Background|NNP Glutamate|NNP decarboxylase|NN (|( GAD|NNP )|) catalyzes|NNS the|DT formation|NN of|IN the|DT inhibitory|NN neurotransmitter|NN γ-amino|JJ butyric|JJ acid|NN (|( GABA|NNP )|) from|IN glutamate|NN .|. 
In|IN mammals|NNS ,|, the|DT two|CD isoforms|NNS of|IN this|DT enzyme|NN ,|, GAD|NNP 67|CD and|CC GAD|NNP 65|CD ,|, are|VBP expressed|VBN from|IN two|CD separate|JJ genes|NNS ,|, Gad|NNP 1|CD and|CC Gad|NNP 2|CD respectively|RB [|NN 1|CD ,|, 2|CD ,|, 3|CD ]|NN .|. 
GABA|NNP signaling|VBG plays|VBZ several|JJ roles|NNS in|IN neuronal|NN development|NN .|. 
Early|RB in|IN CNS|NNP development|NN ,|, GABA|NNP can|MD modulate|VBP neuron|NN progenitor|NN proliferation|NN as|RB well|RB as|IN neuron|NN migration|NN ,|, survival|NN and|CC differentiation|NN [|NN 4|CD ,|, 5|CD ,|, 6|CD ,|, 7|CD ,|, 8|CD ,|, 9|CD ,|, 10|CD ,|, 11|CD ,|, 12|CD ,|, 13|CD ,|, 14|CD ]|NN .|. 
In|IN some|DT classes|NNS of|IN neural|NN progenitors|NNS GABA|NNP stimulates|NNS these|DT processes|VBZ while|IN in|IN others|NNS it|PRP has|VBZ an|DT antagonistic|JJ activity|NN .|. 
For|IN example|NN ,|, recent|JJ work|NN has|VBZ demonstrated|VBN that|IN GABA|NNP acts|NNS in|IN the|DT developing|VBG neocortex|NN to|TO stimulate|VB the|DT proliferation|NN of|IN progenitors|NNS in|IN the|DT ventricular|NN zone|NN while|IN inhibiting|VBG the|DT proliferation|NN of|IN progenitors|NNS in|IN the|DT subventricular|NN zone|NN [|NN 14|CD ]|NN .|. 
Later|RB ,|, during|IN postnatal|NN development|NN ,|, normal|JJ GABAergic|NNP input|NN is|VBZ required|VBN for|IN activity-dependent|JJ plasticity|NN in|IN the|DT visual|JJ cortex|NN as|IN shown|VBN in|IN the|DT Gad|NNP 2|CD knockout|NN mouse|NN [|NN 15|CD ,|, 16|CD ]|NN .|. 
In|IN addition|NN to|TO these|DT functions|NNS in|IN the|DT developing|VBG CNS|NNP ,|, GABA|NNP signaling|VBG is|VBZ also|RB required|VBN for|IN the|DT normal|JJ development|NN of|IN non-neural|JJ tissues|NNS .|. 
Targeted|NNP mutations|NNS of|IN the|DT Gad|NNP 1|CD gene|NN lead|NN to|TO defective|JJ development|NN of|IN the|DT secondary|JJ palate|NN [|NN 17|CD ,|, 18|CD ]|NN .|. 
The|DT cleft|NN palate|NN phenotype|NN of|IN the|DT Gad|NNP 1|CD mutants|NNS suggests|VBZ the|DT involvement|NN of|IN GABA-mediated|NNP signals|NNS in|IN the|DT normal|JJ development|NN and|CC differentiation|NN of|IN a|DT structure|NN derived|VBN from|IN the|DT oral|JJ epithelium|NN and|CC neural|NN crest|NN ecto-mesenchyme|JJ .|. 
This|DT conclusion|NN is|VBZ further|RBR supported|VBN by|IN the|DT similar|JJ cleft|NN palate|NN defect|NN seen|VBN in|IN mice|NNS with|IN a|DT deletion|NN or|CC targeted|VBN mutation|NN in|IN the|DT β|NN 3|CD subunit|NN of|IN the|DT GABA|NNP A|DT receptor|NN [|NN 19|CD ,|, 20|CD ,|, 21|CD ,|, 22|CD ]|NN .|. 

This|DT intriguing|JJ genetic|JJ evidence|NN indicates|VBZ a|DT role|NN for|IN GABA-mediated|NNP signaling|VBG in|IN the|DT development|NN of|IN a|DT non-neural|JJ structure|NN ,|, the|DT secondary|JJ palate|NN .|. 
The|DT potential|NN for|IN this|DT pathway|NN to|TO be|VB involved|VBN in|IN the|DT early|JJ development|NN of|IN additional|JJ non-neural|JJ tissues|NNS has|VBZ not|RB yet|RB been|VBN thoroughly|RB explored|JJ [|NN 23|CD ]|NN .|. 
To|TO address|VB this|DT question|NN ,|, we|PRP surveyed|VBD Gad|NNP 1|CD transcript|NN distribution|NN in|IN the|DT non-|NN CNS|NNP tissues|NNS of|IN the|DT embryo|NN .|. 
Using|VBG a|DT whole|JJ mount|VB in|IN situ|NN hybridization|NN approach|NN ,|, we|PRP found|VBD that|IN Gad|NNP 1|CD is|VBZ indeed|RB expressed|VBN in|IN a|DT number|NN of|IN different|JJ regions|NNS and|CC tissues|NNS .|. 
A|DT notable|JJ feature|NN of|IN this|DT expression|NN pattern|NN is|VBZ that|IN Gad|NNP 1|CD transcripts|NNS accumulate|VBP in|IN the|DT specialized|JJ ectodermal|NN structures|NNS that|WDT are|VBP involved|VBN in|IN the|DT formation|NN of|IN the|DT mystacial|NN vibrissae|NN and|CC in|IN limb|NN outgrowth|NN .|. 
These|DT specialized|JJ ectodermal|NN tissues|NNS are|VBP known|VBN to|TO be|VB sources|NNS of|IN developmental|NN signals|NNS [|NN 24|CD ,|, 25|CD ,|, 26|CD ]|NN .|. 
In|IN addition|NN ,|, transcripts|NNS are|VBP expressed|VBN in|IN the|DT mesenchymal|NN stem|NN cell|NN population|NN of|IN the|DT tailbud|NN and|CC in|IN the|DT pharyngeal|NN endoderm|NN and|CC mesenchyme|NN .|. 
The|DT expression|NN patterns|NNS show|VBP that|IN Gad|NNP 1|CD is|VBZ expressed|VBN in|IN several|JJ non-|NN CNS|NNP structures|NNS that|WDT are|VBP derived|VBN from|IN each|DT of|IN the|DT three|CD germ|NN layers|NNS of|IN the|DT embryo|NN .|. 

Results|NNS The|DT mouse|NN Gad|NNP 1|CD gene|NN is|VBZ widely|RB expressed|VBN in|IN the|DT embryonic|JJ central|JJ nervous|JJ system|NN [|NN 27|CD ]|NN .|. 
To|TO define|VB additional|JJ sites|NNS of|IN expression|NN outside|IN of|IN the|DT CNS|NNP ,|, we|PRP analyzed|VBD the|DT distribution|NN of|IN Gad|NNP 1|CD transcripts|NNS in|IN E|NNP 8.5|CD to|TO E|NNP 14.5|CD mouse|NN embryos|NNS by|IN whole|JJ mount|VB in|IN situ|NN hybridization|NN .|. 

Gad|NNP 1|CD transcripts|NNS were|VBD not|RB detected|VBN in|IN E|NNP 8.5|CD day|NN embryos|NNS (|( data|NNS not|RB shown|VBN )|) .|. 
At|IN E|NNP 9.0|CD Gad|NNP 1|CD was|VBD readily|RB detected|VBN in|IN the|DT tailbud|NN (|( figure|NN 1|CD A|DT )|) .|. 
Expression|NNP in|IN the|DT tail|NN continued|VBD through|IN E|NNP 12.5|CD and|CC was|VBD undetectable|JJ by|IN E|NNP 13.5|CD (|( figure|NN 1|CD B|NNP ,|, C|NNP ,|, Dand|NNP data|NNS not|RB shown|VBN )|) ,|, a|DT period|NN corresponding|JJ to|TO secondary|JJ body|NN axis|NNS formation|NN in|IN the|DT mouse|NN embryo|NN [|NN 28|CD ]|NN .|. 
Examination|NNP of|IN sections|NNS from|IN an|DT E|NNP 9.5|CD embryo|NN revealed|VBD a|DT high|JJ level|NN of|IN Gad|NNP 1|CD expression|NN throughout|IN the|DT mesenchyme|NN and|CC neural|NN epithelium|NN in|IN the|DT caudal|NN portion|NN of|IN the|DT tailbud|NN (|( figure|NN 1|CD F|NN )|) .|. 
No|DT transcripts|NNS were|VBD detected|VBN in|IN the|DT surface|NN ectoderm|NN surrounding|VBG the|DT tailbud|NN mesenchyme|NN (|( figure|NN 1|CD F|NN )|) .|. 
At|IN more|JJR cranial|NN levels|NNS within|IN the|DT tail|NN ,|, expression|NN was|VBD localized|VBN to|TO paraxial|NN mesoderm|NN ,|, ventral|NN neural|NN tube|NN ,|, notochord|NN and|CC cells|NNS of|IN the|DT dorsal|NN hindgut|NN (|( figure|NN 1|CD E|NNP )|) .|. 
In|IN the|DT paraxial|NN mesoderm|NN ,|, the|DT highest|JJS expression|NN levels|NNS were|VBD also|RB localized|JJ ventrally|RB ,|, adjacent|JJ to|TO the|DT notochord|NN (|( figure|NN 1|CD E|NNP )|) .|. 

In|IN the|DT pharyngeal|NN region|NN of|IN E|NNP 9.5|CD embryos|NNS ,|, Gad|NNP 1|CD RNA|NNP was|VBD detected|VBN in|IN and|CC around|IN the|DT second|JJ ,|, third|JJ and|CC fourth|JJ pharyngeal|NN pouches|NNS (|( figure|NN 2|CD A|DT )|) .|. 
Sections|NNP through|IN the|DT third|JJ pouch|NN confirmed|VBD the|DT presence|NN of|IN Gad|NNP 1|CD expression|NN in|IN the|DT pouch|NN endoderm|NN (|( data|NNS not|RB shown|VBN )|) .|. 
Expression|NNP was|VBD particularly|RB strong|JJ in|IN the|DT dorsal|NN portion|NN of|IN this|DT pouch|NN (|( figure|NN 2|CD B|NNP )|) .|. 
The|DT additional|JJ diffuse|NN staining|VBG appeared|VBN to|TO be|VB in|IN the|DT pharyngeal|NN mesenchyme|NN (|( figure|NN 2|CD B|NNP )|) .|. 
The|DT expression|NN in|IN the|DT pharyngeal|NN region|NN was|VBD very|RB transient|JJ ;|: transcripts|NNS were|VBD easily|RB detected|VBN at|IN E|NNP 9.5|CD ,|, but|CC only|RB faintly|RB at|IN E|NNP 9.0|CD and|CC were|VBD not|RB detectable|JJ by|IN E|NNP 10.5|CD .|. 

In|IN the|DT limb|NN buds|NNS ,|, Gad|NNP 1|CD RNA|NNP was|VBD detected|VBN from|IN E|NNP 9.0|CD to|TO E|NNP 11.5|CD (|( figure|NN 3|CD A|DT ,|, B|NNP ,|, C|NNP ,|, D|NNP ,|, E|NNP ,|, F|NN ,|, G|NNP ,|, H|NNP )|) .|. Transcripts|NNP were|VBD initially|RB expressed|VBN in|IN the|DT pre-apical|JJ ectodermal|NN ridge|NN (|( pre-|NN AER|NNP )|) at|IN E|NNP 9.5|CD (|( figure|NN 3|CD A|DT ,|, B|NNP )|) and|CC by|IN E|NNP 10.5|CD were|VBD seen|VBN in|IN the|DT definitive|JJ AER|NNP of|IN the|DT forelimb|NN (|( figure|NN 3|CD D|NNP )|) .|. 
At|IN E|NNP 10.5|CD Gad|NNP 1|CD was|VBD expressed|VBN in|IN a|DT diffuse|NN stripe|NN in|IN the|DT forelimb|NN (|( figure|NN 3|CD D|NNP )|) while|IN in|IN the|DT hindlimb|NN expression|NN was|VBD only|RB detected|VBN in|IN the|DT apical|JJ ectoderm|NN (|( figure|NN 3|CD E|NNP )|) .|. 
By|IN E|NNP 11.5|CD forelimb|NN AER|NNP expression|NN was|VBD fading|VBG and|CC expression|NN was|VBD seen|VBN in|IN a|DT diffuse|NN stripe|NN in|IN the|DT proximal|NN forelimb|NN and|CC a|DT diffuse|NN crescent|NN in|IN the|DT proximal|NN hindlimb|NN (|( figure|NN 3|CD G|NNP ,|, H|NNP )|) .|. 
The|DT earlier|JJR activation|NN of|IN Gad|NNP 1|CD in|IN the|DT forelimb|NN reflects|VBZ the|DT normal|JJ temporal|JJ order|NN of|IN events|NNS in|IN limb|NN development|NN .|. 
Sections|NNP indicate|VBP that|IN the|DT expression|NN within|IN the|DT limb|NN buds|NNS was|VBD in|IN surface|NN ectoderm|NN and|CC adjacent|JJ mesenchyme|NN (|( data|NNS not|RB shown|VBN )|) .|. 
Gad|NNP 1|CD RNA|NNP was|VBD not|RB detected|VBN in|IN the|DT limbs|NNS by|IN whole|JJ mount|VB in|IN situ|NN hybridization|NN after|IN E|NNP 11.5|CD .|. 

A|DT dynamic|JJ pattern|NN of|IN Gad|NNP 1|CD expression|NN was|VBD detected|VBN in|IN the|DT developing|VBG vibrissae|NN from|IN E|NNP 12.5|CD to|TO E|NNP 14.5|CD (|( figure|NN 4|CD A|DT ,|, B|NNP ,|, C|NNP ,|, D|NNP ,|, E|NNP ,|, F|NN ,|, G|NNP ,|, H|NNP )|) .|. 
Expression|NNP was|VBD first|RB detected|VBD in|IN the|DT supra-orbital|JJ ,|, infra-orbital|JJ ,|, and|CC post-oral|JJ vibrissae|NN and|CC in|IN the|DT posterior|NN vibrissae|NN in|IN the|DT lateral|NN nasal|NN and|CC maxillary|JJ rows|NNS (|( figure|NN 4|CD A|DT ,|, B|NNP ;|: nomenclature|NN as|IN in|IN [|NN 29|CD ]|NN )|) .|. 
Gad|NNP 1|CD RNA|NNP was|VBD also|RB detected|VBN in|IN some|DT of|IN the|DT posterior|NN labial|NN vibrissae|NN at|IN this|DT stage|NN .|. 
Expression|NNP was|VBD activated|VBN in|IN a|DT posterior|NN to|TO anterior|NN (|( towards|IN the|DT nose|NN )|) progression|NN in|IN the|DT lateral|NN nasal|NN and|CC maxillary|JJ rows|NNS ,|, reflecting|VBG the|DT pattern|NN of|IN vibrissal|NN development|NN [|NN 29|CD ]|NN .|. 
By|IN E|NNP 13.5|CD ,|, Gad|NNP 1|CD expression|NN was|VBD detected|VBN in|IN the|DT anterior|NN lateral|NN nasal|NN and|CC maxillary|JJ rows|NNS and|CC was|VBD activated|VBN in|IN the|DT rhinal|NN ,|, labial|NN and|CC submental|NN vibrissae|NN (|( figure|NN 4|CD C|NNP ,|, D|NNP )|) .|. 
By|IN E|NNP 14.5|CD ,|, expression|NN was|VBD strong|JJ in|IN the|DT labial|NN ,|, submental|NN and|CC rhinal|NN vibrissae|NN (|( figure|NN 4|CD E|NNP ,|, F|NN )|) .|. 
Sections|NNP of|IN E|NNP 12.5|CD whole|JJ mounts|VBZ show|NN that|IN Gad|NNP 1|CD expression|NN was|VBD localized|VBN to|TO the|DT epidermal|NN placodes|NNS of|IN the|DT mystacial|NN vibrissae|NN (|( figure|NN 4|CD G|NNP ,|, H|NNP )|) and|CC was|VBD maintained|VBN as|IN the|DT placodes|NNS begin|VBP to|TO invaginate|NN (|( figure|NN 4|CD G|NNP )|) .|. 

Control|NN hybridizations|NNS using|VBG a|DT sense|NN strand|NN Gad|NNP 1|CD probe|NN were|VBD also|RB performed|VBN .|. 
Embryos|NNP hybridized|JJ to|TO the|DT sense|NN probe|NN did|VBD not|RB reveal|VB any|DT staining|VBG pattern|NN at|IN any|DT of|IN the|DT stages|NNS tested|VBN (|( E|NNP 8.5|CD -|: E|NNP 14.5|CD )|) .|. 
Sense|NN strand|NN hybridization|NN results|NNS for|IN E|NNP 10.5|CD and|CC E|NNP 11.5|CD embryos|NNS are|VBP shown|VBN in|IN figure|NN 5|CD .|. 

Discussion|NNP The|DT expression|NN results|NNS reported|VBD here|RB show|NN that|IN Gad|NNP 1|CD was|VBD activated|VBN in|IN several|JJ tissues|NNS outside|IN of|IN the|DT central|JJ nervous|JJ system|NN during|IN mouse|NN development|NN .|. 
Transcripts|NNP were|VBD not|RB seen|VBN at|IN E|NNP 8.5|CD and|CC were|VBD first|RB detected|VBD at|IN E|NNP 9.0|CD .|. 
It|PRP was|VBD surprising|VBG that|IN this|DT very|RB early|JJ phase|NN of|IN Gad|NNP 1|CD expression|NN was|VBD largely|RB outside|IN of|IN the|DT developing|VBG CNS|NNP and|CC was|VBD localized|JJ in|IN the|DT tail|NN bud|NN mesenchyme|NN and|CC in|IN the|DT pre-apical|JJ ectodermal|NN ridge|NN (|( pre-|NN AER|NNP )|) of|IN the|DT forelimb|NN bud|NN .|. 
As|IN development|NN proceeded|VBD Gad|NNP 1|CD was|VBD detected|VBN in|IN pharyngeal|NN endoderm|NN and|CC in|IN the|DT ectodermal|NN placodes|NNS of|IN the|DT vibrissae|NN .|. 
The|DT data|NNS demonstrate|VBP that|IN Gad|NNP 1|CD is|VBZ expressed|VBN in|IN several|JJ sites|NNS outside|IN of|IN the|DT developing|VBG CNS|NNP and|CC in|IN derivatives|NNS of|IN all|DT three|CD germ|NN layers|NNS .|. 
We|PRP have|VBP also|RB detected|VBN the|DT expression|NN of|IN Gad|NNP 1|CD -|: lacZ|NN transgenes|NNS in|IN the|DT developing|VBG vibrissae|NN and|CC limbs|NNS supporting|VBG the|DT novel|NN and|CC surprising|JJ in|IN situ|NN hybridization|NN results|NNS we|PRP report|VBP here|RB (|( J.J.|NN Westmoreland|NNP and|CC B.G.C.|NN ,|, unpublished|JJ results|NNS )|) .|. 

Previous|JJ studies|NNS have|VBP shown|VBN that|IN Gad|NNP 1|CD can|MD be|VB regulated|VBN at|IN the|DT post-transcriptional|JJ and|CC translational|NN level|NN .|. 
Gad|NNP 1|CD mRNA|NN translation|NN or|CC protein|NN stability|NN can|MD be|VB regulated|VBN in|IN mature|VBP neurons|NNS by|IN the|DT level|NN of|IN GABA|NNP [|NN 30|CD ,|, 31|CD ]|NN .|. 
During|IN embryogenesis|NNS ,|, post-transcriptional|JJ regulation|NN occurs|VBZ by|IN alternative|NN splicing|VBG during|IN embryonic|JJ development|NN in|IN rats|NNS and|CC mice|NNS [|NN 32|CD ,|, 33|CD ]|NN .|. 
This|DT alternate|JJ embryonic|JJ transcript|NN inserts|NNS a|DT stop|NN codon|NN into|IN the|DT Gad|NNP 1|CD mRNA|NN and|CC can|MD produce|VB the|DT truncated|JJ proteins|NNS ,|, GAD|NNP 25|CD and|CC GAD|NNP 44|CD ,|, from|IN its|PRP$ 5|CD '|POS ;|: and|CC 3|CD '|POS ends|VBZ respectively|RB .|. 
The|DT studies|NNS reported|VBD here|RB used|VBN a|DT probe|NN that|WDT will|MD detect|VB the|DT adult|JJ Gad|NNP 1|CD mRNA|NN that|IN encodes|NNS GAD|NNP 67|CD as|RB well|RB as|IN the|DT embryonic|JJ alternatively|RB spliced|JJ mRNA|NN that|WDT can|MD encode|NN GAD|NNP 25|CD and|CC GAD|NNP 44|CD .|. 
These|DT additional|JJ mechanisms|NNS of|IN Gad|NNP 1|CD regulation|NN may|MD control|VB the|DT production|NN of|IN GAD|NNP proteins|NNS and|CC the|DT synthesis|NN of|IN GABA|NNP in|IN the|DT non-neural|JJ cell|NN types|NNS detected|VBD in|IN our|PRP$ study|NN .|. 

The|DT whole|JJ mount|VB in|IN situ|NN hybridization|NN data|NNS reported|VBD here|RB extends|VBZ the|DT results|NNS of|IN a|DT recently|RB published|VBN section|NN in|IN situ|NN hybridization|NN study|NN on|IN E|NNP 10.5|CD -|: E|NNP 12.5|CD mouse|NN embryos|NNS [|NN 27|CD ]|NN .|. 
Our|PRP$ analysis|NN showed|VBD that|IN Gad|NNP 1|CD expression|NN is|VBZ first|JJ detectable|JJ earlier|RBR at|IN E|NNP 9.0|CD and|CC revealed|VBD novel|NN non-|NN CNS|NNP sites|NNS of|IN expression|NN in|IN the|DT pharyngeal|NN region|NN ,|, vibrissae|NN ,|, tail|NN bud|NN and|CC limb|NN bud|NN .|. The|DT results|NNS of|IN the|DT previous|JJ study|NN [|NN 27|CD ]|NN ,|, together|RB with|IN the|DT data|NNS reported|VBD herein|NN ,|, provide|VBP a|DT comprehensive|JJ picture|NN of|IN Gad|NNP 1|CD expression|NN in|IN the|DT E|NNP 9.0|CD -|: E|NNP 12.5|CD mouse|NN embryo|NN .|. 

Previous|JJ studies|NNS have|VBP noted|VBN Gad|NNP expression|NN outside|IN of|IN the|DT CNS|NNP .|. 
In|IN adults|NNS Gad|NNP 1|CD and|CC Gad|NNP 2|CD have|VBP been|VBN detected|VBN in|IN a|DT number|NN of|IN tissues|NNS including|VBG kidney|NN ,|, testis|NNS ,|, oviduct|NN ,|, pancreatic|JJ islets|NNS and|CC adrenal|NN cortex|NN [|NN 34|CD ,|, 35|CD ,|, 36|CD ,|, 37|CD ]|NN .|. 
Previously|RB reported|VBD sites|NNS of|IN embryonic|JJ Gad|NNP 1|CD expression|NN outside|IN of|IN the|DT brain|NN and|CC spinal|JJ cord|NN during|IN rodent|NN development|NN include|VBP the|DT lens|NN fibers|NNS and|CC the|DT olfactory|NN pit|NN [|NN 38|CD ,|, 39|CD ]|NN .|. 
In|IN E|NNP 10.5|CD -|: E|NNP 12.5|CD mouse|NN embryos|NNS Gad|NNP 1|CD is|VBZ expressed|VBN in|IN the|DT olfactory|NN and|CC the|DT lens|NN placodes|NNS ,|, the|DT anlagen|NN of|IN the|DT olfactory|NN pit|NN and|CC lens|NN fibers|NNS [|NN 27|CD ]|NN .|. 
We|PRP also|RB detected|VBD Gad|NNP 1|CD expression|NN in|IN these|DT tissues|NNS (|( please|VB see|VB figure|NN 3|CD Aand|NNP data|NNS not|RB shown|VBN )|) .|. 
Expression|NNP of|IN Gad|NNP in|IN the|DT developing|VBG heart|NN and|CC blood|NN vessels|NNS has|VBZ also|RB been|VBN reported|VBN [|NN 27|CD ]|NN .|. 
We|PRP detected|VBD weak|JJ staining|VBG in|IN the|DT heart|NN and|CC did|VBD not|RB detect|VB blood|NN vessel|NN expression|NN ,|, perhaps|RB due|JJ to|TO the|DT very|RB low|JJ levels|NNS of|IN expression|NN in|IN developing|VBG vasculature|NN [|NN 27|CD ]|NN .|. 
Our|PRP$ results|NNS document|NN localized|JJ expression|NN of|IN Gad|NNP 1|CD at|IN additional|JJ non-|NN CNS|NNP sites|NNS in|IN the|DT mouse|NN embryo|NN ,|, suggesting|VBG a|DT potential|JJ role|NN for|IN GABA|NNP signaling|VBG in|IN the|DT development|NN of|IN these|DT structures|NNS .|. 

Our|PRP$ interest|NN in|IN the|DT role|NN of|IN GABA|NNP signaling|VBG in|IN developing|VBG tissues|NNS outside|IN of|IN the|DT central|JJ nervous|JJ system|NN stems|VBZ from|IN the|DT cleft|NN palate|NN phenotype|NN of|IN the|DT Gad|NNP 1|CD and|CC the|DT β|NN 3|CD GABA|NNP A|DT receptor|NN subunit|NN mutants|NNS [|NN 17|CD ,|, 18|CD ,|, 19|CD ,|, 21|CD ,|, 22|CD ]|NN .|. 
The|DT genetic|JJ data|NN strongly|RB suggest|VBP that|IN GABA|NNP acts|NNS through|IN GABA|NNP A|DT receptors|NNS to|TO modulate|VBP the|DT development|NN of|IN this|DT tissue|NN .|. 
Although|IN the|DT data|NNS reported|VBD here|RB do|VBP not|RB explain|VB the|DT origin|NN of|IN the|DT cleft|NN palate|NN phenotype|NN ,|, they|PRP do|VBP indicate|VBP that|IN Gad|NNP 1|CD is|VBZ expressed|VBN in|IN several|JJ additional|JJ non-|NN CNS|NNP tissues|NNS in|IN the|DT mouse|NN embryo|NN .|. 
It|PRP is|VBZ particularly|RB noteworthy|JJ that|IN these|DT include|VBP the|DT AER|NNP of|IN the|DT limb|NN buds|NNS and|CC the|DT ectodermal|NN placodes|NNS of|IN the|DT vibrissae|NN .|. 
Both|DT are|VBP ectodermal|NN structures|NNS known|VBN to|TO be|VB sources|NNS of|IN developmental|NN signals|NNS required|VBN for|IN morphogenesis|NNS and|CC patterning|VBG [|NN 24|CD ,|, 25|CD ,|, 26|CD ,|, 40|CD ]|NN .|. 
It|PRP will|MD be|VB of|IN interest|NN to|TO examine|VB the|DT expression|NN pattern|NN of|IN GABA|NNP receptors|NNS in|IN the|DT mesenchyme|NN adjacent|JJ to|TO these|DT ectodermal|NN signaling|VBG centers|NNS .|. 
Expression|NNP of|IN GABA|NNP receptor|NN subunits|NNS in|IN adjacent|JJ tissues|NNS would|MD indicate|VB that|IN these|DT receptors|NNS read|VBP the|DT developmental|NN signals|NNS mediated|JJ by|IN GABA|NNP in|IN these|DT structures|NNS and|CC tissues|NNS .|. 

Conclusions|NNP The|DT mouse|NN gene|NN encoding|VBG the|DT 67|CD kDa|NN isoform|NN of|IN glutamate|NN decarboxylase|NN (|( Gad|NNP 1|CD )|) is|VBZ expressed|VBN in|IN the|DT tail|NN bud|NN mesenchyme|NN ,|, vibrissal|NN placodes|NNS ,|, pharyngeal|NN arches|NNS and|CC pouches|NNS and|CC the|DT apical|JJ ectodermal|NN ridge|NN (|( AER|NNP )|) ,|, mesenchyme|NN and|CC ectoderm|NN of|IN the|DT limb|NN buds|NNS in|IN mouse|NN embryos|NNS from|IN E|NNP 9.0|CD -|: E|NNP 14.5|CD .|. Some|DT of|IN the|DT Gad|NNP 1|CD expressing|VBG tissues|NNS (|( vibrissal|NN placodes|NNS ,|, AER|NNP )|) are|VBP known|VBN sources|NNS of|IN developmental|NN signals|NNS .|. 
Other|JJ sites|NNS of|IN expression|NN correspond|VB to|TO stem|VB cell|NN populations|NNS that|WDT give|VBP rise|NN to|TO multiple|JJ differentiated|JJ tissues|NNS (|( tail|NN bud|NN mesenchyme|NN ,|, pharyngeal|NN endoderm|NN and|CC mesenchyme|NN )|) .|. 
The|DT localized|JJ and|CC dynamic|JJ expression|NN pattern|NN of|IN Gad|NNP 1|CD suggests|VBZ a|DT wider|JJR role|NN for|IN GAD|NNP and|CC GABA|NNP in|IN the|DT development|NN of|IN non-neural|JJ tissues|NNS than|IN was|VBD previously|RB known|VBN .|. 

Materials|NNS and|CC Methods|NNP Whole|JJ mount|VB in|IN situ|NN hybridizations|NNS were|VBD performed|VBN on|IN Swiss|NNP Webster|NNP embryos|NNS as|IN described|VBN [|NN 41|CD ,|, 42|CD ]|NN .|. 
The|DT morning|NN that|IN the|DT vaginal|JJ plug|NN was|VBD found|VBN was|VBD considered|VBN 0.5|CD days|NNS of|IN gestation|NN .|. 
The|DT Gad|NNP 1|CD probe|NN was|VBD derived|VBN from|IN an|DT EST|NNP clone|NN (|( accession|NN W|NNP 59173|CD )|) .|. 
Its|PRP$ 5|CD '|POS end|NN corresponds|NNS to|TO nucleotide|NN 142|CD in|IN exon|NN 1|CD [|NN 43|CD ]|NN and|CC the|DT 3|CD '|POS end|NN is|VBZ at|IN nucleotide|NN 2041|CD in|IN the|DT cDNA|NN sequence|NN [|NN 44|CD ]|NN .|. 
Digoxygenin|NNP sense|NN and|CC antisense|NN RNA|NNP probes|NNS were|VBD generated|VBN by|IN labeling|VBG with|IN digoxygenin-|NN UTP|NNP during|IN transcription|NN .|. 
Embryos|NNP were|VBD removed|VBN and|CC fixed|VBN in|IN 4|CD %|NN paraformaldehyde|NN /|NN PBS|NNP overnight|JJ and|CC used|VBN immediately|RB for|IN the|DT in|IN situ|NN hybridization|NN .|. 
The|DT embryos|NNS were|VBD processed|VBN as|IN described|VBN previously|RB [|NN 41|CD ]|NN and|CC hybridized|JJ to|TO the|DT probe|NN overnight|JJ in|IN 50|CD %|NN formamide|NN ,|, 5|CD X|NNP SSC|NNP (|( pH|NN 5.0|CD )|) ,|, 50|CD μg|NN /|NN ml|NN torula|NN RNA|NNP ,|, 50|CD μg|NN /|NN ml|NN heparin|NN at|IN 70|CD °|NN C.|NN 
The|DT final|JJ concentration|NN of|IN probe|NN in|IN the|DT hybridization|NN was|VBD 1|CD μg|NN /|NN ml|NN .|. 
After|IN an|DT overnight|JJ hybridization|NN ,|, the|DT embryos|NNS were|VBD washed|VBN at|IN high|JJ stringency|NN in|IN prewarmed|JJ 50|CD %|NN formamide|NN ,|, 5|CD X|NNP SSC|NNP ,|, 1|CD %|NN SDS|NNP (|( wash|VB I|PRP )|) at|IN 70|CD °|NN C|NNP for|IN 90|CD minutes|NNS .|. 
The|DT embryos|NNS were|VBD then|RB washed|VBN in|IN a|DT 1|CD :|: 1|CD mix|NN of|IN wash|NN I|PRP and|CC wash|VB II|NNP (|( 0.5|CD M|NNP NaCl|NNP ,|, 10|CD mM|NN Tris|NNP pH|NN 7.5|CD ,|, 0.1|CD %|NN Tween|NNP 20|CD )|) for|IN 10|CD minutes|NNS at|IN 70|CD °|NN C.|NN 
The|DT embryos|NNS were|VBD washed|VBN several|JJ times|NNS in|IN wash|NN II|NNP at|IN room|NN temperature|NN to|TO remove|VB the|DT formamide|NN and|CC then|RB treated|VBN with|IN 100|CD μg|NN /|NN ml|NN RNase|NNP A|DT ,|, 100|CD units|NNS /|NN ml|NN RNase|NNP T|NN 1|CD in|IN wash|NN II|NNP for|IN 1|CD hour|NN at|IN 37|CD °|NN C.|NN 
Following|VBG the|DT RNase|NNP treatment|NN the|DT embryos|NNS were|VBD washed|VBN in|IN three|CD changes|NNS of|IN 50|CD %|NN formamide|NN ,|, 2|CD X|NNP SSC|NNP pH|NN 5.0|CD at|IN 70|CD °|NN C|NNP for|IN a|DT total|NN of|IN 90|CD minutes|NNS .|. 
Detection|NNP of|IN the|DT hybridized|JJ RNA|NNP probe|NN was|VBD as|IN described|VBN previously|RB [|NN 41|CD ]|NN .|. 
The|DT embryos|NNS were|VBD photographed|VBN without|IN clearing|NN using|VBG a|DT Leica|NNP model|NN MZFL|NNP III|NNP dissecting|VBG scope|NN ,|, a|DT Hamamatsu|NNP model|NN C|NNP 4742|CD -|: 95|CD digital|JJ camera|NN and|CC Openlab|NNP 2.0|CD .7|CD software|NN .|. 

For|IN sectioning|VBG ,|, embryos|NNS were|VBD embedded|VBN in|IN Immunobed|NNP (|( Polysciences|NNP )|) resin|NN and|CC sectioned|JJ at|IN 10|CD μm|NN .|. 
Sections|NNP were|VBD phtotographed|JJ using|VBG an|DT Olympus|NNP BX|NNP 60|CD microscope|NN fitted|JJ with|IN a|DT SPOT|NNP digital|JJ camera|NN (|( Diagnostic|NNP Instruments|NNP Inc.|NN )|) .|. 

