lefty expression during early zebrafish development. (A-P) In situ localization of lft1 and lft2 transcripts at blastula through late somite stages (A,B, animal pole views; C-N, lateral views, dorsal at right; O,P, dorsoanterior views, anterior at left). lft1 and lft2 localize to the blastoderm margin at sphere (A,B) and dome stages (C,D). At shield stage (E,F), and at 90% epiboly (G,H) both genes localize to the dorsal hypoblast; lft1 is also expressed in dorsal forerunner cells (dfc). 1- to 3-somite embryos express both genes in the polster (po) and prechordal plate (pp, I,J); lft1 is uniquely expressed in Kupffer’s vesicle (Kv) (I), lft2 is uniquely expressed in floorplate precursors (fp) (J). At 6-8 somites lft1 expression is maintained (K) while lft2 is downregulated (L). 13-15 somite embryos express lft1 in the posterior notochord and left habenula (arrowhead) (M); lft2 is not expressed (N). 22-24 somite embryos showing lft1 in the anterior notochord (an), left habenula (arrowhead) and left lateral plate mesoderm (lpm) (O); lft2 is expressed in left lateral plate mesoderm (P).

lefty1 and lefty2 expression domains are disrupted in zebrafish midline mutants. (A-H) In situ localization of lft1 and lft2 in mutant embryos at 85- 90% epiboly (dorsal views). Embryos from heterozygous flh parents show wild-type expression of lft1 (A) and lft2 (B). cyc mutant embryos do not express lft1 or lft2 in the anterior midline (C,D) and lft2 is only expressed in a few cells near the dorsal margin. In oep mutants lft1 and lft2 expression in the prechordal plate is limited to a few cells (E,F); lft2 expression is also reduced in floorplate precursor cells (F). In ntl mutants lft1 expression is lost in dorsal forerunner and marginal cells (G), and lft2 expression in floorplate precursors is expanded laterally (H). (I) lefty expression domains at 85% epiboly. lft1 is expressed at the margin, and in dorsal forerunner cells. lft2 is expressed in the posterior midline in floorplate precursors. Both genes are expressed in the anterior midline in the polster and prechordal plate. Expression domains affected in different midline mutants are summarized at right.

Ectopic expression of Lefty1 or Lefty2 results in loss of anterior mesendoderm and ventral neurectoderm. (A-D) Morphological phenotypes of embryos injected with 25 pg Lft2-MT RNA (lateral views). At 95% epiboly, injected embryos accumulate cells on the dorsal side (B, arrowhead) compared to uninjected controls (A). At 24 hpf, injected embryos (D) lack anterior mesendoderm, notochord, and trunk somites and the anteroposterior axis is shortened relative to uninjected controls (C). The neural tube is present and retains some anteroposterior pattern. (E-T) Effect of ectopic Lefty expression on gene expression in 24 hpf (E-J, lateral views) and gastrula-stage embryos (K-P, S,T, 90% epiboly, dorsal view; Q,R, 50% epiboly, animal pole views). Upper panels in each series are uninjected controls, lower panels are embryos injected with 25 pg RNA encoding Lft1-MT (J,L,P,R) or Lft2-MT (F,H,N,T). Embryos were processed by in situ hybridization (E-T), followed by antimyc immunohistochemistry to identify clones of cells expressing Lft1-MT or Lft2-MT (K-T). At 24 hpf, krx-20 (E,F) is expressed at the normal anteroposterior level in the hindbrain of injected embryos although the bands of expression within rhombomeres 3 and 5 are compressed. shh expression (G,H) is abolished from the ventral brain and floorplate of injected embryos. col2a, expression in the floorplate, notochord and hypochord (I) is reduced to a few cells in injected embryos (J). In gastrulae, ntl expression is reduced in the midline and dorsal margin (K,L) and axial expression is reduced in the midline and abolished in endodermal precursors (arrows, M,N) of injected embryos. tbx6 expression is suppressed in much of the ventrolateral margin (O,P), and pitx2 expression in the margin is abolished (Q,R). The otx2 expression domain is shifted toward the margin (S,T) in injected embryos.

Ectopic expression of Lefty1, Lefty2 or Cyclops affects the endogenous expression of each other. (A-C) cyc expression at 85% epiboly. In uninjected embryos cyc is expressed in the midline (A). Expression is abolished in embryos injected with 25 pg of RNA encoding either Lft1-MT (B) or Lft2-MT (C). (D-F) lft2 expression at 85% epiboly. In control embryos lft2 is expressed in the midline (D). Expression of lft2 is abolished in embryos injected with 25 pg of RNA encoding Lft1-MT (E) and is expanded in embryos injected with 10 pg of cyc RNA (F). Embryos (dorsal views) were processed by in situ hybridization followed by anti-myc immunohistochemistry to identify clones of cells expressing Lft1-MT or Lft2-MT.

Cyclops and Lefty2 have opposite and mutually antagonistic effects on mesendodermal gene expression. (A-D) gsc expression at 50% epiboly. Control embryos (A) express gsc in a 90 degree arc at the margin. gsc expression is abolished by injection of 25 pg lft2-MT RNA (B) and is expanded by injection of 10 pg cyc RNA (C). Co-injection of 25 pg lft2-MT RNA and 10 pg ntl expression at 50% epiboly. Uninjected embryos (E) express ntl throughout the margin. In response to injection of 25 pg lft2-MT RNA, ntl expression is slightly downregulated at the site of ectopic expression (F). ntl expression is expanded in embryos injected with 5 pg cyc RNA (G). Embryos co-injected with 25 pg lft2-MT RNA and 5 pg cyc RNA (H) express ntl in a similar pattern to uninjected embryos. Embryos (animal pole view) were processed by in situ hybridization followed by anti-myc immunohistochemistry to identify clones of cells expressing Lft2-MT.