Whole mount in situ hybridizations for ndr1. (A, B) Dome stage, animal pole (A) and lateral (B) views show asymmetric expression of ndr1 at the blastoderm margin. (C) 30% epiboly, lateral view. (D–F) Shield stage. (D) lateral view (dorsal to the right). Arrow marks the site of ndr1 expression in the shield, specifically within the area of the dorsal forerunner cells; the inset shows an enlarged view of this area. (E,F) dorsal view of ndr1 expression in forerunner cells (arrow) at two magnifications. Arrowhead shows traces of ndr1 RNA in more anterior aspects of the shield.

Whole mount in situ hybridization of ndr2 expression. (A) Dome stage, lateral view. (B) 30% epiboly, animal view; ndr2 is expressed at the blastoderm margin. (C) Shield stage, lateral view (dorsal to the right); ndr2 expression is restricted to the hypoblast layer of the shield. (D) 80% epiboly, lateral view, and (E) 70% epiboly, dorsal view; ndr2 expression is seen in involuting hypoblast. (F–H) Bud stage; anterior view (F), lateral view with anterior to the left (G), higher magnification lateral view (H). ndr2 is present in an anterior domain including the prechordal plate, anterior notochord, and ventral neuroectoderm (arrow); open triangles mark a gap in the anterior domain, asterisks indicate the polster, and a dotted line traces Brachet’s cleft separating anterior mesoderm and neurectoderm. ndr2 also is expressed in the tail bud (G). (I–L) 20-somite stage. Lateral view (I; anterior to the left), and dorsal view of the head (J; anterior to the top); ndr2 is expressed to the left of the midline within the posterior diencephalon (arrow) just ventral to the epiphysis (e). Dorsal view (K), and dorsal–lateral view (L); ndr2 is expressed in the left lateral plate (filled triangle), starting at a position next to the anterior hindbrain and extending caudally at decreased width and intensity of expression.

ndr2 expression in ntl and flh mutant embryos. The left column shows wild-type siblings, the right column shows ntl homozygotes (B, D, F, H, J) or a flh homozygote (L). (A–F) Bud stage. (A, B) Lateral view, anterior to the left. (C, D) Anterior view. ndr2 is expressed in anterior notochord and prechordal plate. The embryonic stage shown here is slightly earlier than that shown in Figs. 4F and 4G, and therefore the gap within the anterior expression domain is not yet evident. ndr2 expression is slightly wider in the ntl embryo. (E, F) Posterior view. ndr2 staining is found in the tail bud of wild-type siblings (arrow), while in mutant embryos, tail bud staining is absent but a broader lateral and posterior region is positive (asterisk); 16 (24%) of 66 embryos in a cross between heterozygous ntl zebrafish showed the mutant pattern. (G–L) 20-somite stage. In wild-type embryos, ndr2 is expressed asymmetrically in the lateral plate and diencephalon, but expression is present bilaterally in both ntl (H, J) and flh mutants (L).

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