GSE99211
RNA-seq data analysis of Smad5 knockout and wild type individual mouse amnion samples collected during early embryonic development
Mice deficient in the BMP-effector, Smad5 (Smad5 KO), develop severe defects in embryonic morphogenesis as well as a delay in amnion-chorion separation, important extraembryonic tissues. After closure of the proamniotic canal, a remarkable ectopic primitive streak-like aggregate develops in the amnion of these mutants. We investigated the earliest steps of mutant amnion misdifferentiation by RNAseq of single Control (Ctrl) and Smad5 KO amnion samples collected before the appearance of the aggregate. The transcriptome analysis revealed two separate sets of non-squamous amnion defects. One set of mutants (KO-SetA) robustly overexpressed streak mesoderm-related genes conform former analyses (Pereira et al., 2012). The other set overexpressed extraembryonic ectoderm markers suggestive of chorionic inclusion in amnion (KO-SetB). Tetraploid chimera analyses confirmed that SMAD5 deficiency in the epiblast can result in two distinct sets of amnion defects: one with impaired anterior amnion expansion and differentiation, and another with inclusion of chorionic extraembryonic ectoderm in the space normally occupied by amnion. Overall design: To investigate the molecular differences between WT and Smad5 mutant amnion, we assessed global gene expression by RNA-Seq before morphological differences were apparent between individual control and knockout amnion samples. Amnion was microdissected of control (n=6), and stage-matched littermate Smad5 knockout (n=6)(Chang et al., 1999 Development, 126, 1631–1642) embryos ranging between early pre-headfold (EPHF) and 3-somites (3S) stages (Table S1).  cDNA was synthesized and amplified from individual amnion samples. cDNA for RNA-seq was prepared and amplified following adaptation of a procedure originally designed for RNA-seq of single-cells (Tang et al., 2009) on the SOLiD platform (“Double stranded cDNA synthesis from a single cell”, Applied Biosystems demonstrated protocol). Briefly, reverse transcription and second-strand cDNA synthesis was performed in the sample lysate without RNA isolation using SuperScript III Reverse Transcriptase (Invitrogen) and poly-dT universal primers. This was followed by two rounds of PCR amplification (18 and 12 cycles, respectively) and column purifications (QIAquick, QIAGEN) generating up to 3 kb coverage at the 3' end of all poly-adenylated transcripts. The resulting cDNA was used for RT-qPCR and RNA-seq. The cDNA yield for the 12 RNA-seq samples is shown in Table S1.