The embryonic mouse thalamus contains a large number of basally dividing cells
We found numerous cells away from the surface of the third ventricle that express the M-phase marker phosphorylated histone H3 (PH3), which we define as dividing basal progenitor cells (Figure 1A-E, arrowheads). These cells were found as early as at embryonic day (E)10.5 (Figure 1A,B) and persisted until at least E14.5 (Figure 1E). Double/triple immunohistochemistry showed that most of these basal progenitor cells are within the progenitor domain pTH-C, which gives rise to all of the thalamic nuclei that project to the cerebral cortex [19] (Figure 1B, marked as 'C'). Within the pTH-C domain, the ratio of basal PH3-positive cells to total PH3-positive cells was highest at E12.5 and declined at E14.5, when thalamic neurogenesis is largely complete, except in the most dorsal location (Figure 1E-G) [25]. In contrast, fewer PH3-positive cells were found in the progenitor domain pTH-R, which produces neurons that do not project to the cortex [19] (Figure 1B, marked as 'R') and in the ZLI, the border cell population abutting the thalamus and the prethalamus (Figure 1B, marked as 'ZLI'). The ratio of PH3-positive cells in the basal location to the total PH3-positive cells was significantly higher in pTH-C than the other two domains analyzed (pTH-R and ZLI; Figure 1H). Figure 1I shows the average number of PH3-positive cells in each of the 20 μm-wide medial-lateral bins within pTH-C at E12.5. In addition to the high peak at the ventricular (apical) surface (bin 1), there was another peak of PH3-expressing cells away from the third ventricle (bin 6), indicating the presence of a discrete population of thalamic progenitor cells (Figure 1I). These initial analyses demonstrate the presence of basally dividing progenitor cells in the thalamus throughout neurogenesis and that they are particularly enriched in the progenitor domain pTH-C.
The embryonic mouse thalamus has a defined subventricular zone
We next asked if the basally dividing cells comprise a distinct zone in the mouse thalamus that is not populated by RGs. Such a zone, the SVZ, emerges in mice by E14.5 in the neocortex, and by E13.5 in the ganglionic eminences [26]; however, it has not been evaluated in other brain regions. We used NICD (intracellular domain of Notch) and Pax6 as markers of RGs. NICD is a cleavage product of Notch1 [27], which is co-expressed with Nestin within the neocortical VZ, but not in the SVZ [28]. Notch activity inhibits the formation of IPCs from RGs in the neocortex, indicating that the presence of NICD is a marker for RGs within the VZ. Pax6 is also highly expressed in neocortical RGs in the VZ [29], although low-levels of Pax6 expression are detectable in many IPCs [29] and a recent report identified a new class of Pax6-expressing progenitor cells that divide away from the lateral ventricle in the mouse neocortex [30].
We found that NICD is expressed in a cluster of cells near the third ventricle at both E11.5 (Figure 2A,C,C', left of dashed line) and E12.5 (Figure 2E,G,G', left of dashed line). Basal PH3-positive cells were located on both sides of the lateral margin of this NICD cluster at E11.5 and E12.5 (Figure 2A,E; arrows indicate the outside population and arrowheads indicate the inside population). The outer population became more evident at E12.5 (Figure 2E). PH3-positive cells were also observed on both sides of the Pax6 domain (Figure 2B,F, arrowheads and arrows). Similar to the neocortex, more laterally located basal progenitor cells expressed low levels of Pax6 (Figure 2B,D,D' at E11.5; 2F,H,H' at E12.5). In addition, Pax6 expression was generally lower in the rostral part of the pTH-C domain at both E11.5 and E12.5 (Figure 2B,F, bracket). Labeling of S-phase cells with a 0.5-hour ethynyl deoxyuridine (EdU) pulse showed that some thalamic progenitor cells reside outside the NICD+/Pax6-high zone (Figure 2C',D'G'H'). Based on these results, we propose that, as early as E11.5, a molecularly distinguishable SVZ exists in the pTH-C domain of the thalamus, which we define as the zone where progenitor cells exist outside of the NICD+/Pax6-high VZ. Thalamic basal progenitor cells populate both the VZ and SVZ.
Thalamic and neocortical basal progenitor cells share some molecular properties
We then examined the expression patterns of previously characterized genes that are expressed in thalamic progenitor cells in order to determine the progenitor zone (VZ or SVZ) and progenitor cell types (RGs or basal progenitor cells) in which each gene is expressed (Figures 3, 4 and 5). Thalamic progenitor cells ubiquitously express the bHLH transcription factor Olig3 [19], but the neocortex does not. Double staining with a 0.5-hour EdU pulse showed that the domain of Olig3 expression in the thalamus encompassed the entire medial-lateral extent of thalamic progenitor cells, indicating that Olig3 is expressed in both the VZ and SVZ of the thalamus (Figure 3A,F). In addition, we found that Olig3 heavily overlaps with NICD (Figure 5A), demonstrating that Olig3 is expressed in RGs. Together, these results show that Olig3 is expressed in both the VZ and SVZ and in both RGs and basal progenitor cells in the thalamus.
We next determined if NeuroD1 and Insm1 (insulinoma-associated 1), markers for neocortical IPCs, are also expressed in the thalamus. NeuroD1 is a bHLH transcription factor that is expressed in the upper SVZ and lower intermediate zone of the neocortex, presumably being induced following Tbr2 expression [31]. In the pTH-C domain of the thalamus, a densely packed population of NeuroD1-positive cells was found in the middle portion of the diencephalic wall (Figure 3B, arrow). In addition, some NeuroD1-expressing cells were scattered within the VZ. Double immunostaining for NeuroD1 and a 0.5-hour pulse of EdU showed that NeuroD1 is expressed in basally located progenitor cells in S phase of the cell cycle (Figure 3G, arrowheads). These NeuroD1+/EdU+ cells seemed to be predominantly located in the SVZ. NeuroD1 was also clearly expressed outside of the NICD-expressing VZ (Figure 5D, arrow) and the scattered NeuroD1-positive cells within the VZ did not express NICD (Figure 5D, arrowheads), indicating the lack of NeuroD1 expression in RGs. Double staining experiments also showed that some basal progenitor cells co-expressed NeuroD1 and PH3 (not shown). These results together demonstrate that NeuroD1 is expressed in thalamic basal progenitor cells at least through S phase to M phase of the cell cycle, but not in NICD-expressing RGs.
Insm1 is a zinc-finger transcription factor expressed broadly in progenitor cells within the embryonic brain and spinal cord located away from the ventricular surface [32]. It is required for the generation of basal progenitor cells in the neocortex [12]. We found that Insm1 is strongly expressed in a lateral band of cells within the thalamus. Comparison of Insm1 with PH3 on the same section shows that Insm1 is indeed expressed in thalamic basal progenitor cells (Figure 3C,H).
Olig2 is a bHLH transcription factor expressed in the pTH-C domain of the thalamus in a rostro-ventral high to caudo-dorsal low gradient at E11.5 and E12.5 [19]. We found that Olig2 is not only expressed in the VZ (Figure 3D, arrowhead) but also in a more lateral region (Figure 3D, arrow). Olig2 expression overlapped with a 0.5-hour EdU pulse (Figure 3I), and extended further laterally (Figure 3I, arrow; Figure 5E). Within the VZ, Olig2 co-localized with NICD (Figure 5E, arrowheads), suggesting that it is expressed in RGs. Thus, similar to Olig3, Olig2 is expressed in both RGs and basal progenitor cells. Olig2 also appeared to be expressed lateral to the SVZ, most likely in the mantle zone.
Finally, Lhx2 and Lhx9 are LIM-homeodomain transcription factors expressed in the thalamus [33, 34]. In the neocortex, Lhx2 is expressed in neural progenitor cells and Lhx9 is expressed in the marginal zone [35, 36]. We found Lhx2/9-positive cells are largely confined outside the VZ, with only a minimum overlap with a 0.5-hour EdU pulse (Figure 3J), indicating that they are expressed mostly in postmitotic cells.
Interestingly, a well-established IPC marker in the neocortex, Tbr2, a T-box transcription factor [15, 29], was undetectable in the thalamus at E11.5 and E12.5 (data not shown).
These results collectively show that although the thalamus has a histologically identifiable SVZ populated by basal progenitor cells and these cells share expression of some genes, such as Insm1 and NeuroD1, with neocortical IPCs, they are clearly distinct from their putative neocortical counterpart. Thalamic basal progenitor cells do not express Tbr2 and express additional markers such as Olig2 and Olig3 that are not expressed in the neocortex.
Proneural bHLH proteins Neurog1 and Neurog2 are expressed in overlapping but different progenitor populations in the thalamus
To further characterize the thalamic basal progenitor cells, we examined the expression of two bHLH proteins, Neurog1 and Neurog2, both of which are expressed in neocortical progenitor cells. In the neocortex, expression of Neurog2 is initiated soon after the division of RGs, preceding the induction of Tbr2 [15]. Britz et al. [37] reported that at E12.5, 95% of Neurog1-expressing progenitor cells in the cortical VZ also express Neurog2, and at E15.5, both Neurog1 and Neurog2 are expressed in the VZ as well as the SVZ.
We previously showed that Neurog1 and Neurog2 are expressed in the pTH-C thalamic progenitor domain [19]. In this study, we examined the patterns of their expression in more detail. Comparison of Neurog1 expression with EdU (0.5-hour pulse) indicated that Neurog1 expression does not extend as laterally as EdU (Figure 4A, arrowheads), although Neurog1 and EdU overlap at the lateral part of the Neurog1 expression domain (Figure 4A, arrows). Neurog2 expression extended more laterally than Neurog1, where it heavily overlapped with EdU (Figure 4B, arrowheads). Neurog2 also overlapped with EdU more medially, in the region where Neurog1 is expressed (Figure 4B, arrows). A direct comparison of Neurog1 and Neurog2 expression indicated that Neurog2 expression extends more laterally than Neurog1 (Figure 4C, arrowheads). The lateral border of the Neurog1 expression domain matched the lateral border of NICD expression (Figure 5B, dashed line). Thus, in contrast to the neocortex, Neurog1 expression in the thalamus is confined to the VZ. Within the VZ, Neurog1 and Neurog2 showed partially overlapping but distinct expression patterns (Figure 4C). Similar to the neocortex [28], neither of these two transcription factors co-localized with NICD within the VZ (Figure 5B,C, arrowheads). This result is consistent with the hypothesis that neurogenin-expressing VZ cells are basal progenitors translocating laterally towards the SVZ. In contrast, Olig2 and Olig3 were expressed in both the thalamic VZ and SVZ and had extensive overlap with NICD within the VZ (Figure 5A,E).
Cell cycle properties of basal progenitor cells in the thalamus
We next examined the cell cycle properties of thalamic basal progenitor cells. First, we pulsed the progenitor cells with an S-phase marker, EdU, and analyzed the distribution of PH3-positive cells at various times after EdU injection. We detected EdU and PH3 on the same section of E11.5 and E12.5 embryos to estimate the time it takes progenitor cells to enter M phase (Figure 6). In E11.5 embryos that had been pulsed with EdU 0.5 hours prior to sacrifice, we detected a large, single cluster of EdU-positive cells that encompassed a broad medial-lateral region of the thalamic progenitor domain, suggesting the close proximity of RGs and basal progenitor cells during S phase (Figure 6A; black curve in Figure 6F,G). As expected, very few mitotic cells expressing PH3 are labeled by EdU.
At 2 hours after EdU injection, we detected some EdU-positive cells at the ventricular surface and the region closer to the ventricle (Figure 6B, arrow; red curve in Figure 6F,G). Many PH3-positive cells both at the ventricular surface and in the basal location were also EdU-positive (Figure 6B, arrowheads). This indicates that, particularly at E11.5, cells start to enter M phase about 2 hours after S phase.
At 4 hours, as many as 60 to 75% of PH3-expressing cells were positive for EdU at both the apical and basal locations (Figure 6C, arrowheads; Figure 6E). In addition, we found two dense clusters of EdU-positive cells that were now separated from each other. One was located close to the ventricle. The other population was located more laterally (green curve in Figure 6F,G). This separation implies a distinct migratory behavior of thalamic basal progenitor cells, which stay in the basal location from S phase to M phase. Conversely, RGs translocate their nuclei medially from S phase to M phase by interkinetic nuclear migration.
At 8 hours, we again detected only a small overlap between EdU and PH3, indicating that a majority of progenitor cells labeled 8 hours before have already divided. A broad cluster of EdU-positive cells was found in the middle of the diencephalic wall (Figure 6D, between the dashed lines), and additional EdU-positive cells were found far laterally, which are likely to be postmitotic cells (blue curve in Figure 6F,G).
In summary, the EdU pulse experiment distinguishes RGs and basal progenitor cells because of their distinct patterns of migration during their cell cycle.
Expression of basal progenitor markers at different stages of the cell cycle
By taking advantage of the EdU pulse labeling, we next examined the expression of NeuroD1, Lhx2/9, Neurog1 and Neurog2 in more detail with regard to the cell cycle status of basal progenitor cells.
As already shown in Figure 3, NeuroD1 was co-localized with 0.5-hour EdU only in the basal location (Figure 7A; Figure 7U, black line). Co-localization of NeuroD1 with EdU continues in the basal location at 2 hours (Figure 7B, arrowheads; Figure 7U, black curve), 4 hours (Figure 7C, arrowheads; Figure 7U, green curve) and 8 hours (Figure 7D, arrowheads; Figure 7U, blue curve), indicating that basal progenitor cells express NeuroD1 throughout their cell cycle after the newly generated cells reach the basal location. NeuroD1 also partially co-localized with p27 (Figure 8H), a cyclin-dependent kinase inhibitor expressed in differentiating neural progenitor cells as well as postmitotic neurons [38, 39], but it did not co-localize with NeuN (Figure 8C), a marker for a subset of postmitotic neurons, suggesting that NeuroD1 expression is transient.
Lhx2/9 was expressed in the lateral part of the thalamus, and showed only a minor overlap with EdU at each of the pulse times (Figure 7E-H,7V). The overlap with neuronal markers NeuN and p27 was robust (Figure 8E,J), indicating that Lhx2/9 expression persists in postmitotic neurons, consistent with a previous study showing widespread expression of Lhx2 and Lhx9 in postmitotic thalamic nuclei [33].
As shown in Figure 4, Neurog1 is co-localized with 0.5-hour EdU in the VZ (Figure 7I, arrowheads; Figure 7W, black curve) but not in the SVZ. In contrast, Neurog2 co-localization with 0.5-hour EdU was found in both the VZ and SVZ (Figure 7M, arrowheads; Figure 7X, black curve). Cells co-expressing EdU and Neurog1 and cells co-expressing EdU and Neurog2 were found more medially with a 2-hour pulse (Figure 7J,N, arrowheads; Figure 7W,X, red curve). At 4 hours, when most PH3-positive cells are also EdU-positive, more cells co-expressing neurogenins and EdU were found near the third ventricle, in addition to the lateral cluster (Figure 7K,O, arrowheads; Figure 7W,X, green curve). With an 8-hour pulse, we detected a single cluster of double-labeled cells, while the lateral cluster of EdU-positive cells, which are likely to be postmitotic neurons, did not express neurogenins (Figure 7L,P, arrowheads; Figure W,X, blue curve). These results indicate that Neurog1 and Neurog2 are both induced as newly formed basal progenitor cells migrate to basal positions, within either the VZ (for both Neurog1 and Neuorg2) or the SVZ (for Neurog2), and their expression is maintained as the basal progenitor cells undergo cell cycles and divide again. Double staining with NeuN (Figure 8A,B) and p27 (Figure 8F,G) showed that neurogenins overlap with p27 but not with NeuN. Thus, the expression of neurogenins is transient.
Neurogenins are required for the formation and/or maintenance of basal progenitor cells in the thalamus
In the neocortex, Neurog1 and Neurog2 together play a role in neuronal differentiation and, at the same time, in the specification of the dorsal telencephalic fate of neural progenitor cells [40]. Microarray analysis shows that the expression levels of Tbr2 and NeuroD1 in the neocortex are decreased in Neurog1/2 double knockout mice [40]. Although histological analysis of cortical IPCs with immunohistochemistry for Tbr2 and NeuroD1 has not been reported in these mutant mice, both PH3-positive mitotic cells and bromodeoxyuridine-labeled S-phase progenitor cells are increased in the SVZ and decreased in the VZ in Neurog2 single as well as Neurog1/2 double knockout mice [37], suggesting that these transcription factors are likely to play an important role in IPC specification and/or differentiation.
In order to determine if neurogenins play a role in the formation or maintenance of basal progenitor cells in the thalamus, we analyzed Neurog1/2 double knockout mice and Neurog1 and Neurog2 single knockout mice in comparison with double heterozygous controls. We found that double knockout mice (Neurog1-/- ; Neurog2-/- ) have fewer PH3-positive, dividing basal progenitor cells in the pTH-C domain at E12.5 (Figure 9D,E). Both the absolute number and the ratio against the total PH3-positive cell number were significantly reduced from the controls. In contrast, the number of apical PH3-positive cells or the total PH3-positive cells did not show a significant difference. The Neurog2 single (Neurog1+/- ; Neurog2-/- ) mutant showed reduction in absolute number of basal PH3-positive cells but not in the ratio against the total PH3-positive cells (Figure 9C,E). The Neurog1 single mutant did not show any significant difference from the control (Figure 9B,E). These results indicate that neurogenins are required for the normal number of basally dividing progenitor cells in the thalamus, and that the role of Neurog2 is only partially compensated by Neurog1.
As already shown previously [41], another bHLH transcription factor, Ascl1 (also known as Mash1) is induced in the neocortex of Neurog2 single and Neurog1/2 double mutant mice. Ascl1 is normally expressed at a high level in the ventral telencephalon, suggesting a role for neurogenins in specifying dorsal telencephalic fate and suppressing ventral telencephalic fate. It has also been shown that neurogenins are required to suppress Ascl1 expression in the thalamus [41, 42]. Consistent with these previous findings, we found robust Ascl1 induction in the thalamus of Neurog1/2 double mutant mice (Figure 9H), whereas Neurog2 single mutants (Neurog1+/- ; Neurog2-/- ) showed much less severe induction of Ascl1 (Figure 9G). Ascl1 was not induced in Neurog1 single mutants (Neurog1-/- ; Neurog2+/- ; data not shown). These results demonstrate that neurogenins, of which Neurog2 is the prominent one, suppresses Ascl1 expression. Reduction of the basal progenitor cell number in the thalamus of neurogenin mutant mice indicates that Ascl1 does not compensate for the function of neurogenins in this cell type. Interestingly, Tbr2, a cortical IPC marker, was normally not expressed in the thalamus but was ectopically induced in the mantle zone of the thalamus of the Neurog1/2 double mutant (Figure 9K,L). Considering the fact that SVZ mitosis was increased in the neocortex [37] but decreased in the thalamus (Figure 9) of Neurog1/2 double knockout mice, we conclude that the roles of neurogenins in basal progenitor cells in the thalamus are likely different from those in the neocortex.
The paired-/homeo-domain transcription factor Pax6 is known to play a critical role in thalamic development [43]. As already shown in Figure 2, high-level expression of Pax6 was detected in the thalamic VZ, although the expression decreased in the rostro-ventral part of the pTH-C domain at E11.5 and later. In Pax6 mutant mice, we detected reduction of Neurog2 expression (Figure 10E,G) and ectopic induction of Ascl1 (Figure 10F,H) in the ventral part of the pTH-C domain, but not in the dorsal part (Figure 10A-D). The ratio of basal PH3-positive cells was specifically reduced in ventral sections, where a large number of Ascl1-expressing cells were intermingled with Neurog2-expressing cells (Figure 10G-I). The decrease in the number of basal PH3-positive cells was accompanied by an increase in the number of apical PH3-positive cells (Figure 10J), indicating the role of Pax6 in generating basal progenitor cells from apical progenitor cells. The total number of basal plus apical PH3-positive cells did not change between wild-type and mutant embryos, at both dorsal and ventral levels (data not shown).
