Fig. S7

Analysis of epistatic position of bsx function in pineal complex development.

(A-H,K-P) Dorsal views of embryos at 2 dpf after whole mount in situ hybridization. Anterior is to the top. Scale bar: 50 µm. (A,B) bsx expression is normal in embryos injected with otx5 morpholino (MO) (n=21/21) when compared to embryos injected with standard control MO (n=35/35). (C,D) otx5 MO function was confirmed through downregulation of irbp expression (n=22/24) in comparison to std ctrl MO injected embryos (n=34/34). (E) bsx is expressed in both pineal gland (PG) and parapineal (PP) in std ctrl MO injected embryos at (n=31/31). (F) In most of tbx2b MO injected embryos only PG bsx expression and no PP bsx expression was observed (n=41/43), in the other two embryos left-sided bsx expression was detected, but was weaker than in control embryos. (G) bsx expression is grossly normal in embryos injected with pitx2c MO (n=50/56). (H) bsx expression is also normal in embryos injected with nr2e3 MO (n=34/41). (I) Splice blocking activity of nr2e3 MO was confirmed through PCR (see Material and Methods section for details). (J) bsx expression in pineal complex of morphologically normal siblings in fgf8a^ti282a heterozygous incross (n=31/33). (K) No PP bsx expression is observed in embryos homozygous for the fgf8a^ti282a allele (n=10/10). (L) opn1sw1 is not expressed in the PG of std ctrl MO injected embryos (n=14/14). (M) otx5 knockdown by MO does not result in PG expression of opn1sw1 (n=26/26). (N) Heterozygous or wildtype siblings of bsx mutants do not express opn1sw1 in the PG (n=32/32). (O) Strong PG opn1sw1 is observed in all bsx mutant embryos (n=12/12).