Comparison of labeling for Mash1 (A, B, E) and GAD65 (C, D, F) in the proliferative zones of GE (A, C), septum (B, D) and dorsal telencephalon (E, F) in coronal sections of monkey fetal telencephalon at E47 (E, F) and E55 (A–D). Figures correspond to magnified regions indicated by boxes in Figure 1A,C,D. (A) E55. In the GE many cells were labeled for Mash1 in the VZ and the SVZ, the intensity of labeling being much higher in the VZ than in the SVZ. Moreover, the cells highly labeled for Mash1 in the VZ of the ventral part of GE were organized in two bands in contrast to the dorsal part where the highly labeled cells for Mash1 were distributed in a homogeneous band (see Fig. 1C). (B) E55. In the prospective septal region (Sept) numerous cells highly labeled for Mash1 were observed in the VZ whereas the cells in the SVZ displayed a moderate level of labeling. (C) E55. Many cells labeled for GAD65 were observed in the GE. These cells were mainly located in the SVZ, only a few cells being present in the VZ. (D) E55. Many cells labeled for GAD65 were present in the SVZ of the septal region. Box: magnified region. (E) E47. Cresyl violet labeled section, in which the different cellular zones of the most dorsal part of the dorsal telencephalon are delineated. (F) E47. Some cells moderately labeled for Mash1 were observed in the proliferative zones of the most dorsal part of the dorsal telencephalon on the roof of the lateral ventricle. Most of these Mash1 positive cells were present in the upper part of the SVZ, on the border to the IZ. Some cells were also observed in the deepest part of the VZ. (G) E47. In contrast to Mash1 no GAD65-containing cells were observed in the VZ and SVZ of the most dorsal part of the dorsal telencephalon. However in this region, some GAD65-containing cells with a characteristic morphology of early postmitotic migrating neurons, including a leading process, and tangentially oriented, were observed in the IZ and the CP (arrows). Scale bars = 50 μm (A–G), 10 μm (box in D).

Labeling patterns for Mash1 and GAD65 in coronal sections at E68, E75 and E88. (A–D) Mash1. (A) At E75, presence of Mash1 immunoreactivity in GE as well as in ventricular (VZ) and subventricular (SVZ) zone of the entire dorsal telencephalon. In both areas, the intensity of labeling for Mash1 decreased from the VZ to the SVZ. Few cells very lightly labeled for Mash1 were observed in the lower part of the IZ. (B) At E68, the same pattern of labeling for Mash1 is already present in the VZ and SVZ of the dorsal telencephalon. No labeling is observed in the upper part of IZ, the SP and the CP. (C) E75 Higher magnification of panel A. The numerous Mash1 highly labeled cells located in the VZ and SVZ of the dorsal telencephalon are round-shaped. (D) At E88, end of neurogenesis, the intensity of labeling for Mash1 was strongly decreased as compared to E68 (B) and E75 (A and C). (E–H) GAD65. (E) At E75, GAD65 immunolabeling was present in the GE, but also in the proliferative VZ and SVZ of the dorsal telencephalon in contrast to earlier stages (Figs 1D, 3B). The stream of migrating GAD65-containing neurons observed at E55 (Fig. 3B) in the lower part of IZ and upper part of SVZ was still present (arrows). (F) At E68, similar labeling was already observed. In contrast to previous stages (E47–E55 compare with Figs 2F and 3B) many migrating-like postmitotic neurons with one leading process were now observed in the VZ and SVZ of the dorsal telencephalon. (G) At E75, in addition to migrating-like postmitotic neurons, many round-shaped cells without any process were labeled for GAD65 (arrowheads). These cells displayed a similar morphology to that observed in the GE and septal region (compare with Fig. 2C,D, and box in Fig. 2D). (H) At E75, many migrating-like GAD65 neurons in the SVZ of the dorsal telencephalon displayed multidirectional orientations. Scale bar = 550 μm (A, E), 250 μm (B), 60 μm (C, D) and 30 μm (F–H).

Confocal micrographs of coronal sections of monkey fetal telencephalon at E55 (A–D) and E75 (E–G) double-labeled for Mash1 (red, nuclear) and PGDFR-α (green, cytoplasmic). (A) Few cells double-labeled for PGDFR-α and Mash1 were present among the numerous cells only labeled for Mash1 observed in the SVZ of the GE. (B, C, D) Some PDGFR-α cells were observed in IZ and SP of the dorsal telencephalon, these cells contained very low or no detectable level of Mash1 in contrast to PDGFR-α in the GE (compare with A). (E) At E75 many cells double-labeled for PDGFR-α and Mash1 were now observed in the SVZ and IZ of the dorsal telencephalon. (F, G) In the SP (F) and CP (G) presumed PDGFR-α containing oligodendrocytes did not show detectable level of Mash1. Scale bars = 10 μm (A, C, D, F, G), and 50 μm (B, E).

Mash1 (A, C) and GAD65 (D) labeling patterns in coronal sections of monkey fetal telencephalon at E47 (A) and E55 (B–D). (A) At E47 Mash1 was highly expressed in the proliferative zone of the ventral telencephalon, the GE. Mash1 immunoreactivity was also observed in the proliferative zones of the dorsal telencephalon and followed a clear dorso-ventral decreasing gradient in the labeling intensity within the entire cortical wall. Whereas moderate level of labeling was observed in the most dorsal part of the telencephalon, on the roof of the lateral ventricle (vent) (indicated by square and magnified in Fig. 2E), the intensity of staining decreased in the lateral and medial wall. No Mash1 immunoreactivity was observed close to the GE, nor in the most ventral part of the medial telencephalic wall that corresponds at this level to dorsal hippocampus (dhipp). No Mash1 immunoreactivity could be seen in the thalamic nuclei (th). (B–D) Adjacent sections from E55 fetal brain at a rostral level in which the lateral ventricle displayed its rostral dilatation (paleocortical ventricle indicated by a star). (B) Cresyl violet labeled section, in which the following regions were specified: corpus callosum (CC), septum (sept), caudate nucleus (caud), putamen (put), capsula interna (CI), and primordium of the dorsal hippocampus (dhipp). (C) Strong intensity of labeling for Mash1 was present in the GE (box magnified in Fig. 2A) and septal proliferative zone (box magnified in Fig. 2B). (D) GAD65-immunolabeling was observed in the same regions of the ventral telencephalon as Mash1, the GE and the septal eminence (boxes enlarged in Fig. 2C,D). The levels of sections (A, B, C, D) are indicated by dotted lines on the schematic drawing of monkey fetal brain. Crossed arrows indicated the orientation of sections (A, B, C, D). Scale bar = 1 mm (same for all the figures).

Labeling patterns for Mash1 (A) and GAD65 (B–E) in coronal sections at E55. (A) IHC labeling for Mash1 is present in the GE but not in the neighboring structures including the caudate nucleus (caud), capsula interna (CI), and adjacent zones of the dorsal telencephalon: VZ, SVZ, and IZ. Arrows indicated border between GE and adjacent structures. (B) A high level of GAD65 labeling is present in the GE. In addition a stream of postmitotic like GAD65-containing neurons extending from GE to dorsal telencephalon was observed in the lower IZ/upper SVZ. Arrows indicate the deeper border of this stream, showing that at E55 none of the cells leaving GE migrate through VZ and lower SVZ. Boxes: magnified regions in (C) and (D). (C) GAD65-containing cells with a characteristic morphology of postmitotic migrating neurons, including a leading process (arrows) leaving the GE. (D) In the IZ and upper SVZ of dorsal telencephalon, the majority of GAD65 migrating neurons are tangentially (parallel to pia) oriented (arrows). (E) Tangentially oriented GAD65 migrating neurons are also observed in the SP (arrows), CP, and MZ of the dorsal telencephalon. Scale bars = 350 μm (A, B), 20 μm, (C, D, E).

Confocal micrographs of coronal sections of monkey fetal telencephalon at E55 and E75 double-labeled (A–D) for Mash1 (red, nuclear) and GAD65 (green, cytoplasmic) or double-labeled (E–I) for GAD65 (green, cytoplasmic) and Ki67 (red, nuclear). (A) At E55, almost all Mash1-labeled cells in SVZ of the GE showed cytoplasmic labeling for GAD65, whereas none of the migrating GAD65 neurons in the IZ displayed detectable level of Mash1. (B) At E75, in the SVZ of GE many Mash1-containing cells were labeled for GAD65. (C) At E75, in the dorsal telencephalon, most Mash1-labeled cells of SVZ also contained GAD65, whereas many Mash1-labeled cells in the VZ did not. (D) At E75, aggregates of cells double-labeled for Mash1 and GAD65 were frequently observed in the SVZ of the dorsal telencephalon, suggesting cells ongoing division. (E) At E55, in the SVZ of the GE many GAD65-labeled cells were labeled for Ki67 and therefore corresponded to proliferating cells. In contrast none of the migrating GAD65-containing cells in the IZ displayed detectable level of Ki67 as illustrated at higher magnification (inset E′) and therefore likely corresponded to early postmitotic neurons. (F, G) At E55 in the SVZ of GE (F) like at E75 in the SVZ of the dorsal telencephalon (G) most GAD65-containing cells were labeled for Ki67 and therefore likely corresponded to progenitors of GABAergic neurons. (H, I) At E75, many migrating like GAD65-containing cells in the IZ (H) and SP (I) were not labeled for Ki67. Scale bars = 50 μm (A, C, E, F, G, I), 30 μm (B, E′) and 10 μm (D, H).

GAD67 labeling patterns in coronal sections of monkey fetal telencephalon at E55 (A, a, a′), E68 (B, b) and E75 (C, c, c′). (A) At E55 as at earlier stage of cortical development, GAD67-containing neurons were mainly present in the MZ and below the CP in the upper part of the subplate (SPu). In the MZ these neurons were surrounded by dense plexus of stained fibers as illustrated at higher magnification (inset a′). Some neurons were also observed in the IZ (arrows). Many of them in this layer displayed a unipolar migratory-like morphology (inset a). (B) At E68 a general increased density of GAD67-labeled neurons was observed but this increase was particularly striking in the lower part of the subplate (SPL) (arrows). Inset b: In this later layer, many GAD67-labeled cells corresponded to morphologically differentiated neurons, that is, neurons with strong labeling of the cell body and proximal dendrites (arrow). These neurons contrast with migratory-like postmitotic neurons lightly labeled for GAD67 (arrowhead). (C) At E75 the thickness of the SP and the density of GAD67-containing neurons further increased. More GAD67-labeled neurons were also observed in the MZ and the IZ. Some GAD67-containing neurons were now present in the CP (arrows). Higher magnification illustrating morphologically differentiated neurons and axon terminals labeled for GAD67 in the SP (inset c) and in the CP (inset c′). (D) At E75 Adjacent section stained for cresyl violet showed that the SP is at this age up to 3 times thicker than the CP. Scale bars = 200 μm (A–D), 50 μm (a′, b, c, c′), 25 μm (a).