Fig. 14

Pnkd transcripts detected in the retina. a RNAseq profiles expressed in FPKM of three expressed Pnkd RefSeq transcripts from retinal and RGC-derived samples of Brn3a and Brn3b WT and KO mice at P3. b Predicted Pnkd splicing variants. Exons are represented as boxes and numbered. White boxes are untranslated regions, gray boxes are coding regions. Splicing sequences for alternative transcripts are indicated above or below the exons. Green lines indicate connected exons for transcript NM_001039509, blue – for NM_025580, purple – for NM_019999. Gray lines represent a putative transcript (XM_006496167) that was not detected by RNAseq. Exons 6–13 are common to NM_001039509 and NM_019999. Red arrows show the positions of primers made to check the presence of each predicted variant (see also Table 2). c Agarose-gel electrophoresis showing RT-PCR reactions from P3 retina RNA. Indicated primer pairs refer to B. Duplicates are shown for each primer combination. The expected sizes for all primer combinations are indicated in Table 2. All bands generated by RT-PCR were gel-extracted, subcloned and sequenced for confirmation. d in situ hybridization analysis in WT and Brn3a-KO mouse retinas at 5 postnatal ages. RNA probes were designed to detect transcript-specific exons: primers 1 and 2 for NM_001039509 (green) and NM_025580 (blue) isoforms, primers 5 and 6 specifically for NM_019999 (purple), and primer 3 and 4 –specifically for NM_025580. Scale bar – 50 μm