- Research article
- Open Access
Dynamic expression of a glutamate decarboxylase gene in multiple non-neural tissues during mouse development
© Maddox and Condie; licensee BioMed Central Ltd. 2001
- Received: 10 November 2000
- Accepted: 8 January 2001
- Published: 8 January 2001
Glutamate decarboxylase (GAD) is the biosynthetic enzyme for the neurotransmitter γ-aminobutyric acid (GABA). Mouse embryos lacking the 67-kDa isoform of GAD (encoded by the Gad1 gene) develop a complete cleft of the secondary palate. This phenotype suggests that this gene may be involved in the normal development of tissues outside of the CNS. Although Gad1 expression in adult non-CNS tissues has been noted previously, no systematic analysis of its embryonic expression outside of the nervous system has been performed. The objective of this study was to define additional structures outside of the central nervous system that express Gad1, indicating those structures that may require its function for normal development.
Our analysis detected the localized expression of Gad1 transcripts in several developing tissues in the mouse embryo from E9.0-E14.5. Tissues expressing Gad1 included the tail bud mesenchyme, the pharyngeal pouches and arches, the ectodermal placodes of the developing vibrissae, and the apical ectodermal ridge (AER), mesenchyme and ectoderm of the limb buds.
Some of the sites of Gad1 expression are tissues that emit signals required for patterning and differentiation (AER, vibrissal placodes). Other sites correspond to proliferating stem cell populations that give rise to multiple differentiated tissues (tail bud mesenchyme, pharyngeal endoderm and mesenchyme). The dynamic expression of Gad1 in such tissues suggests a wider role for GABA signaling in development than was previously appreciated.
- Glutamate Decarboxylase
- Apical Ectodermal Ridge
- Paraxial Mesoderm
- Gad1 Gene
- Gad1 Expression
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 . 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 GABAA 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 . 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.
The mouse Gad1 gene is widely expressed in the embryonic central nervous system . 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.
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 . 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 , 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 . We also detected Gad1 expression in these tissues (please see figure 3A and data not shown). Expression of Gad in the developing heart and blood vessels has also been reported . 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 . 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 GABAA receptor subunit mutants [17,18,19, 21, 22]. The genetic data strongly suggest that GABA acts through GABAA 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.
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.
Whole mountin 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  and the 3' end is at nucleotide 2041 in the cDNA sequence . 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  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 . 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.).
We thank J.J. Westmoreland and Jyoti Koushik for performing some of the initial Gad1 in situ hybridizations and Drs. Nancy Manley and Audrey Napier for comments on the manuscript. This work was supported by a Medical College of Georgia Research Institute Grant and an MCG Biomedical Research Support Grant.
- Erlander MG, Tillakaratne NJK, Feldblum S, Patel N, Tobin AJ: Two genes encode distinct glutamate decarboxylases. Neuron. 1991, 7: 91-100.View ArticlePubMedGoogle Scholar
- Bu D-F, Erlander MG, Hitz BC, Tillakaratne NJK, Kaufman DL, Wagner-McPherson CB, Evans GA, Tobin AJ: Two Human Glutamate Decarboxylases, 65-kDa GAD and 67-kDa GAD, Are Each Encoded by a Single Gene. Proc. Natl. Acad. Sci. USA. 1992, 89: 2115-2119.PubMed CentralView ArticlePubMedGoogle Scholar
- Pinal CS, Tobin AJ: Uniqueness and redundancy in GABA production. Perspect Dev Neurobiol. 1998, 5: 109-118.PubMedGoogle Scholar
- Barbin G, Pollard H, Gaiarsa JL, Ben-Ari Y: Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons. Neuroscience Letters. 1993, 152: 150-154. 10.1016/0304-3940(93)90505-F.View ArticlePubMedGoogle Scholar
- Ikeda Y, Nishiyama N, Saito H, Katsuki H: GABAA Receptor Stimulation Promotes Survival of Embryonic Rat Striatal Neurons in Culture. Developmental Brain Research. 1997, 98: 253-258. 10.1016/S0165-3806(96)00183-610.1016/S0165-3806(96)00183-610.1016/S0165-3806(96)00183-610.1016/S0165-3806(96)00183-6.View ArticlePubMedGoogle Scholar
- Behar TN, Schaffner AE, Colton CA, Somogyi R, Olah Z, Lehel C, Barker JL: GABA-induced chemokinesis and NGF-induced chemotaxis of embryonic spinal cord neurons. The Journal of Neuroscience. 1994, 14: 29-38.PubMedGoogle Scholar
- Behar TN, Yong-Xin L, Tran HT, Ma W, Dunlap V, Scott C, Barker JL: GABA stimulates chemotaxis and chemokinesis of embryonic cortical neurons via calcium-dependent mechanisms. Journal of Neuroscience. 1996, 16: 1808-1818.PubMedGoogle Scholar
- Behar TN, Schaffner AE, Scott CA, O'Connell C, Barker JL: Differential response of cortical plate and ventricular zone cells to GABA as a migration stimulus. J Neurosci. 1998, 18: 6378-6387.PubMedGoogle Scholar
- Liu J, Morrow L, Devaud L, Grayson DR, Lauder JM: GABAA Receptors mediata Trophic Effects of GABA on Embryonic Brainstem Monoamine Neurons In Vitro. The Journal of Neuroscience. 1997, 17: 2420-2428.PubMedGoogle Scholar
- LoTurco JL, Owens DF, Heath MJS, Davis MBE, Kriegstein AR: GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron. 1995, 15: 1287-1298.View ArticlePubMedGoogle Scholar
- Antonopoulos J, Pappas IS, Parnavelas JG: Activation of the GABAA receptor inhibits the proliferative effects of bFGF in cortical progenitor cells. Eur J Neurosci. 1997, 9: 291-298.View ArticlePubMedGoogle Scholar
- Fueshko SM, Key S, Wray S: GABA inhibits migration of luteinizing hormone-releasing hormone neurons in embryonic olfactory explants. J Neurosci. 1998, 18: 2560-2569.PubMedGoogle Scholar
- Behar TN, Schaffner AE, Scott CA, Greene CL, Barker JL: GABA receptor antagonists modulate postmitotic cell migration in slice cultures of embryonic rat cortex. Cereb Cortex. 2000, 10: 899-909. 10.1093/cercor/10.9.899.View ArticlePubMedGoogle Scholar
- Haydar TF, Wang F, Schwartz ML, Rakic P: Differential modulation of proliferation in the neocortical ventricular and subventricular zones. J Neurosci. 2000, 20: 5764-5774.PubMed CentralPubMedGoogle Scholar
- Hensch TK, Fagiolini M, Mataga N, Stryker M, Baekkeskov S, Kash SF: Local GABA circuit control of experience-dependent plasticity in developing visual cortex. Science. 1998, 282: 1504-1508. 10.1126/science.282.5393.1504.PubMed CentralView ArticlePubMedGoogle Scholar
- Fagiolini M, Hensch TK: Inhibitory threshold for critical-period activation in primary visual cortex. Nature. 2000, 404: 183-186. 10.1038/35004582.View ArticlePubMedGoogle Scholar
- Asada H, Kawamura Y, Maruyama K, Kume H, Ding RG, Kanbara N, Kuzume H, Sanbo M, Yagi T, Obata K: Cleft palate and decreased brain gamma-aminobutyric acid in mice lacking the 67-kDa isoform of glutamic acid decarboxylase. Proc Natl Acad Sci U S A. 1997, 94: 6496-6499. 10.1073/pnas.94.12.6496.PubMed CentralView ArticlePubMedGoogle Scholar
- Condie BG, Bain G, Gottlieb DI, Capecchi MR: Cleft palate in mice with a targeted mutation in the gamma-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67. Proc Natl Acad Sci U S A. 1997, 94: 11451-11455. 10.1073/pnas.94.21.11451.PubMed CentralView ArticlePubMedGoogle Scholar
- Culiat CT, Stubbs L, Nicholls RD, Montgomery CS, Russell LB, Johnson DK, Rinchik EM: Concordance between isolated cleft palate in mice and alterations within a region including the gene encoding the beta-3 subunit of the type A gamma-aminobutyric acid receptor. Proc. Natl. Acad. Sci. USA. 1993, 90: 5105-5109.PubMed CentralView ArticlePubMedGoogle Scholar
- Culiat CT, Stubbs LJ, Montgomery CS, Russell LB, Rinchik EM: Phenotypic consequences of deletion of the gamma-3, alpha-5, or beta-3 subunit of the type A gamma-aminobutyric acid receptor in mice. Proc. natl. Acad. Sci. USA. 1994, 91: 2815-2818.PubMed CentralView ArticlePubMedGoogle Scholar
- Culiat CT, Stubbs LJ, Woychik RP, Russell LB, Johnson DK, Rinchik EM: Deficiency of the beta-3 subunit of the type A gamma-aminobutyric acid receptor causes cleft palate in mice. Nature Genetics. 1995, 11: 344-346.View ArticlePubMedGoogle Scholar
- Homanics GE, DeLorey TM, Firestone LL, Quinlan JJ, Handforth A, Harrison NL, Krasowski MD, Rick CEM, Korpi ER, Makela R, Brilliant MH, Hagiwara N, Ferguson C, Snyder K, Olsen RW: Mice devoid of gamma-aminobutyrate type A receptor beta-3 subunit, have epilepsy, cleft palate and hypersensitive behavior. Proc. Natl. Acad. Sci. USA. 1997, 94: 4143-4148. 10.1073/pnas.94.8.4143.PubMed CentralView ArticlePubMedGoogle Scholar
- Kash SF, Condie BG, Baekkeskov S: Glutamate decarboxylase and GABA in pancreatic islets: lessons from knock-out mice. Horm Metab Res. 1999, 31: 340-344.View ArticlePubMedGoogle Scholar
- Iseki S, Araga A, Ohuchi H, Nohno T, Yoshioka H, Hayashi F, Noji S: Sonic hedgehog is expressed in epithelial cells during development of whisker, hair, and tooth. Biochem Biophys Res Commun. 1996, 218: 688-693. 10.1006/bbrc.1996.0123.View ArticlePubMedGoogle Scholar
- Chiang C, Swan RZ, Grachtchouk M, Bolinger M, Litingtung Y, Robertson EK, Cooper MK, Gaffield W, Westphal H, Beachy PA, Dlugosz AA: Essential role for Sonic hedgehog during hair follicle morphogenesis. Dev Biol. 1999, 205: 1-9. 10.1006/dbio.1998.9103.View ArticlePubMedGoogle Scholar
- Ng JK, Tamura K, Buscher D, Izpisua-Belmonte JC: Molecular and cellular basis of pattern formation during vertebrate limb development. Curr Top Dev Biol. 1999, 41: 37-66.View ArticlePubMedGoogle Scholar
- Katarova Z, Sekerkova G, Prodan S, Mugnaini E, Szabo G: Domain-restricted expression of two glutamic acid decarboxylase genes in midgestation mouse embryos. J Comp Neurol. 2000, 424: 607-627. 10.1002/1096-9861(20000904)424:4<607::AID-CNE4>3.0.CO;2-C.View ArticlePubMedGoogle Scholar
- Goldman DC, Martin GR, Tam PP: Fate and function of the ventral ectodermal ridge during mouse tail development. Development. 2000, 127: 2113-2123.PubMedGoogle Scholar
- Yamakado M, Yohro T: Subdivision of mouse vibrissae on an embryological basis, with descriptions of variations in the number and arrangement of sinus hairs and cortical barrels in BALB/c (nu/+; nude, nu/nu) and hairless (hr/hr) strains. Am J Anat. 1979, 155: 153-173.View ArticlePubMedGoogle Scholar
- Rimvall K, Sheikh SN, Martin DL: Effects of Increased γ-Aminobutyric Acid Levels on GAD67 Portein and mRNA Levels in Rat Cerebral Cortex. J. Neurochemistry. 1993, 60: 714-720.View ArticleGoogle Scholar
- Rimvall K, Martin DL: The Level of GAD67 Protein Is Highly Sensitive to Small Increases in Intraneuronal γ-Aminobutyric Acid Levels. J. Neurochemistry. 1994, 62: 1375-1381.View ArticleGoogle Scholar
- Bond RW, Wyborski RJ, Gottlieb DI: Developmentally Regulated Expression of an Exon Containing a Stop Codon in the Gene for Glutamic Acid Decarboxylase. Proc. Natl. Acad. Sci. USA. 1990, 87: 8771-8775.PubMed CentralView ArticlePubMedGoogle Scholar
- Szabo G, Katarova Z, Greenspan R: Distinct Protein Forms Are Produced from Alternatively Spliced Bicistronic Glutamic Acid Decarboxylase mRNAs during Development. Molecular and Cellular Biology. 1994, 14: 7535-7545.PubMed CentralView ArticlePubMedGoogle Scholar
- Tillakaratne NJ, Erlander MG, Collard MW, Greif KF, Tobin AJ: Glutamate decarboxylases in nonneural cells of rat testis and oviduct: differential expression of GAD65 and GAD67. J Neurochem. 1992, 58: 618-627.View ArticlePubMedGoogle Scholar
- Kim J, Richter W, Aanstoot HJ, Shi Y, Fu Q, Rajotte R, Warnock G, Baekkeskov S: Differential expression of GAD65 and GAD67 in human, rat, and mouse pancreatic islets. Diabetes. 1993, 42: 1799-1808.View ArticlePubMedGoogle Scholar
- Liu ZH, Striker LJ, Hattori M, Yang CW, Striker GE: Localization of glutamic acid decarboxylase in the kidneys of nonobese diabetic mice. Nephron. 1996, 72: 662-666.View ArticlePubMedGoogle Scholar
- Chessler SD, Lernmark A: Alternative splicing of GAD67 results in the synthesis of a third form of glutamic-acid decarboxylase in human islets and other non-neural tissues. J Biol Chem. 2000, 275: 5188-5192. 10.1074/jbc.275.7.5188.View ArticlePubMedGoogle Scholar
- Li X, Ma W, Barker JL, Piatigorsky J: Transient expression of glutamate decarboxylase and gamma amino butyric acid in embryonic lens fibers of the rat. Dev Dyn. 1995, 203: 448-455.View ArticlePubMedGoogle Scholar
- Wray S, Fueshko SM, Kusano K, Gainer H: GABAergic neurons in the embryonic olfactory pit/vomeronasal organ: maintenance of functional GABAergic synapses in olfactory explants. Dev Biol. 1996, 180: 631-645. 10.1006/dbio.1996.0334.View ArticlePubMedGoogle Scholar
- Hardy MH: The secret life of the hair follicle. Trends Genet. 1992, 8: 55-61.View ArticlePubMedGoogle Scholar
- Carpenter EM, Goddard JM, Chisaka O, Manley NR, Capecchi MR: Loss of Hox-A1 (Hox-1.6) function results in the reorganization of the murine hindbrain. Development. 1993, 118: 1063-1075.PubMedGoogle Scholar
- Manley NR, Capecchi MR: The role of hoxa-3 in mouse thymus and thyroid development. Development. 1995, 121: 1989-2003.PubMedGoogle Scholar
- Szabo G, Katarova Z, Kortvely E, Greenspan RJ, Urban Z: Structure and the promoter region of the mouse gene encoding the 67-kD form of glutamic acid decarboxylase. DNA Cell Biol. 1996, 15: 1081-1091.View ArticlePubMedGoogle Scholar
- Katarova Z, Szabo G, Mugnaini E, Greenspan RJ: Molecular identification of the 62 kd form of glutamic acid decarboxylase from the mouse. European Jounal of Neuroscience. 1990, 2: 190-202.View ArticleGoogle Scholar
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