Background
Glutamate decarboxylase (GAD) catalyzes the formation of
the inhibitory neurotransmitter γ-amino butyric acid (GABA)
from glutamate. In mammals, the two isoforms of this
enzyme, GAD67 and GAD65, are expressed from two separate
genes,
Gad1 and
Gad2 respectively [ 1, 2, 3]. GABA
signaling plays several roles in neuronal development.
Early in CNS development, GABA can modulate neuron
progenitor proliferation as well as neuron migration,
survival and differentiation [ 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14]. In some classes of neural progenitors GABA
stimulates these processes while in others it has an
antagonistic activity. For example, recent work has
demonstrated that GABA acts in the developing neocortex to
stimulate the proliferation of progenitors in the
ventricular zone while inhibiting the proliferation of
progenitors in the subventricular zone [ 14]. Later, during
postnatal development, normal GABAergic input is required
for activity-dependent plasticity in the visual cortex as
shown in the
Gad2 knockout mouse [ 15, 16]. In
addition to these functions in the developing CNS, GABA
signaling is also required for the normal development of
non-neural tissues. Targeted mutations of the
Gad1 gene lead to defective
development of the secondary palate [ 17, 18]. The cleft
palate phenotype of the
Gad1 mutants suggests the involvement
of GABA-mediated signals in the normal development and
differentiation of a structure derived from the oral
epithelium and neural crest ecto-mesenchyme. This
conclusion is further supported by the similar cleft palate
defect seen in mice with a deletion or targeted mutation in
the β3 subunit of the GABA
A receptor [ 19, 20, 21, 22].
This intriguing genetic evidence indicates a role for
GABA-mediated signaling in the development of a non-neural
structure, the secondary palate. The potential for this
pathway to be involved in the early development of
additional non-neural tissues has not yet been thoroughly
explored [ 23]. To address this question, we surveyed
Gad1 transcript distribution in the
non-CNS tissues of the embryo. Using a whole mount
in situ hybridization approach, we
found that
Gad1 is indeed expressed in a number
of different regions and tissues. A notable feature of this
expression pattern is that
Gad1 transcripts accumulate in the
specialized ectodermal structures that are involved in the
formation of the mystacial vibrissae and in limb outgrowth.
These specialized ectodermal tissues are known to be
sources of developmental signals [ 24, 25, 26]. In
addition, transcripts are expressed in the mesenchymal stem
cell population of the tailbud and in the pharyngeal
endoderm and mesenchyme. The expression patterns show that
Gad1 is expressed in several non-CNS
structures that are derived from each of the three germ
layers of the embryo.
Results
The mouse
Gad1 gene is widely expressed in the
embryonic central nervous system [ 27]. To define
additional sites of expression outside of the CNS, we
analyzed the distribution of
Gad1 transcripts in E8.5 to E14.5
mouse embryos by whole mount
in situ hybridization.
Gad1 transcripts were not detected in
E8.5 day embryos (data not shown). At E9.0
Gad1 was readily detected in the
tailbud (figure 1A). Expression in the tail continued
through E12.5 and was undetectable by E13.5 (figure
1B,C,Dand data not shown), a period corresponding to
secondary body axis formation in the mouse embryo [ 28].
Examination of sections from an E9.5 embryo revealed a high
level of
Gad1 expression throughout the
mesenchyme and neural epithelium in the caudal portion of
the tailbud (figure 1F). No transcripts were detected in
the surface ectoderm surrounding the tailbud mesenchyme
(figure 1F). At more cranial levels within the tail,
expression was localized to paraxial mesoderm, ventral
neural tube, notochord and cells of the dorsal hindgut
(figure 1E). In the paraxial mesoderm, the highest
expression levels were also localized ventrally, adjacent
to the notochord (figure 1E).
In the pharyngeal region of E9.5 embryos,
Gad1 RNA was detected in and around
the second, third and fourth pharyngeal pouches (figure
2A). Sections through the third pouch confirmed the
presence of
Gad1 expression in the pouch endoderm
(data not shown). Expression was particularly strong in the
dorsal portion of this pouch (figure 2B). The additional
diffuse staining appeared to be in the pharyngeal
mesenchyme (figure 2B). The expression in the pharyngeal
region was very transient; transcripts were easily detected
at E9.5, but only faintly at E9.0 and were not detectable
by E10.5.
In the limb buds,
Gad1 RNA was detected from E9.0 to
E11.5 (figure 3A,B,C,D,E,F,G,H). Transcripts were initially
expressed in the pre-apical ectodermal ridge (pre-AER) at
E9.5 (figure 3A,B) and by E10.5 were seen in the definitive
AER of the forelimb (figure 3D). At E10.5
Gad1 was expressed in a diffuse
stripe in the forelimb (figure 3D) while in the hindlimb
expression was only detected in the apical ectoderm (figure
3E). By E11.5 forelimb AER expression was fading and
expression was seen in a diffuse stripe in the proximal
forelimb and a diffuse crescent in the proximal hindlimb
(figure 3G,H). The earlier activation of
Gad1 in the forelimb reflects the
normal temporal order of events in limb development.
Sections indicate that the expression within the limb buds
was in surface ectoderm and adjacent mesenchyme (data not
shown).
Gad1 RNA was not detected in the
limbs by whole mount
in situ hybridization after
E11.5.
A dynamic pattern of
Gad1 expression was detected in the
developing vibrissae from E12.5 to E14.5 (figure
4A,B,C,D,E,F,G,H). Expression was first detected in the
supra-orbital, infra-orbital, and post-oral vibrissae and
in the posterior vibrissae in the lateral nasal and
maxillary rows (figure 4A,B; nomenclature as in [ 29]).
Gad1 RNA was also detected in some of
the posterior labial vibrissae at this stage. Expression
was activated in a posterior to anterior (towards the nose)
progression in the lateral nasal and maxillary rows,
reflecting the pattern of vibrissal development [ 29]. By
E13.5,
Gad1 expression was detected in the
anterior lateral nasal and maxillary rows and was activated
in the rhinal, labial and submental vibrissae (figure
4C,D). By E14.5, expression was strong in the labial,
submental and rhinal vibrissae (figure 4E,F). Sections of
E12.5 whole mounts show that
Gad1 expression was localized to the
epidermal placodes of the mystacial vibrissae (figure 4G,H)
and was maintained as the placodes begin to invaginate
(figure 4G).
Control hybridizations using a sense strand
Gad1 probe were also performed.
Embryos hybridized to the sense probe did not reveal any
staining pattern at any of the stages tested (E8.5- E14.5).
Sense strand hybridization results for E10.5 and E11.5
embryos are shown in figure 5.
Discussion
The expression results reported here show that
Gad1 was activated in several tissues
outside of the central nervous system during mouse
development. Transcripts were not seen at E8.5 and were
first detected at E9.0. It was surprising that this very
early phase of
Gad1 expression was largely outside
of the developing CNS and was localized in the tail bud
mesenchyme and in the pre-apical ectodermal ridge (pre-AER)
of the forelimb bud. As development proceeded
Gad1 was detected in pharyngeal
endoderm and in the ectodermal placodes of the vibrissae.
The data demonstrate that
Gad1 is expressed in several sites
outside of the developing CNS and in derivatives of all
three germ layers. We have also detected the expression of
Gad1-lacZ transgenes in the
developing vibrissae and limbs supporting the novel and
surprising
in situ hybridization results we
report here (J.J. Westmoreland and B.G.C., unpublished
results).
Previous studies have shown that
Gad1 can be regulated at the
post-transcriptional and translational level.
Gad1 mRNA translation or protein
stability can be regulated in mature neurons by the level
of GABA [ 30, 31]. During embryogenesis,
post-transcriptional regulation occurs by alternative
splicing during embryonic development in rats and mice [
32, 33]. This alternate embryonic transcript inserts a stop
codon into the
Gad1 mRNA and can produce the
truncated proteins, GAD25 and GAD44, from its 5'; and 3'
ends respectively. The studies reported here used a probe
that will detect the adult
Gad1 mRNA that encodes GAD67 as well
as the embryonic alternatively spliced mRNA that can encode
GAD25 and GAD44. These additional mechanisms of
Gad1 regulation may control the
production of GAD proteins and the synthesis of GABA in the
non-neural cell types detected in our study.
The whole mount
in situ hybridization data reported
here extends the results of a recently published section
in situ hybridization study on
E10.5-E12.5 mouse embryos [ 27]. Our analysis showed that
Gad1 expression is first detectable
earlier at E9.0 and revealed novel non-CNS sites of
expression in the pharyngeal region, vibrissae, tail bud
and limb bud. The results of the previous study [ 27],
together with the data reported herein, provide a
comprehensive picture of
Gad1 expression in the E9.0-E12.5
mouse embryo.
Previous studies have noted
Gad expression outside of the CNS. In
adults
Gad1 and
Gad2 have been detected in a number
of tissues including kidney, testis, oviduct, pancreatic
islets and adrenal cortex [ 34, 35, 36, 37]. Previously
reported sites of embryonic
Gad1 expression outside of the brain
and spinal cord during rodent development include the lens
fibers and the olfactory pit [ 38, 39]. In E10.5-E12.5
mouse embryos
Gad1 is expressed in the olfactory
and the lens placodes, the anlagen of the olfactory pit and
lens fibers [ 27]. We also detected
Gad1 expression in these tissues
(please see figure 3Aand data not shown). Expression of
Gad in the developing heart and blood
vessels has also been reported [ 27]. We detected weak
staining in the heart and did not detect blood vessel
expression, perhaps due to the very low levels of
expression in developing vasculature [ 27]. Our results
document localized expression of
Gad1 at additional non-CNS sites in
the mouse embryo, suggesting a potential role for GABA
signaling in the development of these structures.
Our interest in the role of GABA signaling in developing
tissues outside of the central nervous system stems from
the cleft palate phenotype of the
Gad1 and the β3 GABA
A receptor subunit mutants [ 17, 18, 19,
21, 22]. The genetic data strongly suggest that GABA acts
through GABA
A receptors to modulate the development
of this tissue. Although the data reported here do not
explain the origin of the cleft palate phenotype, they do
indicate that
Gad1 is expressed in several
additional non-CNS tissues in the mouse embryo. It is
particularly noteworthy that these include the AER of the
limb buds and the ectodermal placodes of the vibrissae.
Both are ectodermal structures known to be sources of
developmental signals required for morphogenesis and
patterning [ 24, 25, 26, 40]. It will be of interest to
examine the expression pattern of GABA receptors in the
mesenchyme adjacent to these ectodermal signaling centers.
Expression of GABA receptor subunits in adjacent tissues
would indicate that these receptors read the developmental
signals mediated by GABA in these structures and
tissues.
Conclusions
The mouse gene encoding the 67 kDa isoform of glutamate
decarboxylase (
Gad1 ) is expressed in the tail bud
mesenchyme, vibrissal placodes, pharyngeal arches and
pouches and the apical ectodermal ridge (AER), mesenchyme
and ectoderm of the limb buds in mouse embryos from
E9.0-E14.5. Some of the
Gad1 expressing tissues (vibrissal
placodes, AER) are known sources of developmental signals.
Other sites of expression correspond to stem cell
populations that give rise to multiple differentiated
tissues (tail bud mesenchyme, pharyngeal endoderm and
mesenchyme). The localized and dynamic expression pattern
of
Gad1 suggests a wider role for GAD
and GABA in the development of non-neural tissues than was
previously known.
Materials and Methods
Whole mount
in situ hybridizations were performed
on Swiss Webster embryos as described [ 41, 42]. The
morning that the vaginal plug was found was considered 0.5
days of gestation. The
Gad1 probe was derived from an EST
clone (accession W59173). Its 5' end corresponds to
nucleotide 142 in exon 1 [ 43] and the 3' end is at
nucleotide 2041 in the cDNA sequence [ 44]. Digoxygenin
sense and antisense RNA probes were generated by labeling
with digoxygenin-UTP during transcription. Embryos were
removed and fixed in 4% paraformaldehyde/PBS overnight and
used immediately for the
in situ hybridization. The embryos
were processed as described previously [ 41] and hybridized
to the probe overnight in 50% formamide, 5X SSC (pH 5.0),
50 μg/ml torula RNA, 50 μg/ml heparin at 70°C. The final
concentration of probe in the hybridization was 1 μg/ml.
After an overnight hybridization, the embryos were washed
at high stringency in prewarmed 50% formamide, 5X SSC, 1%
SDS (wash I) at 70°C for 90 minutes. The embryos were then
washed in a 1:1 mix of wash I and wash II (0.5 M NaCl, 10
mM Tris pH 7.5, 0.1% Tween 20) for 10 minutes at 70°C. The
embryos were washed several times in wash II at room
temperature to remove the formamide and then treated with
100 μg/ml RNase A, 100 units/ml RNase T1 in wash II for 1
hour at 37°C. Following the RNase treatment the embryos
were washed in three changes of 50% formamide, 2X SSC pH5.0
at 70°C for a total of 90 minutes. Detection of the
hybridized RNA probe was as described previously [ 41]. The
embryos were photographed without clearing using a Leica
model MZFL III dissecting scope, a Hamamatsu model C4742-95
digital camera and Openlab 2.0.7 software.
For sectioning, embryos were embedded in Immunobed
(Polysciences) resin and sectioned at 10 μm. Sections were
phtotographed using an Olympus BX60 microscope fitted with
a SPOT digital camera (Diagnostic Instruments Inc.).