Isl1 and Pou4f2 Form a Complex to Regulate Target Genes in Developing Retinal Ganglion Cells
Figure 8
Isl1 and Pou4f2 contribute quantitatively to the full levels of expression of downstream genes.
A. Luciferase assays indicating that whereas Pou4f2 and Isl1 can each activate transcription from the reporter alone, together they promote transcription to a higher level. N = 3 for all transfections. Significance of differences was evaluated by student t test. Double black asterisks indicate p<0.01 when compared with control, double red asterisks indicate p<0.01 when compared with Pou4f2, double purple asterisks indicates p<0.01 when compared with Isl1. B. In situ hybridization of three downstream genes, Gap43, Nefl, and Stmn2, of Isl1 and Pou4f2. Compared to the wild-type retina, although all three genes have significantly reduced levels of expression in the RGCs of both Isl1- and Pou4f2-null retinas, substantial levels of expression remain. In the Isl1/Pou4f2 double null retinas, Gap43 and Nefl are not detectable, but Stmn2 is still expressed. C. The expression levels of downstream genes of Isl1 and Pou4f2 vary greatly. Shown are the relative expression levels of five downstream genes as revealed by RNA-seq. Y axis is Fragments Per Kilobase of exon per Million fragments mapped (FPKM) based on the number of sequence reads and lengths of the transcripts of individual genes. D. Changes of expression levels of five downstream genes of Isl1 and Pou4f2 in the Isl1- and Pou4f2–null retinas as revealed by RNA-seq. WT: wild-type; P4f2: Pou4f2. The expression levels (Y axis) are calculated based on the FPKM of three replicates and the average values for each gene in the wild-type is set to1. N = 3 for all samples. Significance of differences was evaluated by student's t test. Single asterisk indicates p<0.05, double asterisks indicate p<0.01.