Basal progenitor cells in the embryonic mouse thalamus - their molecular characterization and the role of neurogenins and Pax6.

Abstract

The size and cell number of each brain region are influenced by the organization and behavior of neural progenitor cells during embryonic development. Recent studies on developing neocortex have revealed the presence of neural progenitor cells that divide away from the ventricular surface and undergo symmetric divisions to generate either two neurons or two progenitor cells. These 'basal' progenitor cells form the subventricular zone and are responsible for generating the majority of neocortical neurons. However, not much has been studied on similar types of progenitor cells in other brain regions.
We have identified and characterized basal progenitor cells in the embryonic mouse thalamus. The progenitor domain that generates all of the cortex-projecting thalamic nuclei contained a remarkably high proportion of basally dividing cells. Fewer basal progenitor cells were found in other progenitor domains that generate non-cortex projecting nuclei. By using intracellular domain of Notch1 (NICD) as a marker for radial glial cells, we found that basally dividing cells extended outside the lateral limit of radial glial cells, indicating that, similar to the neocortex and ventral telencephalon, the thalamus has a distinct subventricular zone. Neocortical and thalamic basal progenitor cells shared expression of some molecular markers, including Insm1, Neurog1, Neurog2 and NeuroD1. Additionally, basal progenitor cells in each region also expressed exclusive markers, such as Tbr2 in the neocortex and Olig2 and Olig3 in the thalamus. In Neurog1/Neurog2 double mutant mice, the number of basally dividing progenitor cells in the thalamus was significantly reduced, which demonstrates the roles of neurogenins in the generation and/or maintenance of basal progenitor cells. In Pax6 mutant mice, the part of the thalamus that showed reduced Neurog1/2 expression also had reduced basal mitosis.
Our current study establishes the existence of a unique and significant population of basal progenitor cells in the thalamus and their dependence on neurogenins and Pax6. These progenitor cells may have important roles in enhancing the generation of neurons within the thalamus and may also be critical for generating neuronal diversity in this complex brain region.

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Basal progenitor cells in the embryonic mouse
thalamus - their molecular characterization and
the role of neurogenins and Pax6
Wang et al.
Wang et al. Neural Development 2011, 6:35
http://www.neuraldevelopment.com/content/6/1/35 (11 November 2011)

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RESEARCH ARTICLEOpen Access
Basal progenitor cells in the embryonic mouse
thalamus - their molecular characterization and
the role of neurogenins and Pax6
Lynn Wang1†, Krista K Bluske1,2†, Lauren K Dickel3and Yasushi Nakagawa1,2*
Abstract
Background: The size and cell number of each brain region are influenced by the organization and behavior of
neural progenitor cells during embryonic development. Recent studies on developing neocortex have revealed the
presence of neural progenitor cells that divide away from the ventricular surface and undergo symmetric divisions
to generate either two neurons or two progenitor cells. These ‘basal’ progenitor cells form the subventricular zone
and are responsible for generating the majority of neocortical neurons. However, not much has been studied on
similar types of progenitor cells in other brain regions.
Results: We have identified and characterized basal progenitor cells in the embryonic mouse thalamus. The progenitor
domain that generates all of the cortex-projecting thalamic nuclei contained a remarkably high proportion of basally
dividing cells. Fewer basal progenitor cells were found in other progenitor domains that generate non-cortex projecting
nuclei. By using intracellular domain of Notch1 (NICD) as a marker for radial glial cells, we found that basally dividing
cells extended outside the lateral limit of radial glial cells, indicating that, similar to the neocortex and ventral
telencephalon, the thalamus has a distinct subventricular zone. Neocortical and thalamic basal progenitor cells shared
expression of some molecular markers, including Insm1, Neurog1, Neurog2 and NeuroD1. Additionally, basal progenitor
cells in each region also expressed exclusive markers, such as Tbr2 in the neocortex and Olig2 and Olig3 in the
thalamus. In Neurog1/Neurog2 double mutant mice, the number of basally dividing progenitor cells in the thalamus was
significantly reduced, which demonstrates the roles of neurogenins in the generation and/or maintenance of basal
progenitor cells. In Pax6 mutant mice, the part of the thalamus that showed reduced Neurog1/2 expression also had
reduced basal mitosis.
Conclusions: Our current study establishes the existence of a unique and significant population of basal
progenitor cells in the thalamus and their dependence on neurogenins and Pax6. These progenitor cells may have
important roles in enhancing the generation of neurons within the thalamus and may also be critical for
generating neuronal diversity in this complex brain region.
Background
The immense number of neurons in the mammalian neo-
cortex is thought to be determined during development by
a prominent progenitor cell population that shows a dis-
tinct pattern of migration and division. Unlike the predo-
minant progenitor cell type in other brain regions, the
radial glial cells (RGs), these cells divide basally away from
the ventricular surface and undergo a symmetric division
that generates two neurons or two progenitor cells. It is
thought that the six-layered mammalian neocortex is lar-
gely dependent on division of these basal progenitor cells
that serve as transit amplifying cells or intermediate pro-
genitor cells (IPCs), and that the evolution of the mamma-
lian cortex is correlated with the emergence of progenitor
cell populations that enhance the generation of neurons
[1-4].
Basally dividing progenitor cells have been identified
not only in the cerebral cortex, but also in ganglionic
eminences, thalamus, hindbrain and spinal cord [5-9].
* Correspondence: nakagawa@umn.edu
† Contributed equally
1Department of Neuroscience, Developmental Biology Center and Stem Cell
Institute, University of Minnesota, Minneapolis, MN 55455, USA
Full list of author information is available at the end of the article
Wang et al. Neural Development 2011, 6:35
http://www.neuraldevelopment.com/content/6/1/35
© 2011 Wang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.

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However, only the cerebral cortex and ganglionic emi-
nences have been shown to harbor a robust enough
population of basal progenitor cells to form a distinct
domain, the subventricular zone (SVZ), above the
domain of RGs that comprises the ventricular zone (VZ).
Recent studies identified a number of molecular markers
of basal progenitor cells in the developing neocortex. In
addition, genes such as Tbr2 [10,11], Insm1 [12] and
AP2g [13] or inhibition of Notch signaling [14,15] are
found to be essential for the generation of basal progeni-
tor cells from RGs. Time-lapse analysis of fluorescently
labeled cortical progenitors in slices elucidated the
unique migratory patterns and modes of division of neo-
cortical basal progenitor cells and showed that these cells
function as transit amplifying progenitor cells, or IPCs
[9,16,17].
The mammalian thalamus has an extremely complex
organization with several dozen distinct neuronal popula-
tions called nuclei [18]. During embryogenesis, the thala-
mus is composed of two molecularly distinct domains of
neural progenitor cells, pTH-C and pTH-R, located across
rostro-caudal and dorso-ventral axes [19]. pTH-C is a lar-
ger, caudo-dorsally located domain that expresses the
basic helix-loop-helix (bHLH) transcription factors neuro-
genin 1 (Neurog1) and neurogenin 2 (Neurog2) and gives
rise to all of the thalamic nuclei that project to the cortex.
pTH-R is a smaller domain that expresses another bHLH
protein, Ascl1 (also known as Mash1), and lies between
pTH-C and the zona limitans intrathalamica (ZLI), the
boundary population that abuts the thalamus and the pre-
thalamus [19]. pTH-R likely contributes to the majority of
GABAergic neurons in the thalamus, including part of the
ventral lateral geniculate nucleus and intergeniculate leaf-
let. Recent studies have unveiled critical roles of secreted
signaling molecules in the formation of positional diversity
of thalamic progenitor cells [20-24].
Despite the finding that there are basally dividing cells in
embryonic mouse thalamus [6], their molecular character-
istics and the mechanisms for their generation have not
yet been determined. Considering the extensive connec-
tions between the thalamus and neocortex, we anticipated
that the mammalian thalamus has diversified its progeni-
tor cell populations during evolution to allow generation
of a larger number of neurons comparable to those found
in the six-layered neocortex.
In the study described here, we explored this possibility
by performing a detailed characterization of thalamic
basal progenitor cells in mouse embryos. We found that
the thalamus contains a remarkably large number of
basal progenitor cells, some of which form the SVZ simi-
lar to that found in the neocortex and ventral telencepha-
lon. Thalamic basal progenitor cells do not express the
same molecular markers as neocortical IPCs, such as
Tbr2, but they do share many other aspects with their
putative cortical counterpart, including the expression of
the bHLH transcription factors NeuroD1 and neurogen-
ins (Neurog1 and Neurog2). We also show the first
evidence that Neurog1 and Neurog2 are required for the
normal number of basally dividing cells, which demon-
strates the critical role of these transcription factors in
the formation and/or the maintenance of thalamic basal
progenitor cells.
Results
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 phos-
phorylated 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-posi-
tive 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 (api-
cal) surface (bin 1), there was another peak of PH3-expres-
sing cells away from the third ventricle (bin 6), indicating
the presence of a discrete population of thalamic progeni-
tor cells (Figure 1I). These initial analyses demonstrate
the presence of basally dividing progenitor cells in the tha-
lamus throughout neurogenesis and that they are particu-
larly 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 dis-
tinct 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)
Wang et al. Neural Development 2011, 6:35
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Figure 1 Numerous cells divide away from the third ventricle in the embryonic mouse thalamus. (A-F) Frontal sections of embryonic day
(E)10.5 (A,B), E12.5 (C,D) and E14.5 (E,F) of wild-type mouse forebrain. Midline is to the left. Panels (A,B) and (C,D) each shows the same sections
of triple immunohistochemistry for PH3, Olig3 and Neurog2. Panel (E) is more dorsal to panel (F). Numerous PH3-positive cells exist not only at
the surface of the third ventricle (A, arrow) but also away from the ventricle (A-D, arrowheads). At E14.5, when thalamic neurogenesis is almost
over, a small number of PH3-positive cells are found in dorsal sections (E), but not in more ventral sections (F). Scale bar: 100 μm. PT, pretectum;
C, thalamic progenitor domain pTH-C; R, thalamic progenitor domain pTH-R; ZLI, zona limitans intrathalamica. (G-I) Cell count of PH3-positive
cells in the thalamus. (G) Proportion of PH3-positive cells located more than 40 μm away from ventricular surface (basal PH3+cells) was
calculated for E11.5, E12.5 and E14.5. The ratio of basal PH3+/total PH3+cells was significantly higher at E12.5 than E11.5 or E14.5 (*P < 0.05 and
**P < 0.01, respectively; n = 4 for E11.5, E12.5; n = 6 for E14.5). (H) At E12.5, a significantly higher ratio of basal PH3+/total PH3+cells was found
in pTH-C compared to pTH-R and ZLI domains (***P < 0.001; n = 4 for pTH-C; n = 6 for pTH-R and ZLI) (I) Distribution of PH3-positive cells in
each of the 20-μm-wide medio-lateral bins, showing two distinct populations; one is located closest to the ventricular surface (bin 1) and the
other is located more basally, with the peak at bin 6 (mean ± standard error of the mean; 5.0 ± 0.79).
Wang et al. Neural Development 2011, 6:35
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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 iden-
tified a new class of Pax6-expressing progenitor cells that
divide away from the lateral ventricle in the mouse neo-
cortex [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 pro-
genitor 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 deoxyuri-
dine (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 pro-
genitor cells in order to determine the progenitor zone
(VZ or SVZ) and progenitor cell types (RGs or basal pro-
genitor 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 thala-
mus encompassed the entire medial-lateral extent of thala-
mic 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 progeni-
tor cells in the thalamus.
We next determined if NeuroD1 and Insm1 (insuli-
noma-associated 1), markers for neocortical IPCs, are
also expressed in the thalamus. NeuroD1 is a bHLH tran-
scription 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 popu-
lation 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 scat-
tered 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 tran-
scription factors expressed in the thalamus [33,34]. In the
neocortex, Lhx2 is expressed in neural progenitor cells
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