Comment[ArrayExpressAccession]	E-GEOD-64556
MAGE-TAB Version	1.1					
Public Release Date	2015-12-21
Investigation Title	Retinoic acid signaling coordinates the behavior of multiple cell lineages during cardiac outflow morphogenesis					
Comment[Submitted Name]	Retinoic acid signaling coordinates the behavior of multiple cell lineages during cardiac outflow morphogenesis
Experiment Description	Retinoic acid (RA), the bioactive derivative of vitamin A, is essential for vertebrate heart development. Both excess and reduced RA signaling lead to cardiovascular malformations, particularly affecting the cardiac outflow tract (OFT). The cellular mechanisms underlying the effects of RA signaling during OFT morphogenesis are not fully understood. To address this question, we used transient maternal RA supplementation to rescue the early lethality resulting from inactivation of the murine retinaldehyde dehydrogenase 2 (Raldh2) gene. By embryonic day 13.5, Raldh2-/- hearts exhibited OFT septation defects. Although cardiac neural crest cells (cNCC) were present in OFT cushions of Raldh2-/- mutant embryos, we observed that cNCC were ectopically located in the peripheral region of the endocardial cushions, closer to the myocardium rather than immediately subjacent to the endocardium. Mislocated cNCC were aligned in parallel instead of perpendicular arrays to the endocardial surface within the proximal OFT. Supernumerary mesenchyme was generated both in and ex vivo within the cushions by Raldh2-/- mutant endocardium and found in place of the cNCC in a subendocardial, medial position. Although mislocated and misoriented, mutant cNCC resembled wildtype cells in their compaction density and individually elongated shape. Our data show that RA signaling acts on cNCC orientation relative to the septation plane and position within OFT cushions during septation process. Transcriptomic analysis and pathway-specific readout suggest that up-regulation of the Bmp pathway may be responsible for increased endothelial-to-mesenchymal transition, leading thereby to displacement of cNCC. E10.5 stage embryonic mouse outflow tracts were microdissected. Four pools of four outflow tracts were established for wildtype (WT) and mutant (Raldh2-/-) embryos derived from dams whose food had been supplemented with all-trans-RA (Sigma) directly mixed into powdered food at 0.1 mg/g food and offered between E7.5 and E8.5. Total RNA was extracted using Macherey-Nagel NucleoSpin® RNA columns without on-column DNase digestion.					
Term Source Name	ArrayExpress	EFO
Term Source File	http://www.ebi.ac.uk/arrayexpress/	http://www.ebi.ac.uk/efo/efo.owl				
Person Last Name	Etchevers	Robrini	Etchevers	Zaffran	Bertrand	
Person First Name	Heather	Nicolas	Heather	Stéphane	Nicolas	
Person Mid Initials	C	E	C			
Person Email	heather.etchevers@inserm.fr					
Person Affiliation	INSERM					
Person Phone	(33)491324937					
Person Address	INSERM UMR_S910, INSERM, 27 boulevard Jean Moulin, Marseille, France					
Person Roles	submitter					
Protocol Name	P-GSE64556-1	P-GSE64556-4	P-GSE64556-5	P-GSE64556-2	P-GSE64556-3	P-GSE64556-6
Protocol Description	Data were filtered and analyzed on GeneSpring using the standard analysis protocol GE1_107_Sep09. ID_REF =  VALUE = gProcessedSignal - green channel linearly scaled, normalized processed intensity values	50 ng total RNA input labeled with Cy3 using the Agilent One-color Low Input Quick Amp labeling kit. Labeled and purified cRNA quantities were assessed on a Nanodrop spectrophotometer.	Labeled cRNAs were hybridized on a single Agilent 8x60K SurePrint G3 Mouse GE 8x60K microarray for 40 hours at 65°C in an ozone-free environment.	Four pooled samples of four microdissected outflow tracts from both conditions (1 and 2 - retinoic acid-rescued embryos of Raldh2 KO or WT genotype respectively) were collected.	Total RNA was extracted without DNase treatment using Macherey-Nagel Nucleospin RNA columns. After assessing quality with an Agilent Bioanalyzer 2100, samples with an RNA Integrity Number (RIN) over 8.0 were selected for each condition (mutant or wildtype littermates).	The slide was then scanned on Agilent DNA scanner G2505C at 3 µm using one color scan setting for 1x44k array slides (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%). Raw data were extracted using Feature Extraction software.
Protocol Type	normalization data transformation protocol	labelling protocol	hybridization protocol	sample treatment protocol	nucleic acid extraction protocol	array scanning protocol
Experimental Factor Name	genotype					
Experimental Factor Type	genotype					
Publication Title	Cardiac outflow morphogenesis depends on effects of retinoic acid signaling on multiple cell lineages.					
Publication Author List	El Robrini N, Etchevers HC, Ryckeb�sch L, Faure E, Eudes N, Niederreither K, Zaffran S, Bertrand N					
PubMed ID	26442704					
Publication DOI	10.1002/dvdy.24357					
Comment[SecondaryAccession]	GSE64556					
Comment[GEOReleaseDate]	2015-12-21					
Comment[ArrayExpressSubmissionDate]	2014-12-29					
Comment[GEOLastUpdateDate]	2015-12-23					
Comment[AEExperimentType]	transcription profiling by array					
SDRF File	E-GEOD-64556.sdrf.txt