islet2 is a downstream target of Prdm14. (A) CaP axons are shortened in islet2 morphants and slg mutants. The embryos are stained with znp1 antibody. Arrows indicate shortened CaP axons. (B) islet2 RNA expression is only downregulated in CaP (black arrowheads) in slg mutant and splmo morphant embryos, whereas islet2 expression in RB (black arrows) and cloaca (white arrowhead) is unaffected. islet1 and mnx1 expression are unaffected by Prdm14 reduction. (C) Islet antibody staining indicates that the signal intensity of Islet2 (circled) in the CaP neurons is substantially reduced in slg mutants and splmo morphants compared with heterozygotes. Islet1 signal (arrowheads) in GFP-negative neurons (non-CaP) showed comparable staining intensity in embryos of all genotypes. (D) (a-a′′ ′) islet2 RNA expression is decreased specifically in CaP (black arrowheads) in slg mutants (a′). islet2 is specifically reduced in RB (black arrows) in prdm1a morphants (a3). prdm1a MO injection into slg mutants results in greatly decreased islet2 expression in both RB and CaP (a′′ ′). islet2 in cloaca remains unchanged (white arrowheads). (b-c3′) islet1 and mnx1 expression in RB and CaP remains largely unchanged when prdm14 and prdm1a are downregulated. (B,D) The number of embryos showing this phenotype out of the total examined is indicated bottom right. (E) The islet2 promoter contains a putative Prdm14 binding site (pBS). The conserved sequence among zebrafish, mouse and human is indicated in red; mutated nucleotides are in green. TSS, transcription start site. (F) ChIP-PCR shows that Prdm14 antibodies specifically precipitate an islet2 promoter fragment containing the pBS in wild-type embryos. (G) The ChIP signal from the Prdm14 antibodies is greatly decreased in slg mutants. (H) Zebrafish Prdm14 expressed in HCT116 cells efficiently pulls down the pBS-containing fragment when an islet2 promoter (Pisl2) plasmid is cotransfected. The PCR signal is greatly reduced when a pBS mutant (Pisl2 mut) plasmid is transfected or Prdm14 protein is not expressed in the cells. Scale bars: 20 μm in A,C; 200 μm in B,D.