
  
    
      
        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.).
      
    
  
