Research Article

Live imaging of neurogenesis in the adult mouse hippocampus

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Science  09 Feb 2018:
Vol. 359, Issue 6376, pp. 658-662
DOI: 10.1126/science.aao5056
  • Fig. 1 Chronic in vivo imaging of neurogenesis in the adult DG.

    (A) Scheme illustrating the experimental approach allowing for chronic in vivo imaging of NSPCs in the adult DG of Ascl1-tdTomato mice. (B) Representative in vivo images of R and NR cells at 2 days post-induction (dpi). (C) Immunostained images showing Sox2-positive (green), Ascl1-tdTomato–labeled (red) R cells with GFAP (glial fibrillary acidic protein)–positive (white) radial processes and NR cells (Sox2-positive and GFAP-negative) in Ascl1-tdTomato mice at 2 dpi. (D) Selected imaging time points for two R cells (respectively indicated with open and filled arrowheads) over the course of 2 months, showing the emergence of two neuronal clones. Time points after Tam injection are indicated in each panel. Shown are collapsed z-stacks. The clonal expansion of individual R cell progeny and subsequent neuronal maturation can be seen. (E) Lineage tree deduced from tracking one R cell [open arrowhead in (D)] and its progeny. Identified cell types are color-coded, and lineage transitions are depicted depending on their certainty (methods). Each circle in the lineage tree represents an imaging time point. The y axis shows the duration of the imaging. (F) Post hoc immunohistochemical analyses of the clone shown in (D) (boxed area, day 59) confirm neuronal progeny with newborn cells positive for Prox1 (green) and negative for Sox2 (white). The horizontal view of the DG corresponds to the view obtained during in vivo imaging. Scale bars, 20 μm [(B) and (C)] and 50 μm [(D) and (F)]. d, days; GCL, granule cell layer.

  • Fig. 2 The mode of NSPC division is associated with individual cell division history.

    (A) Self-renewal duration (time between first and last division in each lineage) of R cells (9.6 ± 1.3 days; n = 39). (B) Distribution of the final number of cells per active clone (n = 42 lineages). Open circles represent individual clones. (C) Chronic in vivo imaging before and after cell division illustrates asymmetric cell division of R cells. A large overlap is evident in the cellular morphology before (red arrowhead and red outline) and after (green arrowhead and green outline) R cell division. The black arrowhead points at the asymmetrically generated daughter cell. (D) A morphometric index (including circularity and process length; details are given in the methods) shows little deviation in cell morphology before and after cell division (9.6 ± 1.8%; n = 9). (E) Heat map representing the frequencies of modes of division of R cells (all divisions and division rounds 1 to 3; n = 68 divisions total). The division mode changes from predominantly asymmetric (division 1) to a more symmetric differentiating division (divisions 2 and 3). N, neuron; A, astrocyte. (F) Example of an asymmetric division of an R cell (lineage 40; fig. S3). (G) Example of a symmetric division of an R cell (lineage 13). Mother cells are indicated with arrowheads and daughter cells with arrows. (H) Heat map representing the frequencies of cell division modes of NR cells (all divisions and division rounds 1, 3, and 5; n = 153 divisions). (I) Example of a symmetric NR cell division (lineage 1). (J) Example of an asymmetric NR cell division (lineage 3). The NR daughter continues to divide. Mother and daughter cells are indicated as in (F) and (G). (K) Cell division time (TD) of R and NR cells for different divisions. (L) The times until next cell division of sister cells originating from a single R mother cell are correlated (Pearson’s ρ = 0.77; *P < 0.00001; n = 22 pairs). (M) The TD of sister cells originating from a single NR mother are not correlated (Pearson’s ρ = 0.44; P = 0.08; n = 16 pairs). Each plus sign represents a pair of sister cells. Red lines, means; error bars, SEM. Scale bars, 20 μm [(C), (F), (G), (I), and (J)].

  • Fig. 3 Chronic in vivo imaging reveals variable susceptibility to cell death.

    (A) The frequency of cell death in all chronically imaged lineages ranges from 0 to 100% (mean = 59.6%, n = 42; error bars, SEM). (B) Time point of cell death after last cell division. Two peaks of cell death occur before and after 7 days, with almost no cell death occurring >20 days after birth (n = 242). (C) Pictogram depicting the comparison of cell death frequencies in subtrees 1 and 2, derived after the initial R cell division, plus subsequent progeny within that subtree (Div2plus). (D) Difference in cell death frequencies in subtrees 1 and 2 in comparison with cell death frequency in the whole lineage [lineages with >25% difference in cell death rate are colored red; only early cell death (until day 7 after birth) was included]. (E) Pictogram depicting the comparison of cell death frequencies in the Div2plus subtrees and the subtrees derived from the second division after initial R cell division plus subsequent progeny within those subtrees (Div3plus). (F) Cell death frequencies are more asymmetrically distributed among Div3plus than among Div2plus sublineages, as demonstrated by the higher weighted standard deviation to the Div2plus subtree death frequency [Div2plus difference, 13.6 ± 1.6 (n = 34); Div3plus difference, 27 ± 2.2 (n = 33); *P < 0.0001, Wilcoxon rank sum test]. Weighted standard deviation was used to account for differences in subtree sizes within each clone. Red lines, means; error bars, SEM. (G) Locations of surviving newborn neurons (blue circles) relative to cells that underwent cell death (red circles). The observed times until death (calculated from the birth of the individual cell) for dying cells are 2.5 days (cells 1 and 2 in example 1) and 13, 3, and 4 days (cell 1, 2, and 3 in example 2). Spatial localizations of surviving and dying cells overlap. Scale bar, 100 μm.

  • Fig. 4 Modeling-based analysis suggests a developmental-like program for R cell fate behavior.

    (A) In the model paradigm, R cells follow a defined program comprising a proliferative phase (duplications) that switches irreversibly into a neurogenic phase (asymmetric divisions and terminal differentiation). (B) Total number of newborn cells in successive time intervals of 5 days after induction from experiments (dark data set) and from simulations (light data set) for R and NR cells and neurons (N) (methods). Cell counts were pooled over 55 lineage trees with nonquiescent R cells and averaged over 500 realizations for each lineage tree, resulting in a total of 27,500 simulations (model parameters are given in table S2). Error bars indicate the range within which 95% of the simulation results fall. (C) Relative frequencies of different cell fates in successive time intervals of 5 days after induction from experiments and simulations, as in (B). Later time intervals (20 to 50 days) with small numbers of events have been pooled together. SD, symmetric differentiating divisions; A, asymmetric divisions; SR, symmetric self-renewing divisions; D, cell death. (D) Average clone content as a function of time from experiments (dots) and from simulations (lines) for different indicated cell types. Shaded areas indicate the regions within which 95% of the simulation results fall.

Supplementary Materials

  • Live imaging of neurogenesis in the adult mouse hippocampus

    Gregor-Alexander Pilz, Sara Bottes, Marion Betizeau, David J. Jörg, Stefano Carta, Benjamin D. Simons, Fritjof Helmchen, Sebastian Jessberger

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S7
    • Tables S1 and S2
    • Captions for Movies S1 to S3
    • References

    Images, Video, and Other Media

    Movie S1
    Example analyses of in vivo imaged R cells. Shown are examples of 3 individual R cells obtained during in vivo imaging sessions. Shown are in order: all single z-planes that contain information on the individual R cell repeated twice (sequence is indicated at the bottom left; zstepsize is 5�m), the maximum projection of all z-planes, and a rotating 3D reconstruction of the R cell to illustrate the overall morphology of imaged R cells, including the extension of a typical long basal process.
    Movie S2
    Illustration of image-based cell annotation. This movie illustrates the annotation of cell types using the information of all z-planes during all imaging time points. Shown is a clonal lineage starting with an R cell at 2dpi followed until 63dpi (corresponding to lineage 40 in Figure S3). For four time points in the lineage (d2.5, d3, d6 and d12), sequences of all single z-planes are shown in addition to the maximum projections, to illustrate that the cell type judgement is based on detailed inspection of cell morphology through all z-planes. After each scan through a z-stack, color-coding on the maximum projection illustrates the annotated cell types (R cell: red, NR cell: orange, Neuron: blue). Cells not belonging to the clone are not annotated. Note the asymmetric division of the R cell (d2.5 to d3; NR cell generated) and the persistence of the R cell until d8 (visible basal process of the R cell remains; see z stack at d6). Small shifts in x and y at different time points have been corrected.
    Movie S3
    Chronic in vivo imaging of two individual R cells. Shown is the lineage progression of two initial R cells that get activated at distinct time points and generate neuronal progeny (corresponding to Figure 1D, E and lineages 2 and 3 in Figure S3). Note the occurrence of cell death, cell migration and maturation of granule cells during the time course. Small shifts in x and y at different time points have been corrected.

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