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Fig. 4 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.