S1Supplemental Data Laterotopic Representation of Left-Right Information onto the Dorso-Ventral Axis of a Zebrafish Midbrain Target Nucleus

Supplemental Experimental Procedures To generate the Tg(lefty1:GFP) line of fish, we used regulatory elements from the lefty1 gene to drive GFP expression. lefty1 cDNA [S6] was used for screening zebrafish genomic PAC libraries (IncyteZebrafish Lines Embryos and larvae were obtained by natural spawning from wild- Genomics) and one clone, approximately 125 kbp in length, was isolated. We generated the 13.5-lefty1:GFP plasmid by subcloningtype, one-eyed pinheadtz57, about facerw715 (abf, see below), Tg(lefty1:GFP)rw017 (see below), Tg(foxD3:GFP), Tg(isl1:GFP)rw0, and a 13.5-kbp 5-upstream genomic fragment of the lefty1 gene in the pEGFP-1 vector (Clontech). Preparation of DNA and microinjectionTg(brn3a-hsp70:GFP)rw0110b (see below) fish [S1, S2]. For some exper-iments, a morpholino directed against ntlmRNAwas used to knock- were performed as described previously [S7]. Injected embryos were raised, and pairwise crosses identified a fish that passed thedown activity of the Ntl protein in wild-type and Tg(foxD3:GFP) em-bryos as previously described [S3, S4]. Embryos were reared and transgene on to its progeny. Transgene expression recapitulated expression of the endogenous lefty1 gene in the diencephalon.staged according to standard procedures [S5], and occasionally 0.002 % of phenylthiourea was added to the fish water from 12 hpf To generate the Tg(brn3a-hsp70:GFP) line of fish, we first isolated the zebrafish brn3a gene. We used a PCR-amplified DNA fragmentto inhibit pigment formation. containing sequence encoding the POU domain of zebrafish brn3c [S8] to screen gt11 cDNA library from 20- to 26-hpf zebrafish em