Progressive divisions of multipotent neural progenitors generate late-born chandelier cells in the neocortex

Abstract

Diverse γ-aminobutyric acid (GABA)-ergic interneurons provide different modes of inhibition to support circuit operation in the neocortex. However, the cellular and molecular mechanisms underlying the systematic generation of assorted neocortical interneurons remain largely unclear. Here we show that NKX2.1-expressing radial glial progenitors (RGPs) in the mouse embryonic ventral telencephalon divide progressively to generate distinct groups of interneurons, which occupy the neocortex in a time-dependent, early inside-out and late outside-in, manner. Notably, the late-born chandelier cells, one of the morphologically and physiologically highly distinguishable GABAergic interneurons, arise reliably from continuously dividing RGPs that produce non-chandelier cells initially. Selective removal of Partition defective 3, an evolutionarily conserved cell polarity protein, impairs RGP asymmetric cell division, resulting in premature depletion of RGPs towards the late embryonic stages and a consequent loss of chandelier cells. These results suggest that consecutive asymmetric divisions of multipotent RGPs generate diverse neocortical interneurons in a progressive manner.

Fig. 1

Progressive changes in neocortical interneuron output of dividing NKX2.1⁺ MGE/PoA RGPs. a Images of the somatosensory cortex of representative P21 mouse brains that received in utero intraventricular injection of high-titer EGFP-expressing retrovirus at E12-E17. EGFP-labeled interneurons are shown in black. L, layer; WM, white matter. Scale bar: 150 μm. b 3D reconstruction images of P21 brains that received in utero retrovirus injection at different embryonic stages. Blue lines indicate the contours of the whole brain. Red and blue dots represent the cell bodies of the EGFP-expressing interneurons in the superficial (L2-4) and deep (L5/6) layers of the neocortex, respectively. A anterior, P posterior, D dorsal; V ventral. c Quantification of the total number of EGFP-expressing interneurons per hemisphere of the neocortex. (n = 6 hemispheres per time point from at least three different animals). Center line, median; box, interquartile range; whiskers, minimum and maximum. Red dots indicate individual data points. ****P < 0.0001 (P = 3.4e-13, Jonckheere–Terpstra test). d Quantification of the laminar distribution of EGFP-expressing interneurons. Data are presented as mean ± SEM (n = 6 hemispheres per time point from at least three different animals). *** P = 0.0001; **** P < 0.0001 (Kruskal-Wallis test)

Fig. 2

Dividing NKX2.1⁺ MGE/PoA RGPs at E12 generate distinct neocortical interneurons at different time points. a Schematic of the experimental design. Animals received in utero injection of EGFP-expressing retrovirus, as well as EdU injection at E12 and BrdU injection at E15 were analyzed at P21. b Image of the somatosensory cortex of P21 mouse brain stained for EGFP (green), EdU (red), and BrdU (green), and counterstained with DAPI (blue). Broken lines represent the pial surface and white matter boundaries. L layer, WM white matter. High magnification images of representative EGFP-expressing interneurons in different layers (arrows) are shown to the right. Scale bars: 150 μm and 25 μm. c Percentage of EGFP-expressing interneurons in different layers labeled with EdU or BrdU. Data are presented as mean ± SEM (n = 3 brains)

Fig. 3

Dividing NKX2.1⁺ MGE/PoA RGPs at E12 generate superficial layer chandelier cells at the late embryonic stage. a Images of P21 neocortices injected with EGFP-expressing retrovirus at E12, stained for EGFP (green), and counterstained with DAPI (blue). EGFP-expressing chandelier cells were frequently found at the border of superficial layers 1 and 2. High magnification images of the vertical arrays of axonal cartridges characteristic of the chandelier cell (broken rectangles) are shown in the insets. Broken lines indicate the pial surface. Scale bar: 50 µm. b 3D reconstruction image of a neocortical hemisphere showing EGFP-expressing chandelier cells and non-chandelier cells. Black and gray dots represent the cell bodies of EGFP-expressing chandelier and non-chandelier cells, respectively. D dorsal, P posterior, L lateral. c Percentage of chandelier cells among EGFP-expressing neocortical interneurons. Gray lines represent mean ± SEM. Black dots represent individual brains (n = 3). d 3D reconstruction image of a neocortical hemisphere showing EGFP-expressing chandelier and non-chandelier cells in layer 2 only. Red and gray dots represent the cell bodies of EGFP-expressing chandelier and non-chandelier cells, respectively. e Percentage of chandelier cells among EGFP-expressing interneurons in L2. Gray lines indicate mean ± SEM. Black dots represent individual brains (n = 3). f Schematic of the experimental design for birth dating analysis of EGFP-expressing chandelier cells. Animals received in utero retrovirus injection at E12 and EdU at E13, or E14, or E15 were analyzed at P21. g Image of P21 neocortex injected with EGFP-expressing retrovirus at E12 followed by EdU injection at E15, stained for EGFP (green) and EdU (red). The arrow indicates an EGFP-expressing chandelier cell labeled by EdU injection at E15. High magnification images of the chandelier cell are shown to the right. The broken line indicates the pial surface. Scale bars: 150 μm and 10 µm. h Percentage of EGFP-expressing chandelier cells that are labeled by EdU. Gray lines represent mean ± SEM. Black dots represent individual brains (n = 3 per time point). **P = 0.005 (E13-E15); **P = 0.002 (E14-E15) (unpaired t-test with Welch’s correction)

Fig. 4

Consecutive divisions of NKX2.1⁺ MGE/PoA RGPs generate superficial layer chandelier cells. a Schematic of the experimental design. Animals received in utero injection of mCherry-retroviruses and EGFP-retroviruses at E12 and E14, respectively, were analyzed at P21. b Images of P21 neocortex stained for mCherry (red) and EGFP (green). The arrows indicate a superficial layer chandelier cell expressing both mCherry and EGFP. High magnification images of the chandelier cells are shown to the right. Broken lines indicate the pial surface and white matter (WM) boundaries. L, layer. Scale bars: 150 µm and 50 µm. c 3D reconstruction images of the neocortical hemispheres that received in utero retrovirus injection at E12-E17. Red and gray dots represent EGFP-expressing chandelier and non-chandelier cells in L2, respectively. A anterior, P posterior, D dorsal, V ventral, M medial, L lateral. d Percentage of chandelier cells among EGFP-expressing interneurons in L2. Gray lines represent mean ± SEM. Black dots represent individual brains (n = 3 per time point). ***P = 0.0003 (E16 vs. E17); n.s., not significant (E12 vs. E13, P = 0.2258; E13 vs. E14, P = 0.4920; E14 vs. E15, P = 0.3375; E15 vs. E16, P = 0.1069); unpaired t-test with Welch’s correction)

Fig. 5

Late outside-in neurogenesis by NKX2.1⁺ MGE/PoA RGPs generates deep layer chandelier cells. a Images of P21 neocortices that received in utero EGFP-expressing retrovirus injection, stained for EGFP (green), and counterstained with DAPI (blue). EGFP-expressing chandelier cells were found in layers 5 (left two cells) and 6 (right two cells). High magnification images of the vertical arrays of axonal cartridges characteristic of the chandelier cell (broken rectangles) are shown in the insets. Broken lines represent the white matter (WM) boundary. Scale bar: 50 µm. b 3D reconstruction images of the neocortical hemispheres showing EGFP-expressing chandelier cells and non-chandelier cells in layers 5/6. Blue and gray dots represent the cell bodies of L5/6 chandelier and non-chandelier cells, respectively. c Percentage of chandelier cells among EGFP-expressing interneurons in layers 5/6. Gray lines represent mean ± SEM. Black dots represent individual brains (n = 3 per time point). ****P < 0.0001 (one-way ANOVA). d 3D reconstruction images of the neocortical hemispheres showing EGFP-expressing chandelier cells in layers 2 and 5/6. Red and blue dots represent the cell bodies of EGFP-expressing chandelier cells in the superficial layer 2 and deep layers 5/6, respectively. A anterior, P posterior, D dorsal; V ventral, M medial, L lateral. e Ratios of EGFP-expressing chandelier cells in the superficial layer 2 and deep layers 5/6. **** P < 0.0001 (one-way ANOVA)

Fig. 6

PARD3 regulates NKX2.1⁺ MGP/PoA RGP asymmetric division. a Images of E14, E15, and E16 MGE/PoA in control (Ctrl) and Pard3 cKO mouse brains stained for OLIG2 (green) and counterstained with DAPI (blue). The arrows indicate the loss of OLIG2⁺ cells in the ventricular zone (VZ) of Pard3 cKO MGE. Scale bar: 100 µm. b Quantification of the number of OLIG2⁺ cells in the VZ per 250 µm column in the MGE/PoA (E14: n = 4 sections from 3 control brains and n = 4 sections from 3 Pard3 cKO brains; E15: n = 7 sections from 3 control brains and n = 10 sections from 7 Pard3 cKO brains; E16: n = 6 sections from 3 control brains and n = 6 sections from 3 Pard3 cKO brains). Center line, median; box, interquartile range; whiskers, minimum and maximum. **P < 0.01, *** P < 0.001, n.s., not significant(P = 0.38) (unpaired t-test with Welch’s correction). c Schematic of in vivo clonal analysis to assess RGP division pattern. d Images of E15 control and Pard3 cKO mouse brains injected with low-titer EGFP-expressing retrovirus (green) at E14 and stained for Ki67 (white) and OLIG2 (red), and counterstained with DAPI (blue). High magnification images of the EGFP-expressing cell pairs (broken rectangles) are shown to the bottom and right. Scale bars: 50 µm, 20 µm, and 15 µm. e 3D reconstruction images of control and Pard3 cKO MGE/PoA showing EGFP-expressing cell pairs containing RGP (green), IP (yellow), or interneurons (IN, red). f Percentage of EGFP-expressing cell pairs representing symmetric proliferative division, asymmetric neurogenic division, or symmetric terminal division in the MGE/PoA of control (n = 6) and Pard3 cKO (n = 6) brains. *** P = 0.0004 (black); *** P = 0.0009 (cyan) (Chi-square test)

Fig. 7

Chandelier cell production depends on RGP asymmetric division. a Schematic of the experimental design. Animals received in utero injection of Cre-dependent EGFP-expressing retrovirus at E15 were analyzed at P21. b 3D reconstruction images of P21 control (Ctrl) and Pard3 cKO neocortical hemispheres with EGFP-expressing chandelier cells and non-chandelier cells. Black and gray dots represent the cell bodies of EGFP-expressing chandelier cells and non-chandelier cells, respectively. A anterior, P posterior, D dorsal, V ventral, M medial, L lateral. c Quantification of the number (left) and percentage (right) of chandelier cells among EGFP-expressing interneurons per neocortical hemisphere (control, n = 10 hemispheres from 5 animals; Pard3 cKO, n = 16 hemispheres from 8 animals). Center line, median; box, interquartile range; whiskers, minimum and maximum. ** P = 0.003 (Mann–Whitney test). d 3D reconstruction images of P21 control and Pard3 cKO neocortical hemispheres with EGFP-expressing chandelier cells and non-chandelier cells in layer 2. Red and gray dots represent the cell bodies of EGFP-expressing chandelier cells and non-chandelier cells, respectively, in layer 2. e Quantification of the number (left) and percentage (right) of chandelier cells among EGFP-expressing interneurons in layer 2 per neocortical hemisphere (control, n = 10 hemispheres from 5 animals; Pard3 cKO, n = 16 hemispheres from 8 animals). Center line, median; box, interquartile range; whiskers, minimum and maximum. *P = 0.04 (left); *P = 0.03 (right) (Mann–Whitney test). f 3D reconstruction images of P21 control and Pard3 cKO neocortical hemispheres with EGFP-expressing chandelier cells and non-chandelier cells in layers 5/6. Blue and gray dots represent the cell bodies of EGFP-expressing chandelier cells and non-chandelier cells, respectively, in layers 5/6. g Quantification of the number (left) and percentage (right) of chandelier cells among EGFP-expressing interneurons in layers 5/6 per neocortical hemisphere (control, n = 10 hemispheres from 5 animals; Pard3 cKO, n = 16 hemispheres from 8 animals). Center line, median; box, interquartile range; whiskers, minimum and maximum. * P = 0.01; *** P = 0.0007 (Mann–Whitney test)