Tbr2 only (C–C4, F–F4). Type 1 (A3 and D3) and type 2a (B3 and E3) cells formed extensive syncytial connections with other cells from the same developmental stage, as indicated by the gap-junction-permeable avidin-neurobiotin (A–B) staining. Scale bar = 20 µm. (G) Type 1 and type 2a Fezf2-GFP-positive neural progenitor cells were hyperpolarized; by contrast, type 2b cells lacking Kv1.1 channels were significantly more depolarized than the wild-type (WT) cells (n = 14, 14, 9, 10, 9 (WT) and 28, 14, 10, 11, 8 (Kv1.1KO) for type 1 cells (Fezf2-GFP+/Sox2+), type 2a cells (Fezf2-GFP+/Sox2+/Tbr2+), type 2b cells (Fezf2-GFP+/Tbr2+), immature neurons (POMC-GFP+), and label-free mature neurons; (two-way ANOVA followed by Sidak's multiple comparisons test; p=0.02, for type 2b cells)).
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