Multiple origins and modularity in the spatiotemporal emergence of cerebellar astrocyte heterogeneity
Fig 8
Proliferation and cell cycle exit of cerebellar astrocytes.
(A-D) Analysis of active proliferation of astrocytes in different layers during early postnatal development. (A) Percentage of EdU-incorporating astrocyte precursors among total hGFAP+ cells subdivided per layers. EdU was administered 6 h before killing at P1, P4, or P7 to detect actively proliferating cells. At all analyzed time points, proliferation varies among layers and declines over time in both vermis and hemispheres. In B-D”, arrowheads point to double-labeled proliferating precursors, highlighting the overall reduction over time in the proliferation activity and the higher number of proliferating cells in the PCL compared to the other layers. (E-G”) Birthdating of astrocytes retaining a strong BrdU signal (BrdUhigh) after the completion of the maturation process (P30). The histogram in (E) shows that the highest percentages of BrdUhigh+ cells are observed in the first postnatal week, with minimal labeling before and afterwards. In detail, numerous WMAs exit the cell cycle already at P1, while GLAs and BG differentiate later. On the whole, this layer-specific pattern applies to both the vermis and the hemispheres (P = 0.139), where, however, differentiation delayed (PCL, P < 0.001). (F-F”,G-G”) Full and empty arrowheads point to astrocytes retaining a strong (BrdUhigh) or a diluted (BrdUlow) BrdU signal, respectively. *, P < 0.05; **, P < 0.01; ***, P < 0.001; P values are calculated with GEE analysis. Plots represent data averaged from distinct animals. Scale bars: 30 μm. The numerical data used in panels (A,E) are included in S1 Data. BG, Bergmann glia; BrdU, bromodeoxyuridine; EdU, 5-ethynyl-2′-deoxyuridine; GEE, generalized estimated equations; GL, granular layer; GLA, granular layer astrocyte; hGFAP, human glial fibrillary acidic protein; P, postnatal day; PCL, Purkinje cell layer; PWM, prospective white matter; WM, white matter; WMA, white matter astrocyte.