Comparative analysis of the subventricular zone in rat, ferret and macaque: evidence for an outer subventricular zone in rodents

Abstract

The mammalian cerebral cortex arises from precursor cells that reside in a proliferative region surrounding the lateral ventricles of the developing brain. Recent work has shown that precursor cells in the subventricular zone (SVZ) provide a major contribution to prenatal cortical neurogenesis, and that the SVZ is significantly thicker in gyrencephalic mammals such as primates than it is in lissencephalic mammals including rodents. Identifying characteristics that are shared by or that distinguish cortical precursor cells across mammalian species will shed light on factors that regulate cortical neurogenesis and may point toward mechanisms that underlie the evolutionary expansion of the neocortex in gyrencephalic mammals. We immunostained sections of the developing cerebral cortex from lissencephalic rats, and from gyrencephalic ferrets and macaques to compare the distribution of precursor cell types in each species. We also performed time-lapse imaging of precursor cells in the developing rat neocortex. We show that the distribution of Pax6+ and Tbr2+ precursor cells is similar in lissencephalic rat and gyrencephalic ferret, and different in the gyrencephalic cortex of macaque. We show that mitotic Pax6+ translocating radial glial cells (tRG) are present in the cerebral cortex of each species during and after neurogenesis, demonstrating that the function of Pax6+ tRG cells is not restricted to neurogenesis. Furthermore, we show that Olig2 expression distinguishes two distinct subtypes of Pax6+ tRG cells. Finally we present a novel method for discriminating the inner and outer SVZ across mammalian species and show that the key cytoarchitectural features and cell types that define the outer SVZ in developing primates are present in the developing rat neocortex. Our data demonstrate that the developing rat cerebral cortex possesses an outer subventricular zone during late stages of cortical neurogenesis and that the developing rodent cortex shares important features with that of primates.

Figure 1. The distribution of mitoses during cortical development is similar in ferrets and rats, but different in macaques.

(a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for phosphorylated vimentin (4A4, green) to label dividing cells and counterstained with DAPI (blue). Images are displayed at the same scale. There are a significant number of abventricular mitoses in each species. (d–f) Graphs showing changes in the distribution of mitoses during development of the neocortex. The stage of development is shown at the bottom of the graphs. The approximate cortical layer generated at each stage of development is indicated along the top of the macaque graph, and applies to each graph. (d) Early in macaque neurogenesis the majority of mitoses shifted from the ventricular zone (VZ) to abventricular locations. By E80, when layer 4 neurons are being generated , less than 20% of mitoses remained in the VZ and 57% were located in the outer subventricular zone (oSVZ). (e) The distribution of mitoses in the gyrencephalic ferret did not shift away from the VZ during early stages of neurogenesis as in macaque. At E34 during generation of layer 4 neurons , greater than half of all mitoses were still located in the VZ. (f) The distribution of mitoses in the lissencephalic rat cortex was similar to that in the gyrencephalic ferret cortex. At E21, which represents the genesis of layer 2 neurons in somatosensory cortex , 35% of mitoses were located superficial to the SVZ in a zone that we term the oSVZ. Legend indicates histological zones: VZ: blue; inner SVZ (iSVZ): red; oSVZ: green; cortical plate (CP)/preplate (PP)/subplate (SP)/marginal zone (MZ): purple. Scale bar in (a) applies to (a–c).

Figure 2. The distribution of Tbr2+ cells during cortical development is similar in ferrets and rats, but different in macaques.

(a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for Tbr2 (red) and counterstained with DAPI (blue). Images are displayed at the same scale. In each species a dense inner band of Tbr2+ cells was located within the cell dense zone surrounding the lateral ventricle visualized with DAPI. The diffuse outer band of Tbr2 expression was located within a striated zone marked by streams of cells organized in tangential clusters. (d–f) Graphs showing changes in the distribution of Tbr2+ cells during development of the cerebral cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) In macaque the distribution of Tbr2+ cells progressively shifted to the outer subventricular zone (oSVZ) during development. By E80, during genesis of layer 4 neurons , 59% of Tbr2+ cells were located in the oSVZ. (e) The distribution of Tbr2+ cells in the gyrencephalic ferret shifted to the oSVZ more slowly in comparison to macaque. At P2 during genesis of layer 2 neurons , 62% of Tbr2+ cells remained in the inner SVZ (iSVZ). (f) The distribution of Tbr2+ cells in the lissencephalic rat was similar to that of the gyrencephalic ferret. At E20 during genesis of upper layer neurons , the majority of Tbr2+ cells (59%) were located in the iSVZ, and 17% were located in the oSVZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. Scale bar in (a) applies to (a–c).

Figure 3. Density of Tbr2+ cells in the dense inner band and diffuse outer band of macaque, ferret and rat.

(a–c) Images taken from coronal sections of E65 macaque, P2 ferret and E20 rat immunostained for Tbr2 (red) and counterstained with DAPI (blue). The dense inner band and diffuse outer band of Tbr2+ cells are indicated. (d–f) Histograms displaying the number of Tbr2+ cells per 2500 µm² in the dense inner band (blue) and diffuse outer band (red) for each species. The diffuse outer band of Tbr2+ cells appears at an early age in macaque in comparison to ferret and rat. In macaque the diffuse outer band was present at E65 during generation of deep layer neurons while the diffuse outer band first appeared at later stages of development in ferret and rat. Nonetheless, the relative density of Tbr2+ cells in the dense and diffuse bands was similar in each species. Scale bar in (a) applies to (a–c).

Figure 4. The dense inner band of Tbr2+ cells corresponds to the inner subventricular zone (iSVZ) and the diffuse outer band of Tbr2+ cells corresponds to the outer SVZ (oSVZ) in macaque visual cortex.

(a) Nissl stained E80 macaque visual cortex. (b) An adjacent section immunostained for Tbr2 (red). The dense inner band and diffuse outer band are indicated. (c) Image of the same adjacent section showing Tbr2 (red) and DAPI stain (blue). The different compartments, such as the outer fiber layer (OFL) can be visualized with DAPI stain. VZ, ventricular zone; IFL, inner fiber layer. Scale bar in (b) applies to (a–c).

Figure 5. Inner and outer fiber layers are not apparent in macaque cortical areas rostral to the occipital lobe.

(a) Nissl stained coronal section of E80 macaque frontal lobe at the level of the genu of the corpus callosum (CC). (b–c) An adjacent section taken from the E80 macaque frontal lobe stained with DAPI (blue) and Tbr2 (red). (b) DAPI staining highlights the different cell densities within the inner subventricular zone (iSVZ) and outer SVZ (oSVZ). The cell dense region surrounding the lateral ventricle includes the very thin ventricular zone and the iSVZ. Tangential streams of cells can be visualized within the oSVZ in DAPI staining. Inner and outer fiber layers are not apparent. (c) The same section showing Tbr2 (red) and DAPI. As in visual cortex, the dense inner band of Tbr2 cells corresponds to the iSVZ and the diffuse outer band of Tbr2+ cells corresponds to the oSVZ. Scale bar in (c) applies to (b). IC, internal capsule.

Figure 6. Tbr2+ cells are located superficial to the developing white matter near the corpus callosum (CC) in the prenatal medial cortex of macaque, ferret and rat.

(a–d) Coronal sections from rat, ferret, and macaque immunostained for Tbr2 (red) and counterstained with DAPI (blue). (a) Image from E20 rat showing clusters of Tbr2+ cells superficial to developing callosal fibers along the medial wall of the cortex (arrowheads). (b) Image from P3 rat showing an isolated pocket of Tbr2+ cells (arrowheads) located deep within the developing cingulate cortex (Cg Cx) superficial to the developing white matter. (c) Tbr2+ cells located superficial to the developing callosal fibers along the medial wall of the P2 ferret cortex (arrowheads). (d) Tbr2+ cells positioned above callosal fibers (arrowheads) in the cingulate cortex of E80 macaque. The supracallosal Tbr2+ cells appear to be oSVZ cells that were separated from underlying precursor cells by growing callosal axons. LV, lateral ventricle.

Figure 7. Graphs showing the total number of Tbr2+ cells in the ventricular zone (VZ), inner subventricular zone (iSVZ) and outer SVZ (oSVZ) within a 200 µm wide radial unit of macaque, ferret and rat somatosensory cortex.

(a–c) There was a similar number of Tbr2+ cells in the iSVZ of each species, but macaque had a much larger number of Tbr2+ cells in the oSVZ. The stage of development is shown at the bottom of each graph, and the approximate cortical layer generated during each stage of development is indicated along the top of each graph. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green.

Figure 8. Histograms comparing the thickness of the subventricular zone in the developing cortex of rat, ferret, and macaque.

Measurements of the thickness of the dense inner band (blue) and diffuse outer band (red) of Tbr2+ cells were made in radial bins stretching from the ventricle to the pial surface in the dorsal somatosensory cortex of rat (a), ferret (b), and macaque (c). In each species the dense inner band appeared first in development followed by the diffuse outer band. In macaque the dense inner band and diffuse outer band were both significantly thicker than in ferret or rat. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph.

Figure 9. The distribution of Pax6+ cells during cortical development is similar in ferrets and rats, but different in macaques.

(a–c) Coronal sections of somatosensory cortex from E80 macaque, P2 ferret and E20 rat immunostained for Pax6 (red) and counterstained with DAPI (blue). Images are displayed at the same scale. In each species a dense inner band of Pax6+ cells was colocalized with the ventricular zone (VZ), and a diffuse band of Pax6-expressing cells extended outward through the inner subventricular zone (iSVZ) and the outer SVZ (oSVZ). The iSVZ and oSVZ were identified based on the pattern of DAPI staining as described above. (d–f) Graphs showing changes in the distribution of Pax6+ cells during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) In macaque the distribution of Pax6+ cells rapidly shifted to the oSVZ. At E50 nearly 90% of Pax6+ cells were located in the VZ. But by E65 during production of layer 5 neurons , the majority of Pax6+ cells (60%) were located in the oSVZ. (e) The distribution of Pax6+ cells in the gyrencephalic ferret shifted to the oSVZ much more slowly than in macaque. At P2 during genesis of layer 2 neurons , 52% of Pax6+ cells remained in the VZ. (f) The distribution of Pax6+ cells in the lissencephalic rat was similar to that of the gyrencephalic ferret. At the end of neurogenesis on E20 during production of layer 2 neurons , the majority of Pax6+ cells (64%) were still located in the VZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green. Cortical plate (CP)/preplate (PP)/subplate (SP)/marginal zone (MZ): purple. Scale bar in (a) applies to (a–c).

Figure 10. Graphs showing the total number of Pax6+ cells in the ventricular zone (VZ), inner subventricular zone (iSVZ), outer SVZ (oSVZ), and cortical plate (CP) within a 200 µm wide radial unit of macaque, ferret and rat somatosensory cortex.

(a–c) There was a similar number of Pax6+ cells in the iSVZ of each species, but macaque had a much larger number of Pax6+ cells in the oSVZ. The stage of development is shown at the bottom of each graph, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green; CP: purple. PP, preplate; SP, subplate; MZ, marginal zone.

Figure 11. The distribution of Tbr2+ mitoses shifts to the outer subventricular zone (oSVZ) during early stages of cortical development in macaque, but not in ferret or rat.

(a–c) Images taken from coronal sections of the E80 macaque, P2 ferret, and E20 rat oSVZ. Tissue was immunostained for 4A4 (green), Tbr2 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ mitotic cells that either do not express Tbr2 (#1) or that do express Tbr2 (#2). (d–f) Graphs showing changes in the distribution of Tbr2+ mitoses during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque all Tbr2+ mitoses were located in the inner SVZ (iSVZ). There was a steady decrease in the proportion of Tbr2+ divisions occurring in the iSVZ. By E80 the majority of Tbr2+ mitoses (76%) had shifted to the oSVZ. (e) In ferret the majority of Tbr2+ mitoses were located in the iSVZ throughout neurogenesis in the somatosensory cortex. At P2, during neurogenesis of layer 2 neurons , 73% of Tbr2+ mitoses were still located in the iSVZ. (f) The pattern observed in rat was similar to that of the ferret. The majority of Tbr2+ mitoses were located in the iSVZ throughout the period of cortical neurogenesis. At P2, during production of layer 2 neurons , there was a similar number of Tbr2+ mitoses located in the rat iSVZ (74%) as was observed in the ferret. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green.

Figure 12. The distribution of Pax6+ mitoses shifts from the ventricular zone (VZ) to the subventricular zone (oSVZ) during early stages of cortical development in macaque, but not in ferret or rat.

(a–c) Images taken from coronal sections of the E80 macaque, P2 ferret and E20 rat oSVZ. Tissue was immunostained for 4A4 (green), Pax6 (red) and counterstained with DAPI (blue). Boxes to the right highlight examples of 4A4+ mitotic cells that express Pax6. (d–f) Graphs showing changes in the distribution of Pax6+ mitoses during development of the somatosensory cortex. The stage of development is shown at the bottom of the graphs, and the approximate cortical layer generated at each stage of development is indicated along the top of each graph. (d) At E50 in the macaque nearly 90% of Pax6+ mitoses were located in the VZ. There was a steady shift in the distribution of mitoses to the outer SVZ (oSVZ). By E80 the majority of Pax6+ mitoses (61%) were located in the oSVZ. (e) In ferret the largest proportion of Pax6+ mitoses were located in the VZ until late stages of neurogenesis. At E38, during neurogenesis of layer 2 neurons , 48% of Pax6+ mitoses were still located in the VZ, 23% were located in the iSVZ and 26% in the oSVZ. (f) A similar pattern was observed in the rat. The majority of Pax6+ mitoses were located in the VZ until E20. At E20 40% of Pax6+ mitoses were located in the VZ, 45% were in the iSVZ and 13% in the oSVZ. Legend indicates histological zones: VZ: blue; iSVZ: red; oSVZ: green; CP: purple. PP, preplate; SP, subplate; MZ, marginal zone.

Figure 13. A subpopulation of Pax6+ mitotic cells coexpress Tbr2 in the outer subventricular zone (oSVZ) of macaque, ferret, and rat dorsal somatosensory cortex.

(a) Image from E80 macaque oSVZ immunostained for Pax6 (green) and Tbr2 (red), and counterstained with DAPI (blue). Boxes highlight examples of Pax6+ mitoses. (b) Examples of Pax6+ mitotic cells from insets in (a) demonstrating two examples of Pax6+ mitoses that coexpress Tbr2 (1 and 2) and one example that does not (3). (c) E34 ferret oSVZ. An example of a Pax6+ mitosis that coexpresses Tbr2 and a second example that does not. (d) E20 rat oSVZ. An example of a Pax6+ mitosis that coexpresses Tbr2 and a second example that does not. In all three species most Tbr2+ mitotic cells express Pax6, although in most cases the level of Pax6 expression is low in Tbr2+ cells. There are significant numbers of Pax6+ mitotic cells in the oSVZ of each species that do not express Tbr2 (see Table 13). Scale bar applies to all images.

Figure 14. Mitotic translocating radial glial (tRG) cells in rodent cortex express Pax6.

(a) Example of a mitotic tRG cell labeled with 4A4 (green). The pial fiber of the dividing cell is seen coursing to the pial surface. All mitotic tRG cells coexpressed Pax6 (red). DAPI stained nuclei are shown (blue). (b) An example of a GFP+ tRG cell (green) labeled through in utero retroviral injection at E16. The tRG cell (arrowhead) expresses Pax6 (red). A daughter cell can be seen migrating along the pial fiber (arrow). All tRG cells labeled with retroviral injections expressed Pax6. (c) tRG cells labeled in the E18 mouse through in utero electroporation with a GFP expressing plasmid (green) expressed both Pax6 (red) and Sox2 (blue). Left panel shows a low magnification merged image of the tRG cell. Middle images show high magnification individual images of GFP (green), Pax6 (red) and Sox2 (blue). Right image shows high magnification merged image of GFP (green), Pax6 (red) and Sox2 (blue). All GFP+ cells with tRG morphology in mouse were Pax6+ and Sox2+.

Figure 15. Translocating RG (tRG) cells in the embryonic rat oSVZ divide and express Pax6.

(a) Time lapse recording of a GFP-labeled tRG cell in the rat somatosensory cortex after an E16 retroviral injection. At the start of the experiment the labeled cell (red arrowhead) has RG morphology including a ventricular process and a pial process. The first timepoints of this movie focused on the RG cell body in the VZ and did not capture the pial process. An RG daughter cell (white arrowhead) is in contact with the pial process. The RG cell lost contact with the ventricle at t = 17 h, acquired tRG morphology, and began translocating toward the cortical plate. The first daughter cell (white arrowhead) remained in close proximity to its parent tRG cell for the duration of the experiment and did not divide. The tRG cell divided again at approximately t = 47 h (asterisk), producing a self-renewed tRG cell (red arrowhead) and a second daughter cell (white arrow). The tRG cell continued to translocate and divided once again at approximately t = 78 h, producing a third daughter cell (white arrowhead with red border). In addition, the second daughter cell also divided at approximately t = 78 h, producing two daughter cells with similar morphology. After an additional two hours the section was fixed in 4% PFA, sectioned on a cryostat and immunostained for Pax6 and NeuN. The clonal cells are shown on the far right image at higher magnification after fixation. (b) Lineage tree depicting the progeny of the RG cell in this time-lapse recording. (c) Immunostaining shows that the tRG cell expresses Pax6. The first daughter cell did not express Pax6. The daughter cells that were produced by division of the tRG second daughter cell (white arrow) expressed weak levels of Pax6. The tRG third daughter cell (white arrowhead with red border) expressed a moderate level of Pax6 that was lower in comparison to the level of Pax6 expression by the tRG mother cell. All cells in this clone were NeuN-negative. White arrowheads indicate the locations of clonal cells in each panel. This data shows that mitotically active RG cells and tRG cells maintain high levels of Pax6 expression, and suggests that non-RG daughter cells slowly downregulate Pax6 expression as they migrate toward the cortical plate. Scale bar in the right panel of (a) applies to all images in (c). VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone.

Figure 16. tRG cells express Pax6 during and after neurogenesis.

(a–c) 4A4+ tRG cells (green) in the E17 rat somatosensory cortex that express Pax6 (red). Blue channel shows DAPI staining. (d–f) Examples of 4A4+ tRG cells (green) in post-neurogenic somatosensory cortex of macaque (d), ferret (e), and rat (f) that also express Pax6 (red). Blue channel shows DAPI staining. Scale bar in left panels applies to all panels in each set.

Figure 17. Most mitotic Pax6+ cells express Sox2 during and after neurogenesis.

(a) Coronal section from E17 rat somatosensory cortex immunostained for Pax6 (red) and Sox2 (green) and counterstained with DAPI (blue). Boxes (1 and 2) show examples of Pax6+ mitotic cells that also express Sox2. (b) Coronal section from P3 rat somatosensory cortex immunostained for Pax6 (red) and Sox2 (green) and counterstained with DAPI (blue). Boxes (1 and 2) show examples of Pax6+ mitotic cells that also express Sox2. There was greater overlap of Pax6 and Sox2 expression at E17 than at P3, but the extent of colabeling in mitotic cells was similar at each age (see Table 14). Scale bar in (a) applies to all images.

Figure 18. Olig2 expression distinguishes two subtypes of tRG cells in macaque, ferret and rat.

(a–c) Examples of 4A4+ tRG cells (green) in the E80 macaque (a), P2 ferret (b), and E21 rat (c), that express Pax6 (red). Shown are examples of Pax6+ tRG cells that are Olig2-negative (top row of panels), or Olig2+ (lower row of panels, Olig2 immunostaining: blue). (d–f) Graphs showing changes in the proportion of Pax6+ tRG cells that express Olig2 during cortical development. During early stages of cortical development most Pax6+ cells were Olig2-negative in each species. The proportion of Pax6+ tRG cells that expressed Olig2 increased as development proceeded. Graphs also show Pax6(−)/Olig2+, or Pax6(−)/Olig2(−) 4A4+ cells with a pial fiber. Scale bar in (a) applies to (b).

Figure 19. The outer fiber layer (OFL) is not present in cortical areas rostral to the occipital lobe.

(a, c) Nissl-stained coronal sections of somatosensory and visual cortex from the E65 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). At E65 the OFL is visible in visual cortex superficial to the outer subventricular zone (oSVZ), but the inner fiber layer has not yet developed. The OFL is not apparent in somatosensory cortex. The oSVZ in somatosensory cortex is characterized by a striated appearance created by tangential streams of cells. Scale bar in (b) applies to (d). IC, internal capsule; AC, anterior commissure; VZ, ventricular zone; iSVZ, inner subventricular zone; SP, subplate, CP, cortical plate.

Figure 20. The inner fiber layer (IFL) and outer fiber layer (OFL) are present in the E80 macaque visual cortex but are not apparent in other cortical areas.

(a,c) Nissl-stained coronal sections of somatosensory and visual cortex taken from E80 macaque. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining on adjacent sections (see Fig. 5). The outer fiber layer (OFL) and inner fiber layer (IFL) are visible in the visual cortex but are not apparent in cortical areas rostral to the occipital lobe. In somatosensory cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by differences in cell density. In the visual cortex the IFL divides the iSVZ from the oSVZ. In somatosensory cortex the oSVZ extends much farther from the ventricle than it does in visual cortex and is characterized by a striated appearance. In the E80 macaque the ventricular zone has become very thin and the cell dense proliferative zone that surrounds the lateral ventricle consists almost entirely of iSVZ. The subplate (SP) was identified according to Smart et al 2002 . Scale bar in (b) applies to (d). CC, corpus callosum; IC, internal capsule; AC, anterior commissure; CP, cortical plate.

Figure 21. Inner and outer SVZ in the developing ferret cortex.

(a,c) Nissl-stained coronal sections of somatosensory and visual cortex from P2 ferret. (b,d) Higher magnification images taken from the sections shown in (a) and (c). Dotted line in (b) represents the outer boundary of the outer subventricular zone (oSVZ) in somatosensory cortex determined through Tbr2 immunostaining. The outer fiber layer (OFL) and inner fiber layer are not present in somatosensory cortex, but a structure that resembles the OFL (OFL-like) is apparent in some areas of the ferret occipital lobe. However, note that the OFL-like structure in ferret is located within the oSVZ, in contrast to the OFL in macaque, which is superficial to the oSVZ. In both somatosensory and visual cortex the boundary between the inner SVZ (iSVZ) and oSVZ can be visualized in Nissl stained tissue as a sharp border created by different cellular density. The superficial boundary of the oSVZ can also be visualized in Nissl stained tissue based on cell density (determined through Tbr2 immunostaining). In somatosensory cortex the oSVZ is characterized by a striated appearance of cells organized into clusters that appear to stream at an oblique or tangential angle through the oSVZ. (e–g) The OFL-like structure in ferret resembles the macaque OFL since radially oriented clusters of cells stream through this structure as they do in macaque (see Fig. 4). But the ferret OFL-like structure differs from the macaque OFL since it is within the oSVZ rather than superficial to the oSVZ and because Tbr2+ cells are located throughout the OFL-like structure in ferret but do not penetrate the macaque OFL (See Fig. 4). VZ, ventricular zone; CP, cortical plate. Scale bar in (b) applied to (d). Scale bar in (g) applied to (e, f).

Figure 22. Inner and outer SVZ in the developing rat cortex.

(a, c, e) Nissl-stained sections of somatosensory or visual cortex from the E18 rat. (b, d, e) Higher magnification images taken from the sections shown in (a), (c) and (e). The inner fiber layer and outer fiber layer are not apparent in any cortical areas of rat cortex. At late stages of rat cortical development, such as E18 or E21, the boundary between the inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be discriminated based on differences in cell density. At E17 and younger ages Tbr2 immunostaining is required to visualize the location of dense inner band of Tbr2+ cells to localize the iSVZ and oSVZ. The oSVZ is characterized by a stippled or striated appearance created by oblique and tangential clusters of cells that appear to stream across the oSVZ. At E21, the oSVZ has expanded compared to earlier stages of cortical development. Dotted line indicates the upper boundary of the oSVZ. AC, anterior commissure; IC, internal capsule; VZ, ventricular zone; CP, cortical plate. Scale bar below (f) applies to (b, d, f).

Figure 23. Tau-1 and Tbr2 staining distinguishes the inner subventricular zone (iSVZ) from the outer SVZ (oSVZ).

(a) A coronal section from E80 macaque somatosensory cortex immunostained for Tau-1 (green), Tbr2 (red), and counterstained with DAPI (blue). Tau-1 staining produces dense labeling in the cortical plate, striated staining in the outer subventricular zone (oSVZ) and an absence of label in the inner SVZ (iSVZ). The striated appearance of Tau-1 staining in the oSVZ complements the striated pattern of the oSVZ that is seen in Nissl or DAPI stained tissue. (b) Inset from (a) showing the pattern of cell density (DAPI, blue), Tbr2 staining (red), and Tau-1 staining (green). The Tau-free zone corresponds to the cell dense iSVZ/VZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where the diffuse outer band of Tbr2+ cells is located. (c) Tau-1 and Tbr2 immunostaining produces the same pattern in the E20 rat. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located. (d) Tau-1 and Tbr2 immunostaining produces the same pattern in the P2 ferret. The Tau-free zone corresponds to the cell dense VZ/iSVZ visualized in DAPI staining (blue) and where the dense inner band of Tbr2+ cells (red) is located. The boundary between the Tau-free and the Tau-striated zone delineates the boundary between the iSVZ and oSVZ where diffuse Tbr2+ cells are located.

Figure 24. The boundaries between the ventricular zone (VZ), inner subventricular zone (iSVZ) and outer SVZ (oSVZ) can be identified through a combination of DAPI, Tau-1, Tbr2 and Pax6 staining.

(a) Coronal sections of somatosensory cortex from E20 rat stained with (from left to right) DAPI (blue), and immunostained for Tbr2 (red) and Tau-1 (green). The right panel is an adjacent section that was immunostained for Pax6 (green). DAPI staining identifies the boundary between the iSVZ and oSVZ. The VZ and iSVZ have a near uniform cell density, while the oSVZ is striated and the cell density is lower. Tau-1 staining also identifies the boundary between the iSVZ and oSVZ in macaque, ferret and rat. The iSVZ is located in the Tau-free zone. The border between iSVZ and oSVZ is delineated by the border between the Tau-free zone and the Tau-striated zone. Tau-1+ fibers are largely absent from the VZ and iSVZ. Tbr2 and Pax6 can be used to discriminate the VZ and iSVZ. The dense inner band of Tbr2 expression corresponds to the iSVZ and the dense inner band of Pax6 expression corresponds to the VZ. The dashed lines indicate the boundary between the iSVZ and oSVZ.