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First published online 15 December 2008
doi: 10.1242/dev.029900
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1 Institute of Biotechnology, University of Helsinki, 00014 Helsinki,
Finland.
2 Division of Biochemistry, Faculty of Veterinary Medicine, University of
Helsinki, 00014 Helsinki, Finland.
3 Department of Developmental Biology and Department of Physiology, University
of Tartu, 50090 Tartu, Estonia.
4 Helmholtz Zentrum München, Institute of Developmental Genetics, D-85764
Neuherberg, Germany.
* Author for correspondence (e-mail: Juha.M.Partanen{at}Helsinki.Fi)
Accepted 10 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: GABA, Interneuron, Midbrain, Neurotransmitter, Mouse, Neurogenesis, Ventral tegmental area (VTA), Serotonin, Dorsal raphe, Rhombomere 1
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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GABAergic neurons are found in several regions of the midbrain. They are
thought to operate both as local inhibitory interneurons and as projection
neurons with targets elsewhere in the brain. GABAergic neurons are abundant in
the dorsal superior colliculi and periaquaductal gray matter, where they are
involved in multiple processes, including saccadic eye movements, nociception
and defensive behavior (Behbehani et al.,
1990
; Kaneda et al.,
2008). In the ventral midbrain, GABAergic neurons are thought to
regulate the activity of the dopaminergic (DA) neurons in the substantia nigra
pars compacta and ventral tegmental area (VTA)
(Laviolette and van der Kooy,
2004; Tepper and Lee,
2007). In addition to controlling the DA neurons, GABAergic
neurons located in the VTA and substantia nigra pars reticulata (SNpr) send
their axons to other nuclei in the midbrain, prefrontal cortex and other
limbic areas (Laviolette et al.,
2004; Fields et al.,
2007). Thus, midbrain GABAergic neurons are crucial for neural
processes such as the regulation of voluntary and involuntary movements, mood,
motivation and addiction.
Despite the functional importance of the midbrain GABAergic neurons, their
development remains poorly understood. There appear to be both similarities
and differences in the mechanisms that control the development of GABAergic
neurons in distinct brain regions. Proliferative progenitor cells of the
GABAergic neurons are located in the ventricular zone of both ventral and
dorsal midbrain (Tsunekawa et al.,
2005). This is in contrast to the forebrain, where the GABAergic
neurons are generated in ventral neuroepithelium and reach the cortex by
dorsal tangential migration. As in ventral forebrain and other regions of
GABAergic neurogenesis, the proneural bHLH transcription factor Ascl1
(Mash1) is expressed in the ventricular zone of the midbrain and
plays an important role in GABAergic neuron development
(Horton et al., 1999;
Casarosa et al., 1999;
Miyoshi et al., 2004;
Mizuguchi et al., 2006).
Helt (Heslike, Megane, Mgn) is a bHLH-Orange family
transcription factor that is coexpressed with Ascl1 in the midbrain
ventricular zone (Miyoshi et al.,
2004). Mice lacking Helt function show impaired
development of midbrain GABAergic neurons, especially in the dorsal region
(Guimera et al., 2006;
Nakatani et al., 2007).
Furthermore, Helt has been shown to regulate the GABAergic versus
glutamatergic neuron identity by repressing the proneural genes Ngn1
and Ngn2 (Neurog1 and Neurog2), which in turn
promote glutamatergic neuron development
(Nakatani et al., 2007).
In addition to genes controlling the proliferation, identity and
neurogenesis of the ventricular zone progenitor cells, studies of other parts
of the CNS have revealed transcription factors that are activated only in the
post-mitotic neural precursors. Some of these factors do not regulate the
neuronal differentiation process itself, but are needed to select a particular
neuronal phenotype from among distinct alternatives and are therefore called
selector genes. The bHLH transcription factor Ptf1a appears to act as
a selector of the GABAergic, as opposed to glutamatergic, fate in the spinal
cord and cerebellum (Glasgow et al.,
2005; Hoshino et al.,
2005; Cheng et al.,
2005). However, no such selector gene for the midbrain GABAergic
neurons has been identified so far.
Gata2 and Gata3 are related C4 zinc-finger transcription factors. They are
involved in the development of several organs and tissues, and have been
extensively studied, especially in the hematopoietic system
(Tsai et al., 1994). In the
developing CNS, Gata2 and Gata3 are expressed in similar
patterns in distinct brain regions and Gata2 is often required for
the expression of Gata3 (Nardelli
et al., 1999). Gata2 has been suggested to play a role in
the correct development of cranial motoneurons
(Nardelli et al., 1999),
rostral serotonergic neurons in rhombomere 1
(Craven et al., 2004) and
spinal V2 interneurons (Zhou et al.,
2000; Karunaratne et al.,
2002). However, studies of Gata2 function in neuronal
development have been hampered by the early death of Gata2-null
embryos.
We show here that Gata2 is specifically expressed in the developing midbrain GABAergic neurons as they exit the cell cycle and differentiate. Using conditional mutagenesis and ectopic expression experiments, we show that Gata2 acts as an essential post-mitotic selector gene of the GABAergic identity without affecting proliferating neural progenitors or early neurogenic processes in the embryonic midbrain. We further map the GABAergic progenitor domains in the mouse embryonic midbrain and demonstrate region-specific interactions between Helt and Gata2. Interestingly, our results suggest that GABAergic neurons associated with the ventral DA nuclei in the midbrain use distinct regulatory mechanisms for their development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), HeltKO [MgntZ
(Guimera et al., 2006)] and
Ascl1KO [Mash1null
(Guillemot et al., 1993)]
alleles have been described previously. The conditional Gata2 allele
(Gata2flox) will be described elsewhere (M.H., K.L. and
M.S., unpublished). Briefly, in Gata2flox, exons 1-3,
encoding the N-terminal half of Gata2, are flanked by loxP sites. Their
recombination by Cre recombinase is expected to produce a null allele of
Gata2. Gata2cko embryos were generated from
En1Cre/+; Gata2flox/+ x
Gata2flox/flox mouse crosses. For staging, the day of
vaginal plug was counted as embryonic day 0.5 (E0.5). For BrdU-incorporation
analysis, pregnant females were given an intraperitoneal injection of BrdU (3
mg/100 g body weight) 1-2 hours before dissecting the embryos. All experiments
were approved by the Committee of Experimental Animal Research of the
University of Helsinki, Finland.
In ovo electroporation
In ovo electroporation was performed at Hamburger-Hamilton stage (HH)
14-16. For Gata2 overexpression, pAdRSVGata2HA plasmid
(El Wakil et al., 2006) was
microinjected into the embryonic midbrain (third ventricle) together with the
EGFP expression vector pEGFP-N3 (Clontech). As a control, embryos were
electroporated with the EGFP expression vector only. Electroporation was
performed at 20 mV; 10 x 20 millisecond pulses were applied with 500
millisecond intervals. The embryos were harvested 24 or 48 hours later (at
HH20-22) and EGFP-expressing embryos were embedded in paraffin for analysis.
Five embryos electroporated with pAdRSVGata2HA and two control embryos were
analyzed.
In situ mRNA hybridization and immunohistochemistry
Whole-mount mRNA in situ hybridization (ISH) analysis of E10.5 embryos was
performed by a modified protocol (Henrique
et al., 1995) using a digoxigenin-labeled antisense Gata2
cRNA probe. For ISH and immunohistochemistry (IHC) on sections, embryos or
embryonic brains were fixed in 4% paraformaldehyde (PFA) at room temperature
for 1-5 days. Samples were dehydrated and mounted into paraffin. Sections were
cut at 5 µm, and adjacent sections were collected on separate slides for
parallel stainings. mRNA ISH analyses on paraffin sections were performed as
described (Wilkinson and Green,
1990) using 35S- or digoxigenin-labeled cRNA probes.
Mouse cDNA probes used for ISH analysis were: Gata2, Gata3
(Lillevali et al., 2004),
Ascl1, Fev (Pet1), Ngn2
(Jukkola et al., 2006),
Gad1 (Gad67), Helt, Slc17a6 (Vglut2),
Pitx2 (Guimera et al.,
2006), Pou4f1 (gift from Siew-Lan Ang, National Institute
of Medical Research, London, UK), Nkx2-2 (IMAGE clone 480100),
Pax6 (gift from P. Gruss, Max-Planck-Institute for Biophysical
Chemistry, Goettingen, Germany), Isl1 (gift from V. Pachnis, National
Institute of Medical Research, London, UK) and Lmx1b (gift from H.
Simon, Interdisciplinary Centre for Neuroscience, Universität,
Heidelberg, Germany). In addition, probes for chicken Gata3
(Lillevali et al., 2007),
Ngn2 (Matter-Sadzinski et al.,
2001), Slc17a6 and Gad1
(Cheng et al., 2004) were
used.
IHC was performed as described (Kala et
al., 2008). The following antibodies were used: guinea pig
anti-Heslike (Helt, 1:500; gift from R. Kageyama, Institute for Virus
Research, Kyoto University, Japan), goat anti-HA probe (Santa Cruz sc-805-G,
1:500) and anti-Olig2 (Neuromics GT15132, 1:200), mouse anti-BrdU (GE
Healthcare RPN20AB, 1:400), anti-HuC/D (Molecular Probes A21271, 1:500),
anti-Lim1/2 [Lhx1, Developmental Studies Hybridoma Bank (DSHB) 4F2, 1:10],
anti-Mash1 (Ascl1, BD Biosciences 556604, 1:200), anti-Nkx2-2 (DSHB 74.5A5,
1:250), anti-Nkx6-1 (DSHB F55A10, 1:500) and anti-Pax6 (DSHB PAX6, 1:100),
rabbit anti-caspase 3 active (R&D Systems AF835, 1:500), anti-Gata2 (Santa
Cruz sc-9008, 1:250), anti-5-HT (Immunostar 20080, 1:5000), anti-p57
(NeoMarkers RB-1637-P0, 1:500), anti-phospho-histone H3 (Upstate 06-570,
1:500), anti-Sox2 (Millipore AB5603, 1:500) and anti-TH (Millipore AB152,
1:500).
For combined ISH and IHC, additional primary antibodies were added together with the anti-DIG-POD Fab fragments (Roche). The TSA Fluorescence Palette System (PerkinElmer) was used to visualize ISH signal. Detailed ISH and IHC protocols are available upon request.
Microscopy and quantification
Whole-mount stainings were visualized with a Leica MZFLIII microscope and
photographed using an Olympus DP50-CU camera. Stainings on paraffin sections
were visualized with an Olympus AX70 microscope and photographed using an
Olympus DP70 camera. Images were processed and assembled using Adobe Photoshop
software. Red pseudo-color images representing the ISH results were produced
by replacing the white signal in dark-field images with red, and overlaying
the resulting image with the respective bright-field image.
Confocal images were acquired using the Leica TCS SP5 confocal system and LAS-AF software. Confocal stacks and images were processed and deconvoluted using Imaris 6.1 (Bitplane) and AutoQuantX (AutoQuant) software.
For quantification, cells were counted only from the Helt expression domain. For BrdU incorporation, cells from whole neuroepithelium were counted. For phospho-histone H3 expression, only cells lining the ventricle were counted. A standard Student's t-test was used for comparing the mean values of the data sets.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Zhou et al.,
2000). We compared the pattern of expression of Gata2 and
Gata3 with that of the neural subtype markers Lmx1b (DA
neurons), Isl1 (Islet1, motoneurons), Pou4f1
(glutamatergic neurons) and glutamic acid decarboxylase 1 (Gad1,
GABAergic neurons) in the mouse midbrain from E10.5 to E12.5 by in situ
hybridization (ISH) (Fig. 1;
see Fig. S1 in the supplementary material). Throughout development, expression
of both Gata2 and Gata3 coincided with that of Gad1
and was flanked by Pou4f1 expression domains
(Fig. 1A-L). Strong
Gata2 and Gata3 ISH signal was detected in the intermediate
and marginal zones. Weaker expression of Gata2, but no expression of
Gata3, was also observed in the ventricular zone. We detected no
Gata2 transcripts in Lmx1b- or Isl1-expressing
regions in the mouse midbrain (Fig.
1A-C,M-R). From E12.5 onwards, scattered expression of Gata2,
Gata3 and Gad1 appeared in the dorsal midbrain
(Fig. 1C,F,I) correlating with
the timing of GABAergic neurogenesis in this region
(Tsunekawa et al., 2005).
In order to understand in detail the identity of cells expressing
Gata2, we compared the expression of Gata2 protein with homeodomain
transcription factors expressed in progenitor and precursor cells of the
developing midbrain. Lhx1 (Lim1) is expressed in post-mitotic precursor cells
of all midbrain GABAergic neurons in domains m1-m5, as well as in
glutamatergic neurons of the red nucleus in m6 [for a description of the
midbrain domains m1-m7, see Fig.
8 and Nakatani et al.
(Nakatani et al., 2007)]. At
E12.5, immunohistochemistry (IHC) demonstrated coexpression of Gata2 and Lhx1
in the intermediate zone cells of domains m3-m5, where GABAergic neurons are
produced at this stage of development (Fig.
1S,T). Nkx6-1 and Nkx2-2 are expressed in specific subsets of
GABAergic neuron progenitors and precursors
(Nakatani et al., 2007).
Colocalization of Gata2 and Nkx6-1 was observed in the basal ventricular zone
of domains m5 and m3 (Fig. 1U;
see Fig. S1A in the supplementary material). Also, some progenitor/precursor
cells in m4 expressed both Nkx2-2 and Gata2
(Fig. 1V; see Fig. S1B in the
supplementary material). Thus, we found Gata2 expression in all the midbrain
regions that give rise to GABAergic neurons.
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). By combining ISH and IHC, we showed that Pax6 is
expressed in a subset of Nkx2-2-positive cells in the ventral m4 domain at
E11.5 (see Fig. S1C in the supplementary material). Interestingly, no Gata2
protein was detected in the Pax6-expressing cells
(Fig. 1W). Next, we analyzed
the neurotransmitter identities in the m4 marginal zone. Both ISH and IHC
demonstrated that the Pax6-positive cells express Slc17a6 [vesicular
glutamate transporter 2 (Vglut2)] and are negative for Gad1
(see Fig. S1D-G in the supplementary material). Thus, the m4 domain can be
further divided into a GABAergic Gata2-positive dorsal part (m4-D) and a
glutamatergic Pax6-positive ventral part (m4-V). In addition to the m4-V,
putative glial progenitors expressing Olig2 were also devoid of Gata2
expression at E12.5 (Fig. 1X).
In conclusion, the expression of Gata2 mRNA and protein correlates
with the spatial and temporal appearance of the GABAergic neuron lineage in
the developing mouse midbrain.
Gata2 is activated in the developing GABAergic neurons as the progenitor cells exit the cell cycle and start to differentiate
To gain insight to the possible role of Gata2 in GABAergic
neurogenesis, we compared the expression of the bHLH transcription factors
Ascl1 and Helt with that of Gata2 in the m3-m5 ventricular zone by IHC
(Fig. 2A-C). As reported,
extensive coexpression of Ascl1 and Helt was observed in the progenitors
(Fig. 2D)
(Miyoshi et al., 2004).
Notably, we observed coexpression mostly at the apical and medial regions of
the ventricular zone, while on the basal side, the Ascl1-expressing nuclei
were negative for Helt. Also, Gata2 was detected in the Ascl1-positive cells
(Fig. 2F), but, in contrast to
Helt, its expression was detected primarily in the nuclei on the basal side of
the ventricular zone (Fig.
2E,F). In the medial ventricular zone, nuclei coexpressing Helt
and Gata2 were detected (Fig.
2E). The gradual upregulation of Gata2 in the Ascl1- and
Helt-expressing cells/nuclei as they move further away from the ventricular
surface suggested that Gata2 expression might be switched on in the GABAergic
progenitors that are about to exit the ventricular zone. To confirm this, we
analyzed the Gata2-expressing cells for expression of HuC/D (Elavl3/4), a
marker of post-mitotic neurons, and for DNA synthesis as measured by BrdU
incorporation. Representing mostly intermediate zone cells, a large proportion
of Gata2-expressing cells were also positive for HuC/D
(Fig. 2G, arrows). In addition,
HuC/D-negative, yet Gata2-expressing cells were observed in the ventricular
zone (Fig. 2G, arrowheads).
However, these Gata2-expressing nuclei did not incorporate BrdU during a short
labeling pulse (Fig. 2H).
Together, our results show that Gata2 expression is switched on in GABAergic
progenitors as they become post-mitotic precursor cells and start to
differentiate.
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)] for
Gata2 mRNA expression. Interestingly, Gata2 was undetectable
in E11.5 and E13.5 HeltKO midbrain, except for the
ventral-most GABAergic neurons (Fig.
3A,B; data not shown). The remaining Gata2 expression
correlated with Gad1 expression
(Fig. 3C,D, arrow). We detected
no Gata2 or Gad1 expression in the dorsal midbrain of the
E13.5 HeltKO embryos (data not shown). At the same time,
Ascl1 expression was unaffected by Helt inactivation
(Fig. 3E,F), as shown
previously (Nakatani et al.,
2007).
To test whether Ascl1 also regulates Gata2 expression, we
analyzed E11.5 Ascl1-null mutants
(Guillemot et al., 1993). In
these embryos, expansion of the Sox2- and Helt-expressing layer suggested a
failure in cell cycle exit of GABAergic progenitors
(Fig. 3I-L). However, Gata2 was
still expressed in regions where post-mitotic precursors were produced
(Fig. 3G,H). Altogether, except
for the ventral-most GABAergic precursors, activation of Gata2
requires Helt, but does not require Ascl1.
Conditional Gata2 inactivation leads to specific loss of GABAergic neuron precursors in the embryonic midbrain
To study the role of Gata2 in GABAergic neuron development, we
inactivated Gata2 in the mouse midbrain and rhombomere 1 (r1) by
crossing mice carrying a conditional allele of Gata2 (M.H., K.L. and
M.S., unpublished) with those carrying the En1Cre allele
(Kimmel et al., 2000). In the
En1Cre mouse strain, the Cre recombinase activity has been
demonstrated as early as the 5-10 somite stage
(Chi et al., 2003;
Trokovic et al., 2003), well
before the expression of Gata2 in the midbrain-r1 region. In the
En1Cre; Gata2flox/flox
(Gata2cko) mice, no Gata2 expression was detected
in the midbrain-r1 region at E10.5 or E11.5 (see Fig. S2 in the supplementary
material).
Interestingly, we observed a complete loss of Gad1, Gad2 and Gata3 expression in Gata2cko midbrain at E11.5 and E13.5 (Fig. 4A,A',B,B',E,F; see Fig. S3A-H in the supplementary material). No GABAergic neuron precursors were detected at any dorsoventral or anteroposterior level in the midbrain of Gata2cko mutants at these stages. These results suggested an early and absolute requirement for Gata2 in midbrain GABAergic neuron development that cannot be compensated for over time.
Unaltered progenitor cell proliferation, patterning, neurogenesis, survival and cell cycle exit in the Gata2cko mutant midbrain
Next, we investigated which steps in midbrain GABAergic neuron development
are affected by the loss of Gata2. Despite the loss of GABAergic
precursors, there were no overt anatomical defects in the embryonic
Gata2cko mutant midbrain. Nevertheless, we analyzed in
more detail the properties of the proliferative progenitor cells that give
rise to the midbrain GABAergic precursors. In the Gata2cko
mutants, the ventricular zone progenitors still expressed Sox2 and there was
no difference in the thickness of the Sox2-positive ventricular zone or
HuC/D-positive marginal zone compared with the wild type (see Fig. S4A-E in
the supplementary material). Consistently, we detected no major changes in the
numbers of phospho-histone H3-positive mitotic nuclei or BrdU-incorporating
S-phase nuclei in the GABAergic progenitor domain of the
Gata2cko mutants (see Fig. S4F-K in the supplementary
material). Also, we observed no increase in apoptotic cell numbers (see Fig.
S4N,O in the supplementary material). Thus, we conclude that the loss of
GABAergic neurons is not due to impaired proliferation or survival of their
progenitors.
We then characterized patterning and neurogenesis in the GABAergic neuron progenitors of the Gata2cko mutants. All the analyzed transcription factors, including Nkx6-1, Nkx2-2 and Pax7, involved in the patterning of the ventricular zone progenitor cell layer were correctly expressed (Fig. 4K,L,Q-T; data not shown). The transcription factors showing normal expression in the progenitors also included Ascl1 and Helt (Fig. 4M,N,I,J), as well as Ngn2, which has been suggested to be involved in glutamatergic neurogenesis in the midbrain (Fig. 4O,P). In addition, we observed no change in the expression of p57 (Cdkn1c), a cell cycle inhibitor upregulated at withdrawal from the cell cycle, demonstrating that Gata2-deficient progenitors are still able to exit from the cell cycle and become post-mitotic precursors (see Fig. S4L,M in the supplementary material). Our results suggest that progenitor cell patterning and neurogenic cell cycle exit are not disturbed by the loss of Gata2 in the midbrain.
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In addition to Gad1, Gad2 and Gata3, other genes
characteristic of the post-mitotic GABAergic neuron precursors were also
downregulated in the Gata2cko mutants. At E11.5, Lhx1 was
downregulated in the m5 and m3 domains, but persisted in the m4
(Fig. 4U,V). In addition, the
glutamatergic domain m6 continued to express Lhx1, as expected. Although some
Lhx1-positive cells appeared in the marginal zone of m5
(Fig. 4V), they might have
originated from neighboring domains, as the intermediate zone of m5 is
negative for Lhx1 but both m4 and m6 continued to express it. The m4 domain
also appeared to retain its identity as judged by continued Nkx2-2
expression and lack of Pou4f1 expression in the
Gata2cko mutants (Fig.
4K,L,G,H). Despite continued Lhx1 and Nkx2-2
expression, there was complete loss of GABAergic markers and uniform
glutamatergic marker gene expression in the m4 domain of the mutants. This
discrepancy could be explained by an uncoupling of regional patterning and
neurotransmitter selection, as suggested by Nakatani et al.
(Nakatani et al., 2007).
Alternatively, because m4 produces both GABAergic and glutamatergic precursors
(see above), there could be a transformation within m4 whereby the GABAergic
m4 precursors (m4-D) assume an identity of glutamatergic m4 precursors (m4-V,
Fig. 4A,C). To test this, we
analyzed Pax6 expression in the Gata2cko mutants. Ectopic
Pax6-expressing cells were observed in the m4-D region
(Fig. 4W,X) indicating that in
the absence of Gata2, the whole m4 domain acquired a fate similar to
m4-V glutamatergic neurons. Thus, Gata2 is essential for activating
gene expression patterns typical for post-mitotic GABAergic precursor cells in
the midbrain. Without Gata2, the precursor cells appear to adopt a
phenotype resembling, but not necessarily identical to, that of the adjacent
glutamatergic regions.
Ectopic Gata2 is sufficient to induce GABAergic differentiation in embryonic chicken midbrain
We next examined whether Gata2 alone is sufficient to switch on
the GABAergic differentiation pathway. For this, we employed ectopic
expression of Gata2 in chicken embryos. First, we studied the pattern
of endogenous chicken Gata2 (cGata2) expression and its
relationship with the development of GABAergic neurons in chick midbrain. As
in the mouse embryo at the equivalent stage, expression of Helt,
Gata2, cGata3, Lhx1 and cGad1 coincided in the ventrolateral
chick midbrain, and was flanked by cNgn2- and
cSlc17a6-expressing glutamatergic domains at HH20-22 and HH22-24
(Fig. 5A-I). Similar to in
E12.5 mouse midbrain, we observed scattered cGad1 expression in the
chick dorsal midbrain at a slightly later stage (HH22-24,
Fig. 5H). Unlike in the mouse,
dorsal cNgn2 expression was detected primarily in the marginal zone
and might mark the differentiated glutamatergic neurons rather than their
progenitors (Fig. 5C). However,
our results demonstrate that overall, the pattern, timing and regulatory
mechanisms of midbrain GABAergic neuron production are likely to be conserved
between these two species.
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Normal development of GABAergic, but loss of serotonergic, neurons in rhombomere 1 of Gata2cko mutants
To study GABAergic neuron development in r1, we analyzed E11.5 wild-type
and Gata2cko mouse embryos for Gata3 and
Gad1 expression. In striking contrast to the midbrain, where both
genes were completely downregulated, Gata3 and Gad1
expression in the GABAergic precursors of r1 was not altered in the mutant
(Fig. 6A,B,E,F). Thus,
Gata2 is dispensable for the early development of GABAergic neurons
in r1.
Previous studies have suggested a role for Gata2 and
Gata3 in the development of other neuronal populations in the
midbrain-r1 region, including cranial motoneurons and serotonergic neurons
(Nardelli et al., 1999;
Craven et al., 2004;
Pattyn et al., 2004).
Consistent with the pattern of Gata2 expression, we observed no
defects in the cranial Isl1-expressing motoneurons of nIII and nIV in
the Gata2cko mutants (see Fig. S3I,J in the supplementary
material). Thus, the nIII/nIV defects in the Gata2-null mutants were
likely to be secondary to other developmental abnormalities in these embryos.
By contrast, almost no serotonin (5-HT)-, Lmx1b- or Fev
(Pet1)-positive serotonergic neurons were detected in r1 of E11.5 and
E13.5 Gata2cko mutants
(Fig. 6C,D,G-L). Despite its
continued expression in the GABAergic precursors, Gata3 was
specifically downregulated in the serotonergic neuron precursors in r1
(Fig. 6A,B). These results
confirm that Gata2 is required for the development of rostral
serotonergic neurons. They also further demonstrate the loss of Gata2
function in r1 of Gata2cko mutants.
Analysis of midbrain GABAergic neuron subpopulations in perinatal Gata2cko mutants demonstrates differential requirements for Gata2
To study whether the cells with the transformed neurotransmitter identity
contribute to the maturing brain, we analyzed Gata2cko
mutants shortly before birth. At E17.5 and E18.5, the gross brain morphology
of the Gata2cko mutants was similar to that of the wild
type. In sagittal sections, we observed loss of expression of the GABAergic
markers Gad1, Gata3 and Pitx2 in the
Gata2cko midbrain, in both the dorsal and ventral brain
regions (see Fig. S5 in the supplementary material). By contrast, abundant
expression of GABAergic marker genes was detected in r1. Thus, consistent with
the loss of GABAergic precursor cells in the midbrain at earlier stages of
development, inactivation of Gata2 leads into a specific loss of
GABAergic neurons in the perinatal midbrain but not in r1.
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To test the hypothesis that the GABAergic neurons of VTA and SNpr were
derived from the midbrain neuroepithelium late in development, we analyzed
Gad1 and Gata3 expression in the ventral midbrain at E13.5
and E15.5, after the neurogenic period of this brain region
(Altman and Bayer, 1981). Few
Gad1- or Gata3-expressing cells were detected at E15.5 in
the VTA-SNpr area of wild-type or Gata2cko embryos
(Fig. 7I-L,M-P; see Fig. S3 in
the supplementary material). Thus, GABAergic neurons associated with the DA
nuclei appear late in development.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Fields et al., 2007;
Brandao et al., 2008),
development of the midbrain GABAergic neurons is poorly understood. Here, we
mapped the regions of the embryonic midbrain that give rise to GABAergic
neurons. We identified Gata2 as the first post-mitotic selector gene
required for GABAergic identity in the midbrain. Finally, our results
demonstrate differences in the regulatory pathways and developmental history
of the GABAergic neurons in the midbrain-r1 region.
Gata2 marks regions of midbrain GABAergic neurogenesis
A useful dorsoventral fate map of the developing mouse midbrain was
recently presented by Nakatani et al.
(Nakatani et al., 2007). We
detected Gata2 and Gata3 expression in all midbrain domains
where GABAergic neurons are produced. Our studies suggest a further refinement
to the map of Nakatani et al. We propose that the Nkx2-2-positive m4 domain
gives rise to both GABAergic and glutamatergic neurons, the latter being
negative for Gata2 and positive for Pax6 expression
(Fig. 8A). In the future, it
will be important to further define the mature brain structures to which these
neural progenitor and precursor subpopulations contribute.
In the ventricular zone, the pattern of colocalization of Ascl1, Helt and Gata2 suggests that during their maturation, the Ascl1+ Helt+ progenitors turn into Ascl1+ Gata2+ precursors. Consistent with this, inactivation of Helt resulted in a dramatic reduction in Gata2 expression. Our results thus suggest that Gata2 expression is activated in a Helt-dependent fashion as the neural progenitor cells turn into post-mitotic GABAergic neuron precursors and leave the ventricular zone. The ventral m5 domain appears to be an exception, as here Helt was not essential for Gata2 expression (Fig. 8B). Regulation of Gata2 expression by an as yet unidentified mechanism in m5 is likely to explain the more severe phenotype of Gata2cko, as compared with Helt, mutants.
Gata2 is a selector gene for GABAergic neuron identity of the post-mitotic midbrain precursors
Gata2 has been proposed to inhibit neural progenitor cell
proliferation and promote post-mitotic differentiation, a model consistent
with its pattern of expression (El Wakil
et al., 2006). However, our results suggest that there are no
marked changes in embryonic brain morphology and layering, progenitor
proliferation, or neurogenic cell cycle exit in conditional Gata2
mutants. Thus, although Gata2 may still contribute to the cell cycle
exit of neuronal progenitors, it is dispensable in this respect at least in
the midbrain.
By contrast, the Gata2 mutant neural precursors in the midbrain appear to undergo a complete cell fate transformation. Several genes, the expression of which is normally activated in the post-mitotic GABAergic precursors, failed to be expressed in the Gata2 mutants. In addition, Nkx6-1, the expression of which is normally downregulated upon GABAergic differentiation in the m5 domain, continued to be expressed in the conditional Gata2 mutants. Instead of the GABAergic phenotype, the Gata2-deficient midbrain precursor cells activate expression of glutamatergic marker genes. In the Gata2 mutants, Slc17a6 expression was observed throughout the midbrain, whereas Pou4f1 expression was not ectopically activated in m4. By contrast, expression of Pax6 expanded in the m4 domain of Gata2 mutants. As we showed that Pax6 is specifically expressed in glutamatergic precursors in m4, these results together suggest that in the conditional Gata2 mutants, the identity of m4 is retained but the GABAergic m4 precursors switch to a glutamatergic fate. Also, in other parts of the midbrain, the Gata2-deficient precursors still have regional characteristics despite the transformation of their neurotransmitter identity.
Importantly, all the observed changes in gene expression take place in the
post-mitotic precursors and neurons in the intermediate and marginal zones,
whereas the proliferative progenitor cells and proneural gene expression in
the ventricular zone are unaffected. This is in a clear contrast to the
function of Helt in the ventricular zone, which has been shown to
support GABAergic neurogenesis partly by repressing Ngn2 expression
in the progenitors and thereby glutamatergic neuron production
(Nakatani et al., 2007). In
addition to repressing Ngn2, Helt activates GABAergic gene expression
(Miyoshi et al., 2004;
Guimera et al., 2006;
Nakatani et al., 2007), and
Gata2 appears to be important for this function. Thus, the expression
and function of Gata2 in the midbrain are analogous to those of the
bHLH transcription factor Ptf1a, which acts as a post-mitotic
selector gene of the GABAergic identity in the developing cerebellum and
spinal cord (Glasgow et al.,
2005; Hoshino et al.,
2005; Mizuguchi et al.,
2006).
Developmental diversity of the midbrain GABAergic neurons
In the perinatal Gata2 mutants, all the midbrain GABAergic neuron
subpopulations were transformed to a glutamatergic phenotype, except for the
GABAergic neurons associated with the DA neurons in the VTA and SNpr. This was
highly unexpected because no GABAergic precursor cells were found at any
dorsoventral level of the midbrain at the earlier stages of development. There
are at least three possible explanations for this. Firstly, there could be
incomplete recombination of the Gata2flox allele by
En1Cre. We find this unlikely because we could not see any
Gata2 transcripts or protein in the Gata2cko
mutants analyzed at several stages. Also, no evidence for mosaic inactivation
of Gata2 was observed in VTA, SNpr, or other regions of the midbrain.
Secondly, the remaining GABAergic neurons could be born in a region of the
midbrain that does not require Gata2 and was missed in our analysis.
We cannot completely rule out this possibility, but nor is there any evidence
for it, as no Gad1- or Gata3-positive cells were observed at
any anteroposterior or dorsoventral level of the mutant midbrain at
E11.5-15.5. Finally, the GABAergic neurons of the VTA and SNpr could be
derived from neuroectoderm outside the midbrain. The late appearance of the
VTA and SNpr GABAergic neurons (
E15.5) and their strikingly normal
development in the Gata2cko mutants are consistent with
this hypothesis. Possible sources of the VTA and SNpr GABAergic neurons
include r1 and the diencephalon. However, cell lineage-tracing experiments are
needed to unambiguously determine the origin of these ventral-most GABAergic
neurons.
Gata2 is dispensable for GABAergic, but essential for serotonergic, neuron development in rhombomere 1
Similar to in the midbrain, Gata2 and Gata3 are expressed
in the GABAergic precursors of r1. Strikingly, however, we observed abundant
GABAergic neurons in r1 of the Gata2 conditional mutants, despite the
complete loss of Gata2 in this brain region. Interestingly, the
hindbrain GABAergic precursors continued to express Gata3. This
suggests that Gata3 expression is, perhaps, not directly regulated by
Gata2 in the GABAergic precursors in r1. Analyses of Gata3
mutants and, possibly, Gata2; Gata3 double mutants, are
needed to determine whether Gata3 can possibly compensate for the
loss of Gata2 specifically in the r1 GABAergic precursor cells.
The development of rostral serotonergic neurons has also been shown to be
regulated by Gata2. Craven et al. used explant cultures of
Gata2-null mutant neuroepithelium to demonstrate a requirement for
Gata2 for the differentiation of serotonin-positive cells in r1
(Craven et al., 2004).
Consistent with this, our results show loss of serotonergic neurons in the
conditional Gata2 mutants. In contrast to the Gata2 mutant
explant cultures, our results suggest that loss of Gata2 also leads
to downregulation of Gata3 in the serotonergic neuron precursors.
This discrepancy might be explained by distorted tissue architecture of the
cultured explants and by the continued expression of Gata3 in the
nearby GABAergic precursors in r1. It will be of interest to determine whether
Gata2 also acts as a post-mitotic selector gene in the serotonergic
neuron lineage, similar to its role in the developing midbrain GABAergic
neuron precursors.
Conclusion
GABAergic versus glutamatergic neuron identity appears to be regulated by
different genetic cascades in different regions of the CNS. Here we show
specific requirement for the transcription factor Gata2 as a cell-fate
selector, and locate it in the gene hierarchy regulating development of the
GABAergic neurons in the midbrain (Fig.
8B). Our study also further elucidates how regional identities are
generated in the distinct midbrain GABAergic neuron subpopulations.
Understanding the development, molecular identity and functional
characteristics of these diverse neurons might lead to better diagnostics and
treatment of several forms of neurological and psychiatric disease.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/2/253/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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