Diverse behaviors of outer radial glia in developing ferret and human cortex - PubMed (original) (raw)
Diverse behaviors of outer radial glia in developing ferret and human cortex
Caitlyn C Gertz et al. J Neurosci. 2014.
Abstract
The dramatic increase in neocortical size and folding during mammalian brain evolution has been attributed to the elaboration of the subventricular zone (SVZ) and the associated increase in neural progenitors. However, recent studies have shown that SVZ size and the abundance of resident progenitors do not directly predict cortical topography, suggesting that complex behaviors of the progenitors themselves may contribute to the overall size and shape of the adult cortex. Using time-lapse imaging, we examined the dynamic behaviors of SVZ progenitors in the ferret, a gyrencephalic carnivore, focusing our analysis on outer radial glial cells (oRGs). We identified a substantial population of oRGs by marker expression and their unique mode of division, termed mitotic somal translocation (MST). Ferret oRGs exhibited diverse behaviors in terms of division location, cleavage angle, and MST distance, as well as fiber orientation and dynamics. We then examined the human fetal cortex and found that a subset of human oRGs displayed similar characteristics, suggesting that diversity in oRG behavior may be a general feature. Similar to the human, ferret oRGs underwent multiple rounds of self-renewing divisions but were more likely to undergo symmetric divisions that expanded the oRG population, as opposed to producing intermediate progenitor cells (IPCs). Differences in oRG behaviors, including proliferative potential and daughter cell fates, may contribute to variations in cortical structure between mammalian species.
Figures
Figure 1.
SVZ divisions in the developing ferret cortex. A, P3 ferret cortical slice labeled with AV-GFP and stained for Sox2 (blue), Tbr2 (red), and Olig2 (white) to distinguish the VZ (100 μm thick on average) from the iSVZ (150 μm thick on average) and oSVZ (400 μm thick on average). Boxed region indicates the location of the magnified region, which highlights AV-GFP+ cells within the iSVZ and oSVZ that are Sox2+, Tbr2−, and Olig2− (white closed arrowheads). Scale bars: in merged image, 50 μm; in single channel images, 20 μm. B, Box plot showing median and interquartile range of MST distances in the iSVZ and oSVZ (n = 162). C, D, Time-lapse stills of oRGs labeled with AV-GFP in P3 ferret cortical slices. Time elapsed from start of imaging indicated in the top right of image (hours:minutes, yellow = start of division). VZ surface is down. Scale bars, 20 μm. C, An oRG (closed white arrowhead) maintains the basal fiber (open white arrowheads) during mitosis and exhibits an MST length of 37 μm (white arrow indicates direction of MST) to produce a basal (closed white arrowhead) and apical daughter (closed yellow arrowhead). D, An example of a stationary oRG division in which the cell (closed white arrowhead) maintains the basal fiber (open white arrowheads) during mitosis, and produces a basal (closed white arrowhead) and apical daughter (closed yellow arrowhead).
Figure 2.
MST divisions can be found throughout the VZ and SVZ. A, Percentage of SVZ MST divisions (n = 162) occurring at different distances from the VZ surface (100–650 μm). B, Scatter plot and linear regression line were used to analyze the relationship between MST length and division location measured as distance from the VZ surface (n = 174). The regression line equation is y = 23.585–0.0199x; _r_2 = 0.0296, p = 0.023 (*p < 0.05). C, D, Time-lapse stills of RG cells labeled with AV-GFP in P3 ferret cortical slices. Time elapsed from start of imaging indicated in the top right of image (yellow = division). VZ surface is denoted by a dotted line. Basal daughter indicated with closed white arrowhead and apical daughter with closed yellow arrowhead. Boxed region indicates the location of the basal daughter at the end of time-lapse imaging. Scale bars, 20 μm. C, An RG (closed white arrowhead) divides at the VZ surface with an oblique division angle (36.7° with respect to VZ surface) to produce a basal daughter that inherits the basal process (open white arrowheads) and an apical daughter. Post-time-lapse fate staining reveals that the basal daughter is Sox2+. Shown on the right is a schematic of the time-lapse series. D, An RG (closed white arrowhead) located at the VZ surface undergoes basal MST (10.9 μm, white arrow indicates direction of MST) and divides with a horizontal division angle (4.4° with respect to VZ surface) to produce a basal daughter that inherits the basal process (open white arrowheads) and an apical daughter. Post-time-lapse fate staining reveals that the basal daughter is Sox2+. Schematic of time-lapse series is shown on the right.
Figure 3.
oRG division angles vary with respect to the VZ surface but remain constant with respect to the primary fiber. A, Schematic and quantification of cells undergoing MST with horizontal (0–30°, blue), oblique (30–60°, red), and vertical (60–90°, green) division angles with respect to the VZ surface (n = 174). B, Schematic and quantification of cells undergoing MST with vertical (0–30°, green), oblique (30–60°, red), and horizontal (60–90°, blue) division angles with respect to the primary (1′) fiber (n = 174). C–E, Time-lapse stills of progenitors labeled with AV-GFP in P3 ferret cortical slices. Time elapsed from start of imaging indicated in the top right of image (yellow = division). 1′ daughter is denoted with a closed white arrowhead and non-1′ daughter with a closed yellow arrowhead. Yellow line indicates 1′ fiber orientation before MST. White arrow indicates the direction of MST. VZ surface is down. Scale bars, 20 μm. C, An oRG (closed white arrowhead) undergoes MST (17.1 μm, 100.6 μm from VZ surface) to produce daughters that are Sox2+ Tbr2− Olig2−, and that possess 1′ fibers (open white arrowheads) that are parallel to the VZ surface. D, Example of a progenitor undergoing MST (16 μm, 100 μm from VZ surface) parallel to the VZ surface and dividing 76° relative to the 1′ fiber (open white arrowheads). E, Example of a progenitor undergoing apically directed MST (33 μm, 320 μm from VZ surface) and dividing 76° relative to the 1′ fiber (open white arrowheads).
Figure 4.
oRG fibers are dynamic during the cell cycle and do not always contact the pial surface. Time-lapse stills of progenitors labeled with AV-GFP in P3 ferret cortical slices. Time elapsed from start of imaging indicated in the top right of image (yellow = division). Basal daughter indicated with closed white arrowhead and apical daughter with closed yellow arrowhead. White arrow indicates the direction of MST. VZ surface is down. Scale bars, 20 μm. A, An oRG (closed white arrowhead, 281 μm from VZ surface) exhibits retraction of the basal, 1′ fiber (open white arrowheads, 125.8 μm) during MST (32 μm), which then regrows after cytokinesis. A corresponding _z_-plane analysis of the same time point rotated 90° confirms retraction of the 1′ fiber during mitosis. B, An oRG (closed white arrowhead, 476.7 μm from VZ surface) possesses an apical, non-1′ fiber (open white arrowheads, 62 μm) that retracts during MST (18.3 μm). A corresponding _z_-plane analysis rotated 90° confirms retraction of the apical fiber during mitosis. C, An oRG (closed white arrowhead, 149 μm from VZ surface) with a basally directed 1′ fiber measuring 297 μm (open white arrowheads) maintains the fiber during MST (9.5 μm). Top, A zoomed-in view of the end of the 1′ fiber revealing a growth cone that is motile and dynamic during the cell cycle.
Figure 5.
Human oRGs exhibit diverse behaviors similar to the ferret. Time-lapse stills of progenitors labeled with AV-GFP in human cortical slices from GW 16 (A) and 18 (C–E). Time elapsed from start of imaging indicated in the top right of image (yellow = division). White arrow indicates the direction of MST. VZ surface is down and denoted by a dotted line. Scale bars, 20 μm. A, A vRG (closed white arrowhead) undergoes INM to reach the VZ surface followed by basal MST (64 μm), dividing with a horizontal division angle (3° with respect to VZ surface) to produce a basal (closed white arrowhead) and apical daughter (closed yellow arrowhead). Schematic of time-lapse series is shown on the right. B, Schematic and quantification of cells undergoing MST with horizontal (0–30°, green), oblique (30–60°, red), and vertical (60–90°, blue) division angles with respect to the adjacent fiber scaffold (n = 71). C, A progenitor (open white arrowhead) undergoes MST (29 μm) and divides with a 30° angle with respect to the radial fiber scaffold (yellow line). D, An oRG (closed white arrowhead) displays retraction of the 1′ fiber (open white arrowheads, 103.7 μm) during MST (37.2 μm) after which daughters (closed white and yellow arrowheads) regrow fibers (open white arrowheads). A corresponding _z_-plane analysis rotated 90° confirms retraction of the 1′ fiber during mitosis. E, An oRG (closed white arrowhead) has a basally directed 1′ fiber (open white arrowheads, 202.7 μm) that ends in a growth cone that is motile and dynamic during MST (125 μm). Top, A zoomed-in view of the growth cone.
Figure 6.
oRG daughters can undergo multiple rounds of divisions. Time-lapse stills of progenitors labeled with AV-GFP in P3 ferret cortical slices along with schematics illustrating the divisions shown in the time-lapse series (D = daughter). Time elapsed from start of imaging indicated in the top right of image (yellow = division). White arrow indicates direction of MST. VZ surface is down. Scale bars, 20 μm. A, The 1′ daughter (closed white arrowhead) from an MST division (18 μm) during which the 1′ fiber (open white arrowheads) is retracted, migrates basally (105 μm) and undergoes a second MST division (25 μm) with fiber retraction. B, The 1′ daughter (closed white arrowhead) of an MST division (28 μm), during which the 1′ fiber (open white arrowheads) is retracted, undergoes a second stationary division where the 1′ fiber is maintained (open white arrowheads). C, The non-1′ daughter (closed yellow arrowhead) of an MST division (13 μm), during which the 1′ fiber is retracted (open white arrowheads), regrows a basal fiber (open white arrowheads), and undergoes a second MST division (18 μm) with apparent fiber retraction. D, The non-1′ daughter (closed yellow arrowhead) of an MST division (32 μm), during which the 1′ fiber is retracted (open white arrowheads), regrows a basal fiber, and undergoes a second stationary division where the 1′ fiber is maintained (open white arrowheads).
Figure 7.
The majority of oRG daughters remain undifferentiated. Time-lapse stills of progenitors labeled with AV-GFP in P3 (A–C) or E39 (D) ferret cortical slices. Time elapsed from start of imaging indicated in the top right of image (yellow = division), with fate stains performed at 66:50 (A–C) or 50:00 (D). 1′ fiber indicated with open white arrowheads. White arrow indicates the direction of MST. Basal (1′) daughter indicated with closed white arrowhead and apical (non-1′) daughter with closed yellow arrowhead. VZ surface is down. Scale bars, 20 μm. A, An oRG (closed white arrowhead) undergoes MST (68 μm) to produce a basal and apical daughter. Boxed regions indicate the location of the daughters at the end of time-lapse imaging. Fate staining reveals that both GFP+ daughters are Sox2+Satb2−. B, An oRG (closed white arrowhead) undergoes MST (11.2 μm) to produce a basal and apical daughter. Boxed region indicates the location of the daughters at the end of time-lapse imaging. Fate staining reveals that both GFP+ daughters are Sox2+ Tbr2− Olig2−. C, An oRG (closed white arrowhead) undergoes a stationary division to produce a basal and apical daughter. Fate staining reveals that GFP+ progeny are Sox2+ Tbr2− Olig2−. D, An oRG (closed white arrowhead) undergoes MST (12.3 μm) to produce a basal and apical daughter. Boxed region indicates the location of the daughters at the end of time-lapse imaging. Fate staining reveals that both GFP+ daughters are Sox2+ Tbr2− Olig2−.
Figure 8.
Model of diverse oRG behaviors and daughter cell fates during cortical development. A, During neurogenesis in both the ferret and human, MST divisions can occur throughout the VZ and the SVZ and oRGs exhibit diverse morphologies. Specifically, 1′ fibers do not always contact the pial surface, with some ending in motile growth cones within the SVZ. Moreover, not all oRG 1′ fibers are basally oriented, with some exhibiting curved, horizontal, and apical orientations. However, the vast majority of oRGs divide with a perpendicular division angle with respect to the 1′ fiber. B, The mitotic behavior of oRGs also varies. Although some divide via MST, a subset of oRGs undergoes stationary divisions. In addition, although some oRGs maintain their 1′ fiber during mitosis, which displays varicosities during M phase, some oRGs exhibit retraction of the 1′ fiber upon division. C, oRGs in different species can produce daughters with different cell fates. In the lissencephalic mouse, oRGs appear to make neurons directly. In the gyrencephalic ferret, the majority of oRGs undergo divisions to expand the oRG population, while a small percentage of oRGs produce IPCs. Compared with the ferret, human oRGs appear more likely to produce IPCs that divide symmetrically to produce neurons, and also undergo proliferative, oRG-producing divisions.
References
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