mRNA transcript levels in E15.5, E17.5 and P1 Crb1KOCrb2?RPC against Crb1KO neuroretina, and in E15.5 Crb1KOCrb2?RPC against wild type neuroretina
mRNA transcript levels in embryonic day 15.5 (E15.5), E17.5 and postnatal day 1 (P1) mouse Crb1KOCrb2?RPC against mouse Crb1KO neuroretina (Run1), and in E15.5 mouse Crb1KOCrb2?RPC against wild type neuroretina (Run2) were analyzed. A comparison between Crb1KOCrb2?RPC and Crb1KO retina, at E15.5, or E17.5, or P1 on 100% C57BL/6JOlaHsd, yielded only subtle persistent changes at the transcriptional level over time (Figure 1 G-I, respectively), despite significant differences in morphology. As inter-nal positive control at E15.5, E17.5 and P1 we used ATP-binding cassette, sub-family D (ALD), member 4 (Abcd4), which is substantially upregulated in mouse retina expressing the Cre recombinase fused to the Chx10 promoter, with the Abcd4 gene immediately adja-cent to the Chx10Cre locus in the mouse genome. To further examine potential transcriptional changes at E15.5, we compared Crb1KOCrb2?RPC mice in 100% C57BL/6JOlaHsd genetic background against wild type C57BL/6JRccHsd mice. In this setting, we observed 40 differentially expressed genes (DEGs) compared to the previously 0 DEGs (E15.5; adj. p-val < 0.01 & log2FC > 1.5). The Crb1KOCrb2?RPC mice on C57BL/6JOlaHsd genetic background, due to mutations in the synuclein alpha (Snca), multimerin-1 (Mmrn1), and Crb1 gene, do not express Snca, Mmrn1, or Crb1 gene transcripts, and express high levels of Abcd4 due to the adjacent Chx10Cre transgene on chromosome 6. We made use of all 4 genes as negative and positive controls in the gene expression profiling, since the mice on C57BL/6JRccHsd genetic background have no mutations in Snca, Mmrn1 or Crb1 and express low levels of Abcd4. Analysis of the E15.5 data revealed upregulated expression of the WD repeat and FYVE domain-containing protein 1 (Wdfy1) gene also known as FYVE domain-containing pro-tein localized to endosomes (Fens-1), which encodes a phosphatidylinositol 3-phosphate binding protein that contains a FYVE zinc finger domain and multiple WD-40 repeat do-mains. Overall design: Comparison of E15.5, E17.5 and postnatal day 1 (P1) mouse Crb1KOCrb2?RPC against mouse Crb1KO neuroretina (Run1) on exactly the same genetic background. Comparison of  E15.5, E17.5 mouse Crb1KOCrb2?RPC against wild type neuroretina (Run2) was on different genetic backgrounds of C57BL/6JOlaHsd versus C57BL/6JRccHsd, respectively. Sequencing was performed using Life Technologies SOLiD5500 with single-end, 50 bp reads. Two separate runs were performed. Run1 contained n=30 mice; n=5 Crb1KOCrb2F/F (Crb1KO) and n=5 Crb1KOCrb2?RPC (Tg) per development timepoint of the mouse embryo at E15.5, E17.5 and P1. Run2 contained n=9 mice; n=4 Crb1KOCrb2?RPC mice (C57BL/6JOlaHsd) and n=5 healthy mice (C57BL/6JRccHsd = wildtype, WT) at E15.5. Reads were aligned against mm10, using the 'whole.transcriptome.frag' workflow with bamgen.mqv.threshold set to 20 (Lifescope v2.5). Counts were obtained using the GTF as supplied by Lifescope (transformation of refGene.txt downloaded from UCSC at 25-06-2014) within this workflow.