Cortical astrocytes develop in a plastic manner at both clonal and cellular levels

Abstract

Astrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype.

Fig. 1

MAGIC Markers associated with ChroMS microscopy reveal astrocyte clonal patterns diversity. a MAGIC Markers (MM) constructs for genomic combinatorial labeling: transgenes express a nuclear EBFP2 by default under the control of a CAG promoter. Three recombination possibilities created by alternating pairs of incompatible lox sites each trigger expression of a distinct FP (mCerulean/mTurquoise2, mEYFP, or tdTomato/mCherry) in specific subcellular compartments: cytoplasm (Cytbow) or nucleus (Nucbow). 5′ and 3′ Tol2 (T2) or piggyBac (PB) transposition sequences frame the transgenes. b 63 theoretical color combinations are possible in cells containing 1–3 copies of both ^PBCytbow and ^T2Nucbow. c MM are used to label astrocyte clones arising from distinct cortical progenitors. d E15 IUE of MM and self-excising Cre (SeCre) along with PB and Tol2 transposases labels astrocytes at P7 and P21 in all layers of the mouse cerebral cortex. e Rare nuclear/cytoplasmic color combinations allow the distinction of astrocyte clones. f Chromatic serial multiphoton (ChroMS) microscopy relies on the integration of trichromatic two-photon excitation by wavelength mixing with automated blockface imaging. Adapted from the graphics used in Abdeladim et al., Nat. Commun. 2019 Apr 10;10(1):1662, and licensed under a Creative Commons Attribution 4.0 International License, http://creativecommons.org/licenses/by/4.0/. g, h ChroMS microscopy allows high-resolution 3D imaging of large volumes of MM-labeled cortex, and visualization of astrocyte clones spatial arrangement (i, j). k Examples of single clones plotted in (j) presenting various sizes and spatial distributions. Hipp. hippocampus, DV dorsoventral axis, AP anteroposterior axis, ML mediolateral axis. Scale bars: 100 (d, g, i); 200 (h); 50 (e) µm

Fig. 2

Clonal organization of astrocytes remains stable from P7 to P21 while their arbor expands and complexifies. (a, b) 3D analysis reveals variable PrA clonal dispersion up to 1.25 and 0.5 mm along the DV and ML/AP axis, but no difference between P7 and P21 (n = 113). c Example of a 17-cell clone where red represents nuclei and gray the astrocyte territory (left) along with Delaunay Triangulation (DT, middle) and Convex Hull extraction (CH, right). d DT of random PrA clones highlights their distinct shapes. e CH analysis shows high diversity of the volume covered by clones, but stability of its distribution between P7 and P21. f PrA clones were automatically separated into clusters of apposed cells or isolated cells (elements) using their XYZ coordinates and astrocyte mean diameter + s.d. at each stage. g On average PrA clones are composed of 2.8 disconnected elements and this arrangement is stable from P7 to P21. h Totally, 76.2% of PrA belong to clusters and this proportion is stable from P7 to P21. i Segmentation of color-isolated astrocytes from ChroMS images at P7 (left) and P21 (right). j, k Segmented astrocyte territorial volume increases by 65.7% from P7 to P21 (j) and this expansion occurs in the entire cortical parenchyma, here divided in six equivalent bins (Bin 1 = pial surface, k). l High resolution reconstruction of astrocyte arborizations at P7 and P21 reveals a significant increase in branch number (m), total branch length (n), and volume of the model (o). p Close-ups of neighboring PrA labeled with distinct colors (single optical section) show incomplete filling of cortical space by P7 PrA compared to P21 PrA. DV dorsoventral axis, AP anteroposterior axis, ML mediolateral axis, VZ ventricular zone. Graph values indicate means ± s.e.m. Kruskal–Wallis associated with Dunn’s multiple comparisons (a) and Mann–Whitney (e, g, h, j, k, m–o) statistical tests have been performed. *, ***, **** indicate p value < 0.05, <0.0005, <0.0001, respectively. N = 4 (a, e, g, h, j, k) and 9 (m–o) animals. Scale bars f: 50 (f), 20 (i, l, p) µm

Fig. 3

Clonal expansion based on proliferation and spacing of sister astrocytes lessens during development. a PrA clones of various sizes at P4, P7, and P21 after MM IUE at E15. b PrA clones issued from cortical progenitors targeted at E15 grow in average from 4.5 ± 0.19 cells at P4 to 7.9 ± 0.51 cells at P7 and 8.1 ± 0.47 cells at P21. c PrA clones exhibit various sizes at P4, P7, and P21, with clones composed of 1 to 42 cells. d Schematic view of DV, ML, and AP dispersion on serial sagittal sections. e DV dispersion of PrA clones ≥2 cells as a function of clone size is broad and variable. f DV dispersion per cell expressed as % of cortical thickness (DV clonal dispersion/ clone size) decreases from P4 to P7. g P4 and P7 sagittal sections co-labeled by MM IUE at E15 and EdU injection 48 or 24 h prior to analysis. Proliferating PrA are found throughout the entire cortical wall. h Examples of EdU + PrA astrocyte identified as sister cells with color markers. i Distance between EdU + paired PrA decreases from 48 to 24 h postinjection and from P4 to P7. j Sister cells closer than astrocyte nucleus mean size (6 µm) form doublets. k Doublets of PrA were found at P4, P7, and P21 in stable proportions (20%). l Doublets relative DV positioning (in % of cortical thickness, see Supplementary Fig. 5e) shows that they occur in the entire cortical parenchyma from P4 to P21 whereas EdU + astrocyte pairs are located mostly in upper cortical layers (m). n Doublets found at P4–P7 are not all comprised of Ki67+ cycling cells. o Model of astrocyte clonal maturation through densification between P4 and P7. DV dorsoventral, ML mediolateral, AP anteroposterior. Graph values indicate means ± s.e.m. One-tailed Mann–Whitney (f) and Kruskal–Wallis associated with Dunn’s multiple comparisons (i, k) statistical tests have been performed. **** indicates p value < 0.0001. N = 6 (b), 10 (i), 9 (k) animals. Scale bars: 50 (a), 100 (g), 20 (h, j, n) µm

Fig. 4

Individual cortical progenitors can generate both protoplasmic and pial astrocytes. a–c Examples of homogeneous clones of PrA (a) or PiA (b) and heterogeneous clones containing both astrocyte types (c). d PrA, PiA, and heterogenous clones were found in stable proportions (respectively 76%, 5%, and 19% of clones) throughout postnatal development (P4–P7–P21), with a significant majority of homogeneous PrA. e Heterogeneous astrocyte clones comprise significantly larger number of cells than homogeneous PrA clones. f PrA clones whose barycenter is located in the upper half of the cortex (U) are larger than those in lower layers (L) at P4, P7, and P21. Clone barycenter is calculated using the mean of sister cells relative DV positions. g Examples of intermediate morphologies between PiA and PrA observed at P21, presenting both fibroblast-like features at one extremity and bushy ramifications at the other extremity. Graph values indicate means ± s.e.m. Kruskal–Wallis associated with Dunn’s multiple comparisons statistical tests have been performed. **** indicates p value < 0.0001. N = 9 animals. Scale bars: 50 µm

Fig. 5

Cortical astrocytes arise from Olig2+ seeding units issued from both pre- and postnatal progenitors. a MM electroporated at E15 colocalize with Olig2 marker at P1 (stars in bottom image) but not with S100β (top). b The percentage of Olig2+ cells among MM-labeled cells increases from 5% at E18 to 10% at P0 and is then stable until P1, while no MM+/S100β+ cell is detected at these stages. c IUE of MM in Olig2^Cre mice at E13 results in labeling of astrocyte-like cells at P7 both at the pial surface and in the cortical parenchyma. d Example of a P7 MM-labeled clone occupying three consecutive serial sections comprising S100β+, Aldh1l1+, and Olig2+ cells, revealed by immunostaining. e IUE of MM in E13 Olig2^Cre embryos yields sparse recombined cells at E18. Other cells express nuclear EBFP2, indicating efficient targeting of cortical progenitors. f Olig2+/Aldh1l1+ cells are found scattered in the E18 cerebral cortex after E15 IUE of integrative ^Tol2CAG-mEYFP vector. g P1 co-electroporation of episomal CAG-RFP and integrative ^Tol2CAG-mEYFP vectors labels few astrocytes expressing RFP and markedly more expressing EYFP at P7, among both protoplasmic and pial subtypes (arrows). h P0 electroporation of SeCre plasmid in CAG-Cytbow mice labels clones of PrA and PiA (arrows) at P7. PrA identity is confirmed by S100β immunostaining (stars). i P0 electroporation of integrative ^Tol2CAG-mEYFP plasmid labels YFP+ cells scattered in the entire cortical thickness at P3, several of which coexpress Olig2 and Aldh1l1. Graph values indicate mean ± s.e.m. A two-tailed Mann–Whitney statistical test has been performed. * indicates p -value < 0.05. N = 11 animals. Scale bars: 50 (a, d), 100 (c, e–i) µm

Fig. 6

Comprehensive model for astrocyte development in the mouse cortex. Until now the accepted model of mouse cortical astrocyte development consisted of a first phase where embryonic progenitors colonize the neocortical wall followed by a second step relying on local proliferation of these first settlers after birth, the contribution of postnatal progenitors being debated. Here, we propose that mouse cortical astrocytes are issued from a dual contribution of delaminated embryonic apical progenitors and early postnatal progenitors that both generate pial (PiA) and protoplasmic (PrA) astrocytes. Furthermore, our data show that during the first postnatal week (P0–P7) both pre- and postnatal progenitors scatter throughout the neocortical wall while proliferating. This dynamic phase is followed by a maturation phase (P7–P21) where the clones stop both expansion and proliferation while individual astrocytes increase their volume and the complexity of their processes