Figure 1.
Multiple GFAP antibodies produce equivalent patterns of immunoreactivity in the developing cerebral cortex of macaque.
(A–C) Panels show a coronal section of E80 macaque occipital cortex that was triple immunostained with three anti-GFAP antibodies. (A) Rat anti-GFAP from Virginia Lee, University of Pennsylvania. (B) Goat anti-GFAP from Santa Cruz. (C) Mouse anti-GFAP from Sigma-Aldrich. (D) Higher power magnification images of the ventricular surface from a coronal section of E80 macaque occipital lobe immunostained with the rat (green), goat (red) and mouse (blue) anti-GFAP antibodies. The left panel shows DAPI staining that labels the nuclei of all cells. Mitotic cells are indicated with white arrowheads. Each antibody labeled the same cells and cellular processes. Scale bar in C = 200 µm, applies to all panels A–C. Scale bar in D = 10 µm.
Figure 2.
GFAP expression in the macaque neocortex during neurogenesis of lower layer cortical neurons.
During neurogenesis of the layer 6 neurons (A) and layer 5 neurons (B) [52], GFAP immunostaining encircles the nuclei of ventricular surface cells and labels the pial fibers of radial glial cells. (A) E50 macaque occipital neocortex immunostained for GFAP (red) and costained with DAPI nuclear stain (blue). The left panel shows a coronal section of the E50 macaque occipital neocortex at low magnification. GFAP expression is observed across the cortical wall in the germinal zones, cortical plate and marginal zone. Panels to the right show higher magnification images of the ventricular zone (VZ), inner subventricular zone (iSVZ), intermediate zone (IZ) and cortical plate/marginal zone (CP/MZ). (B) E65 macaque occipital neocortex immunostained for GFAP (red) and costained with DAPI nuclear stain (blue). Left panel shows a coronal section of the E65 macaque occipital neocortex at low magnification. GFAP expression is observed across the entire cortical wall. Panels to the right show higher magnification images of the VZ, iSVZ, outer subventricular zone and outer fiber layer (oSVZ/oFL) and the CP/MZ. Scale bars in A and B, upper left = 100 µm. Scale bars in A and B, lower right = 25 µm.
Figure 3.
GFAP expression in the macaque neocortex during neurogenesis of upper layers.
GFAP immunostaining is continuous along the surface of the ventricle, and dense GFAP-IR is present throughout the proliferative zones during neurogenesis of layer 4 neurons (A) and layer 2/3 neurons [52] (B). (A) E80 macaque occipital neocortex immunostained for GFAP (red) and costained with DAPI (blue). Left panel shows a coronal section of the E80 macaque occipital neocortex at low magnification. Dense GFAP expression is observed throughout the germinal zones. Panels to the right show higher magnification images of the ventricular zone and inner subventricular zone (VZ/iSVZ), outer subventricular zone (oSVZ), outer fiber layer (oFL), cortical plate (CP), and marginal zone (MZ). (B) Coronal section of E100 macaque occipital neocortex immunostained for GFAP (red) and costained with DAPI nuclear stain (blue). At E100 GFAP expression is still dense in the cortical proliferative zones. Panels to the right show higher magnification images of the VZ/iSVZ, oSVZ, oFL, CP, and MZ. At E100 GFAP+ cells with the morphology of astrocytes were present in germinal zones (white arrow in VZ panel), and GFAP+ translocating radial glial cells were apparent (white arrowheads in oSVZ panel). Scale bars in A and B upper left = 500 µm. Scale bar in A, VZ/iSVZ panel = 25 µm.
Figure 4.
GFAP expression in the macaque neocortex after cortical neurogenesis.
Panels show images taken from a coronal section of the E150 macaque neocortex in the occipital lobe immunostained for GFAP (red) and costained with DAPI (blue). Left panel shows a low magnification image. GFAP is expressed across the cortical wall in the ependymal layer (EL), subventricular zone (SVZ), white matter (WM), cortical gray matter, and pial surface. Right panels show higher magnification images of specific cortical zones. Many GFAP+ cells with the morphology of astrocytes were present in abventricular locations (white arrows). GFAP+ processes surrounded blood vessels (white arrowheads). Scale bar at left = 500 µm. Scale bar at right = 25 µm.
Figure 5.
All mitotic cells at the ventricular surface of the macaque neocortical ventricular zone are GFAP+ and Pax6+ during cortical neurogenesis.
Images show coronal sections of fetal macaque occipital neocortex immunostained with antibodies against phosphorylated vimentin (4A4, green) or phosphohistone H3 (PH3, green) to label all mitotic cells, and GFAP (red), and costained with DAPI (blue). (A) E65 macaque occipital cortex stained with 4A4 and GFAP antibodies. Panels A1, A2, and A3 are magnified images of the numbered mitoses (white arrowheads) in Panel A showing colocalization of 4A4 and GFAP. (B) E80 macaque occipital cortex stained with PH3 and GFAP antibodies. Panel B1 shows colocalization of PH3 and GFAP. (C) A coronal section of E80 macaque occipital cortex costained with antibodies against 4A4 (green), Pax6 (blue) and GFAP (red). All mitotic cells at the surface of the ventricle in the prenatal macaque neocortex expressed Pax6 and GFAP. Panels C1, C2, and C3 show colocalization of 4A4, Pax6 and GFAP in surface mitoses. Scale bar in A = 25 µm, in A1 = 5 µm, in B = 10 µm, in C = 10 µm.
Figure 6.
GFAP is expressed by all mitotic cells dividing at the surface of the ventricle in the macaque neocortical ventricular zone.
Analysis of confocal Z-stack optical plane sections of ventricular surface mitoses at 0.5 µm steps from E65 (A) or E80 (B, C) macaque occipital cortex co-immunostained for 4A4 (green), GFAP (red) and DAPI (blue). Panels A–C show serial Z-stack images of the same cell, in each channel, from left to right. GFAP immunoreactivity was visualized in a thin rim of cytoplasm surrounding the nucleus, between sister chromatids, in the initial segment of the pial fiber (white arrowheads), and in the pial fiber. Yellow shows strong co-expression of the mitotic cell marker 4A4 and GFAP. Scale bar = 10 µm.
Figure 7.
Distribution of GFAP in surface dividing VZ precursor cells during M-phase.
Mitotic cells at the ventricular surface of the macaque occipital neocortex at E65 (A–C) and E80 (D) immunostained for the mitotic cell marker 4A4 (green) and GFAP (red), and costained with DAPI nuclear stain (blue). (A) 4A4+/GFAP+ prophase cell in the E65 macaque occipital cortex. During prophase there was strong GFAP immunoreactivity (IR) at the apical and basal poles of the cell. GFAP-IR often extended from the soma into the initial segment of the pial fiber on the basal pole of the cell to give the appearance of a “GFAP cap” (white arrowheads), and also labeled the pial fiber (white arrows). (B) 4A4+/GFAP+ metaphase cell in the E65 macaque occipital cortex. During metaphase GFAP encircled the nucleus in a relatively uniform pattern. The “GFAP cap” (white arrowheads) often appeared on one side of the condensed chromatin during metaphase. (C) 4A4+/GFAP+ anaphase cell in the E65 macaque occipital cortex. GFAP-IR surrounded the dividing chromosomes and was also present between the sister chromatids during anaphase. (D) 4A4+/GFAP+ telophase cell from E80 macaque occipital cortex. GFAP-IR surrounded the dividing sister chromatids of telophase cells and strong GFAP-IR was present in the cleavage furrow. Scale bar = 5 µm.
Figure 8.
GFAP expression by 4A4+ mitotic cells in the macaque subventricular zone.
Images show single optical plane sections from the inner subventricular zone (A, B), and outer subventricular zone (C, D) of the prenatal macaque. (A, B) 4A4+ (green)/GFAP+ (red) mitotic cells in the E65 inner subventricular zone nuclei stained with DAPI (blue). The vast majority of 4A4+ mitotic cells in the inner subventricular zone expressed GFAP. Insets at right show 4A4+/GFAP+ cells from numbered mitoses in the images at left. Merged panels show colocalization of 4A4 and GFAP (yellow) in the mitotic cells. (C) 4A4+ (green) GFAP+ (red) mitotic cells in the E65 outer subventricular zone. GFAP-immunoreactivity extended into the proximal portion of pial-directed processes of oSVZ mitoses (white arrow). Insets at right show 4A4+/GFAP+ cells from the image at left. (D) 4A4+ (green)/GFAP+ (red) mitotic cells in the E80 outer subventricular zone. Insets at right display 4A4+/GFAP+ cells from the image at left. The majority of 4A4+ mitotic cells in the outer subventricular zone expressed GFAP. GFAP-immunoreactivity often extended into the proximal portion of pial-directed processes of oSVZ mitoses (white arrow). Merged panels show colocalization of 4A4 and GFAP (yellow) in the mitotic cells. Scale bars in A–D = 20 µm, in A1–D2 = 10 µm.
Figure 9.
GFAP expression by 4A4+ mitotic cells in the marginal zone, cortical plate, and cortical gray matter during neurogenesis.
Images in panels A–C show coronal sections of prenatal macaque monkey cerebral cortex immunostained for 4A4 (green) and GFAP (red), and stained with DAPI (blue). The majority of 4A4+ mitotic cells in extra-germinal zones expressed GFAP throughout neurogenesis. (A) 4A4+ mitotic cell co-expressing GFAP in the marginal zone (MZ) of E80 macaque occipital cortex. (B) 4A4+ mitotic cell expressing GFAP in the cortical plate (CP) of E100 macaque occipital cortex. Many 4A4+/GFAP+ mitotic cells in the CP had apparent astrocyte morphology. (C) 4A4+ mitotic cell expressing GFAP in the cortical gray matter of E100 macaque occipital cortex. Some 4A4+/GFAP+ cells had the morphology of translocating radial glia (top row). These cells were unipolar with a pial-oriented fiber that often had a large GFAP+ varicosity near the soma (white arrows). Scale bars in left panels (A–C) = 20 µm. Scale bars in 4A4 inset panels = 10 µm and apply to all inset panels.
Figure 10.
GFAP is expressed by the majority of PH3+ abventricular mitotic cells.
(A–I) Coronal sections of the prenatal macaque cerebral cortex immunostained for PH3 (green) and GFAP (red), and stained with DAPI (blue). Most PH3+ mitotic cells in all layers of the developing cerebral cortex expressed GFAP (A–B, D–E, G–H), but some PH3+ mitoses were GFAP-negative (C, F, I). (A) PH3+/GFAP+ mitotic cell with a GFAP+ pial oriented process (white arrow) in the inner subventricular zone of E80 macaque occipital cortex. (A1) Images show colocalization (yellow) of PH3 and GFAP in the mitotic cell shown in panel A to the left. (B) Some PH3+/GFAP+ mitotic cells had no apparent GFAP+ processes but GFAP immunoreactivity encircled the nucleus. (B1) Images show colocalization (yellow) of PH3 and GFAP in the mitotic cell shown in panel B to the left. (C) Example of a PH3+ mitotic cell that did not express GFAP in the inner subventricular zone of E80 macaque occipital cortex. Note the lack of GFAP immunoreactivity between the sister chromatids. (D, E) PH3+/GFAP+ mitotic cells in the outer subventricular zone of E80 macaque occipital cortex. Many PH3+/GFAP+ mitotic cells had the morphology of translocating radial glia. The pial process was GFAP+ (white arrow). (D1, E1) Images show colocalization (yellow) of PH3 and GFAP in the mitotic cells shown in panels D and E to the left. (F) Example of a PH3+ mitotic cell that did not express GFAP in the outer subventricular zone of E80 macaque occipital cortex. (G, H) PH3+/GFAP+ mitotic cell in the cortical plate of E80 macaque occipital cortex. Many GFAP+ mitotic cells in extragerminal zones had the morphology of translocating radial glial cells with pial-oriented processes (white arrow). (G1, H1) Images show colocalization (yellow) of PH3 and GFAP in the mitotic cells shown in panels G and H to the left. (I) Example of a PH3+ mitotic cell in the gray matter of E150 macaque occipital cortex that did not express GFAP. Scale bar in A = 20 µm and applies to all images in left column. Scale bar in A1 = 10 µm applies to all images in right column.
Figure 11.
The proportion of mitotic cells that express GFAP in the prenatal macaque occipital cortex during neurogenic stages (E50, E65, E80, E100) and one postneurogenic stage (E150).
Mitoses were identified in each proliferative zone and cortical layer by 4A4 immunoreactivity, PH3 immunoreactivity and/or condensed chromatin with DAPI. (A) 100% of all mitotic cells at the ventricular surface of the dorsal neocortex expressed GFAP during neurogenic stages. No surface dividing cells were detected at E150. (B) The majority of mitotic cells expressed GFAP in the inner subventricular zone (iSVZ) during cortical neurogenesis. The iSVZ was largely absent at E150. (C) The majority of mitotic cells in the outer SVZ (oSVZ) expressed GFAP during neurogenic stages of development. The oSVZ was not present at E50, and the proportion of mitotic cells that expressed GFAP at E150 fell to 25%. (D). The majority of mitotic cells in extra-germinal zones expressed GFAP during neurogenesis, but not during post-neurogenic stages. SP, subplate; CP, cortical plate; MZ, marginal zone.
Figure 12.
GFAP is expressed by all Pax6+ translocating radial glia (tRG) and all Tbr2+ intermediate progenitor cells.
(A) A coronal section of the E80 macaque occipital cortex immunostained for Pax6 (green), Tbr2 (blue) and GFAP (red). Both tRG cells with GFAP+ pial fibers (Pax6+ Tbr2–, white arrowheads) and Tbr2+ intermediate progenitor cells (Tbr2+/Pax6+, white arrows) expressed GFAP. (B–E) Mitotic cells identified by 4A4 or PH3 (white) are outlined by white dotted lines. All images show single optical planes. (B, C) 4A4+ or PH3+ mitotic cells (white) that express Pax6 (green) but not Tbr2 (blue) expressed high levels of GFAP (red). The merge panel demonstrates co-expression of GFAP in Pax6+ mitotic cells (yellow). (D, E) PH3+ mitotic cells (white) that express Tbr2+ (green) and also Pax6+ (blue) co-expressed GFAP (red). The merge panel demonstrates co-expression of GFAP in Tbr2+ mitotic cells (yellow). We have previously shown that 90% of Tbr2+ cells in the macaque SVZ co-express Pax6 [34]. (D) An example of a mitotic Tbr2+ cell, indicated by white dotted lines, that is GFAP+. (E) A PH3+ mitotic cell (white), indicated by the white dotted line, expressing both Tbr2 (green) and GFAP (red). Some Tbr2+ cells expressed low levels of Pax6. An interphase Tbr2+ intermediate progenitor cell (PH3-negative, white arrowheads) also exhibits strong GFAP expression in the soma and cellular processes. Scale bars in A and B = 10 µm.
Figure 13.
The majority of GFAP-negative mitoses in the prenatal macaque neocortex are Mash1/ASCL1+.
Nevertheless, approximately 94% of ASCL1+ mitotic cells in the prenatal macaque cerebral cortex express GFAP. (A, B) Examples of ASCL1+/GFAP+ mitotic cells in the E80 macaque occipital cortex. Sections were immunostained for PH3 (blue), ASCL1 (green) and GFAP (red). (A, B) Examples of PH3+/ASCL1+/GFAP+ mitotic cells located in the outer subventricular zone. Inset panels below show immunoreactivity for each individual antibody. (C) An example of a PH3+/ASCL1+ mitotic cell that does not express GFAP in the subventricular zone. Scale bars = 10 µm.
Figure 14.
The majority of GFAP-negative mitoses in the prenatal macaque neocortex also express Olig2.
(A) An example of a mitotic PH3+ precursor cell (blue) that expresses Olig2+ (green) but does not express GFAP (red). Inset panels below show immunoreactivity for each individual antibody. (B) An example of PH3+/Olig2+ mitotic precursor cell that expresses GFAP. Inset panels below show immunoreactivity for each individual antibody. Scale bar = 20 µm.
Figure 15.
Iba1+ mitotic microglia do not express GFAP.
Images show Z-stack projection images taken in the E80 macaque occipital cortex immunostained for PH3 (blue), Iba1 (green) and GFAP (red). Inset panels below show immunoreactivity for each individual antibody. The PH3+ mitotic cell expresses the microglial marker Iba1, but does not express GFAP. GFAP+ processes from nearby cells can be seen coursing near the microglial cell. Approximately 10% of the GFAP-negative mitotic cells in the prenatal macaque neocortex expressed Iba1. Scale bar = 20 µm.
Figure 16.
Nearly all mitotic cells at the ventricular surface of the ganglionic eminence (GE) express GFAP, while far fewer abventricular mitoses express GFAP in the GE.
A coronal section through the E80 macaque GE immunostained with PH3 (green) and GFAP (red). Over 90% of mitoses at the ventricular surface of the GE were GFAP+ at E50, E65 and E80. In contrast, less than 60% of abventricular mitoses in the GE expressed GFAP at E50 and E65, and the proportion fell to less than 24% at E80. GFAP immunoreactivity was not as dense in the subventricular zone of the GE compared to that in the neocortical SVZ. Scale bar = 20 µm.