1. Our results thus far are most evident in an experiment injecting 5 mg/kg TMX at
E13.5 and sampling at E20.5 (birth did not occur due to complications). While RFP
expression in the midbrain has been more sparse than expected with 12 µm sections,
these 100 µm sections (Fig. 8) have shown morphologically where E13.5 Tbr2+
neurons become located in the brain. At this point, we may continue by performing
longer-range studies to P10.5 or even full adulthood, and begin colocalizing these
tissues with markers aiding in characterization and identification.
We are performing immunohistochemistry on wild-type tissues using the mitotic
marker PCNA to determine whether the cells are dividing at certain ages. We can
alternately use injection of bromodeoxyuridine (BrdU), a thymidine analog, as a
marker for mitotic cells to determine when these cells are replicating DNA and any
relevant patterns involved. When integrated into DNA in place of thymidine, BrdU
can be detected via immunohistochemistry, indicating that injection occurred during
the synthesis phase. As ourAi14/EoCreERT2 experiments are transitioning to longer-
range, it may be more useful to inject BrdU alongside TMX application, which
would enable detection of RFP+ cells that were also mitotic during their Tbr2
expression. We have found sparse mitotic Tbr2+ midbrain cells at E14.5 using both
markers (Fig. 3), and are extending the experiment to a wider range of ages. Lack of
colocalization indicates the postmitotic stage, which so far appears to be the majority
of Tbr2+ midbrain neurons we have observed at E13.5 or later (Fig. 4, Fig. 5); our
hope is to detect when most of these cells are replicating.
Expression of Transcription Factor
Tbr2 in Embryonic Mouse Midbrain
Michelle AN La1, Ray AM Daza1, and Robert F Hevner1,2
1Children’s Research Institute, Center for Integrative Brain Research, Seattle, WA, USA;
2University of Washington, Department of Neurological Surgery, Seattle, WA, USA
Early neurodevelopment, heavily regulated by transcription factors, structures
fundamental brain processes later in life through neuronal migration, cell-cell
interactions, and cascades of protein expression from cell birth to maturity. The T-
box transcription factor Tbr2 is involved primarily in establishing the progenitor
population that typically produces glutamatergic pyramidal projection neurons in the
cortex1. However, the function and expression patterns of Tbr2 in other brain nuclei
are not well studied. In the developing mouse, we are characterizing a Tbr2+ nucleus
in the midbrain, the seat of functions such as motor control and and motivation once
mature.
Introduction
Figure 1. E13.5 wild type mouse, sagittal section (12 µm). Tbr2 expression (red)
is visible against DAPI nuclear stain (blue); midbrain nucleus indicated with
arrow. Anatomical features of developing brain labeled for clarity.
Wild-Type Expression
EoCreERT2 Lineage Tracer
Immunohistochemistry shows that this nucleus is present from E11.5 through at least
P0. Immunohistochemistry detects the presence of specific antigen peptides through
the use of a primary antibody that binds to the antigen motif, and a fluorescently-
tagged secondary antibody that targets the primary, enabling detection with
fluorescent microscopy. We initially hypothesized that this nucleus was an
acetylcholinergic motor nucleus due to its position and used motor neuron marker
Isl-1 for potential colocalization. Tbr2 and Isl-1 do not colocalize; this nucleus is
therefore not a motor nucleus and is likely glutamatergic, as is the case with other
Tbr2+ neuron populations.2 Our results (Fig. 2) distinguish between two separate
Tbr2+ midbrain nuclei per hemisphere: one lateral of the motor nuclei, and one
medial “bridge” of neurons stretching anteriorly, suggesting a path of migration.
Immunohistochemical Characterization
Figure 2. E14.5 wild type mouse, sagittal section (12 µm). Tbr2 (green) and Isl-1 (red)
are visible against DAPI (blue). Sections moving from lateral at a) to medial at d). At
b), some Tbr2+ cells are present between the motor nuclei. At c), a nucleus of cells
expresses Isl-1 and Tbr2 with no colocalization observed. The motor nuclei appear to
separate two distinct areas of Tbr2+ midbrain expression in one hemisphere.
We are currently performing experiments with Ai14/EoCreERT2 lineage-tracer
reporter mice in order to study these embryonic neurons when they reach maturity,
and identify the nucleus that they become. Upon injection of tamoxifen (TMX), RFP
is activated in any cells currently expressing Tbr2 and remains expressed after Tbr2
is downregulated (Fig. 7).
Figure 7. DNA diagram of EoCreERT2 and Ai14 heterozygote. Wild type alleles shown at top,
construct shown at bottom. CreERT2 and Eomes share the Tbr2 promoter (PTbr2); Ai14, which
codes for RFP, is located under PRosa26, a constitutive proviral promoter. The stop codon
preventing Ai14 transcription is flanked by identical loxP sites (floxed) which can be site-
specifically recombined by Cre. Cre recombinase is expressed whenever Tbr2 is also, but
requires TMX to activate. Therefore, when TMX is injected at an age of interest, Cre is activated
and cleaves out the stop codon, enabling Ai14 RFP expression. The CreERT2 allele was created
through insertion of CreERT2 cDNA into exon 1 of Eomes in embryonic stem cells.4
Figure 8. TE13.5 to
E20, Ai14 CreERT2,
sagittal section (100
µm). A matured
population of cells is
now visible at the
midbrain, and can be
studied more thoroughly
through
immunohistochemistry.
Conclusions
Acknowledgments
This project was supported by the National Institutes of Health (grant
NS085081-01), as well as by the Mary Gates Endowment for Students and the UW
Undergraduate Research Program.
References
1: Englund C, Fink A, Lau C, Pham D, Daza RA, Bulfone A, Kowalczyk T, Hevner RF. 2005. Pax6, Tbr2, and Tbr1 are expressed sequentially by radial glia, intermediate
progenitor cells, and postmitotic neurons in developing neocortex. J Neurosci. 25(1): 247-251.
2: Mizuguchi R, Naritsuka H, Mori K, Klein WH, Yoshihara Y. 2012. Tbr2 deficiency in mitral and tufted cells disrupts excitatory-inhibitory balance of neural circuitry in
the mouse olfactory bulb. J Neurosci. 32(26): 8831-44.
3: Hevner RF, Hodge RD, Daza RA, Englund C. 2006. Transcription factors in glutamatergic neurogenesis: Conserved programs in neocortex, cerebellum, and adult
hippocampus. Neurosci Research. 55(3): 223-233.
4: Pimeisi IM, Tanriver Y, Daza RA, Vauti F, Hevner RF, Arnold HH, Arnold SJ. 2013. Generation and characterization of a tamoxifen-inducible Eomes(CreER) mouse line.
Genesis. 51(10): 725-33.
1. During development, particularly at E14.5, there are at least two distinct
populations of Tbr2+ cells, lateral and medial of cranial nuclei III and IV .
2. While the age of the embryo and related research in the cortex suggests that these
are immature neuronal progenitors, our immunohistochemistry results with Tbr2
and PCNA (from E13.5 to P0) suggest that these cells are no longer mitotic. The
Tbr2+ midbrain nucleus may follow a conserved program of transcription factors
found in other regions of the brain, Pax6 à Tbr2 à Tbr1, that indicates a
glutamatergic fate.
3. Using the Ai14/EoCreERT2, the Tbr2+ population at earlier ages can be
visualized at maturity. Longer-range experiments are needed to identify the adult
midbrain nucleus.
Figure 4. E13.5 wild type
mouse, coronal sections (12
µm); anterior to posterior
from a) to c). Tbr2 (green) and
PCNA (red) are visible against
DAPI (blue); midbrain
nucleus indicated with arrow.
Figure 5. E13.5 wild type
mouse, sagittal sections (12
µm); lateral to medial from a)
to d). Tbr2 (green) and PCNA
(red) are visible against DAPI
(blue); midbrain nucleus
indicated with arrow.
Figure 3. E14.5 wild type acute-BrdU mouse, cell
from sagittal section (12 µm). a) shows the full cell,
while b), c), and d) separate the channels into BrdU
(red), Tbr2 (green), and DAPI (blue), respectively,
demonstrating colocalization.
We are also studying the potential transcription factor cascade Pax6 à Tbr2 à
Tbr1, which has been previously shown to be conserved in glutamatergic
neurogenesis of neocortex, cerebellum, and hippocampus.3 We hypothesize that this
mechanism may be present in the midbrain, and have found colocalization of Pax6
and Tbr2 in E14.5 (Fig. 6), suggesting a) this development scheme, and b) a
glutamatergic fate. This raises questions of whether the neurons originate at the
ventricular surface and migrate inward, as well as whether Tbr1 participates in
development of the nucleus. Tbr1 is expressed in deep cerebellar nuclei, and those
neurons extend processes located in the midbrain; however, Tbr1+ cell bodies may
not be present in the midbrain.
Figure 6. E14.5 wild type mouse,
sagittal sections (12 µm); lateral to
medial from a) to d). Tbr2 (red)
and Pax6 (green) are visible
against DAPI (blue). Confocal Z-
stack microscopy confirms
colocalization.