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First published online 1 October 2003
doi: 10.1242/dev.00803

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Mixer/Bon and FoxH1/Sur have overlapping and divergent roles in Nodal signaling and mesendoderm induction

Prabhat S. Kunwar1,*, Steven Zimmerman1,*, James T. Bennett1, Yu Chen1, Malcolm Whitman2 and Alexander F. Schier1,{dagger}

1 Developmental Genetics Program, Skirball Institute of Biomolecular Medicine, Department of Cell Biology, New York University School of Medicine, New York, NY 10016, USA
2 Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA



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  		Fig. 1. Regulation of bon and sur expression by Nodal signaling. Expression of sur (A,B) and bon (C-G) at 50% epiboly. Expression of bon at dome stage (H,I) and 30% epiboly (J,K). Wild-type (A,C,H,J), MZoep (B,D,I,K), bon (E), MZsur (F) and MZsur;bon (G) embryos. Expression of sur is not affected by loss of Nodal signaling (A,B). Expression of bon is abolished at 50% epiboly in MZoep embryos (C,D) but weak bon expression can be detected in dome stage (H,I) and 30% epiboly (J,K). Expression of bon is not affected in bon mutants (E) but reduced in MZsur and MZsur;bon mutant embryos at 50% epiboly (F,G).

 


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  		Fig. 2. Generation of embryos lacking both bon and sur activity by two different approaches. (A) In the genetic approach, bon and sur heterozygous fish are crossed to generate bon and sur double heterozygotes, which are crossed to sur homozygous fish. The sur homozygous and bon heterozygous embryos are injected with sur mRNA to rescue sur mutants. The intercrossing of these fish results in embryos lacking both bon and maternal and zygotic sur. (B) In the morpholino approach, bonMO is injected into MZsur mutants at the one- to two-cell stage to give rise to embryos lacking both bon activity and maternal and zygotic sur.

 


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  		Fig. 3. Phenotypes associated with lack of both bon and sur activity. Wild-type (A1-4), bon (B1-4), bonMO (C1-4), MZsur (D1-4), MZsur;bon (E1-4) and MZsur;bonMO (F1-4) embryos at 30 hpf. All images are lateral views except A3-F3, which are ventral views. (A1-F1) Comparison of embryos in a lateral view. The bon (B1) and bonMO (C1) embryos have pericardial edema (arrowhead) and enlarged yolk extension (arrow). MZsur;bon (E1) and MZsur;bonMO (F1) embryos have an anterior bulb-like structure (arrowhead; also arrow in E3 and F3). (A2-F2) Lateral view of head region, showing the absence of the hatching gland (arrowhead) in MZsur;bon (E2) and MZsur;bonMO (F2) embryos but present in others. (A3-F3) Ventral view of head region, presence of two hearts (cardiac bifida; arrowheads), in bon (B3) and bonMO (C3), single heart in MZsur (arrowhead in D3) and no heart in MZsur;bon (E3) and MZsur;bonMO (F3) embryos. (A4-F4) Lateral view of trunk and tail region, all embryos display normal notochords (arrow). MZsur;bon and MZsur;bonMO embryos exhibit accumulated blood (arrowhead), owing to defects in circulation. 100% of MZsur;bon embryos lacked hatching gland, heart and ventral CNS (n=41). By contrast, 81% of MZsur embryos had a heart, hatching gland and two eyes, 15% had a heart and hatching gland and fused eyes, and 3% had a heart, no hatching gland and fused eyes (n=85).

 


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  		Fig. 4. Roles of bon and sur in heart and nervous system development. Wild-type (A-C), bonMO (D-F), bon (G-I), MZsur (J-L), MZsur;bonMO (M-O) and MZsur;bon (P-R) embryos at 30 hpf. Ventral view of cardiac myosin light chain 1 (cmlc1) expression (A,D,G,J,M,P) in a single normal heart in wild-type (A), in two reduced hearts in bonMO (D) and bon (G), in a single heart in MZsur (J), but not in MZsur;bonMO (M) and MZsur;bon (P) embryos (arrows). Lateral view, anterior to the left, dorsal up, of emx1 expression in telencephalon (arrowhead in B,E,H,K,N,Q); normal emx1 expression in wild-type (B), bonMO (E), bon (H) and MZsur (K), and anterior expression in bulb-like structure in MZsur;bonMO (N) and MZsur;bon (Q). Expression of shh in head and trunk region (C,F,I,L,O,R), normal expression in wild-type (C), bon (F) and bonMO (I) but discontinuous weak expression in MZsur (L) and absence of staining in MZsur;bonMO (O) and MZsur;bon (R), indicating absence of ventral CNS. Also notice the expression in endoderm in wild-type and MZsur embryos.

 


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  		Fig. 5. Roles of bon and sur in hatching gland, heart and nervous system development. Wild-type (A,E,I), bonMO (B,F,J) MZsur (C,G,K), MZsur;bonMO (D,H,L) embryos at 5S (A-D), 20S (E-H) and 18S (I-L) stages. (A-D) Dorsal view of islet-1 expression in developing hatching gland (arrow). (E-H) Ventral view of cardiac myosin light chain 2 (cmlc2) expression in a single domain in wild-type (E), in two domains in bonMO (F), in a single domain in MZsur (G) but not in MZsur;bonMO (H). Lateral view, anterior to the left, dorsal up, of nk2.2 expression in ventral neuroectoderm; normal expression in wild-type (I) and bonMO (J), reduced anterior expression in MZsur (arrow in K), and lack of expression in MZsur;bonMO (L).

 


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  		Fig. 6. Roles of bon and sur in the development of endoderm and prechordal plate. (A-D) Dorsal view of 90% epiboly embryos. (E-L) Dorsal view of 80-90% epiboly embryos. (M-P) Dorsal view of 90% epiboly embryos. (Q-T) Lateral view of three-somite-stage embryos. (A-D) Expression of goosecoid in prechordal plate in wild-type (A), bonMO (B), MZsur (C) but not in MZsur;bonMO (D). Notice the recovery of gsc expression in MZsur at 90% (C) compared with 50% epiboly (Fig. 7C). (E-H) Expression of axial in dorsal midline and endoderm progenitors. Endodermal cells are reduced dramatically in bon (F) and MZsur;bon mutant (H) embryos but are less reduced in the MZsur mutant embryo (G). In the MZsur;bon mutant embryo (H), anterior expression corresponding to prechordal plate is missing and the remaining midline expression is compressed along the anterior-posterior axis and broadened laterally. (I-L) Expression of the endodermal marker sox17 is partially reduced in MZsur (K), strongly reduced in bon (J) and almost absent in the MZsur;bon mutant embryo (L). Expression of the endodermal marker casanova (M-P) is strongly reduced in bonMO (N), and almost absent in MZsur;bonMO (P). Notice that the expression of sox17 and casanova in dorsal forerunner cells (vegetal-dorsal expression domain) is not affected. Notochord expression of ntl is reduced and discontinuous in MZsur (S) and reduced and compressed in MZsur;bon (T).

 


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  		Fig. 7. Different regulation of Nodal target genes by bon and sur. All embryos are at 50% epiboly; animal pole view except M-P, which are lateral views. (A-D) Expression of gsc in prechordal plate progenitors is weakly reduced in bon (B), strongly reduced in MZsur (C) and absent in MZsur;bon mutant embryos (D). (E-H) Expression of bik is not affected in bon (F) but downregulated in MZsur (G) and MZsur;bon (H) mutant embryos. (I-L) Expression of mezzo is not affected in bonMO (J) but downregulated in MZsur (K) and MZsur;bonMO (L) embryos. (M-P) Expression of cas in endodermal progenitors (arrows) is strongly reduced in bon (N) and MZsur;bon (P) mutant embryos. Notice that cas expression in the yolk syncytial layer is not regulated by Nodal signaling. Expression of snail (Q-T) and flh (U-X) are not affected in any of the mutants.

 


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  		Fig. 8. Regulation of ectopic activation of Nodal target genes by bon and sur. (A-L) squint RNA was injected in different genetic backgrounds and assayed for gene activation at 50% epiboly. (A-D) gsc is induced in bon (B) and MZsur (C) but not in MZsur;bon mutants (D). (E-H) cas is induced in MZsur (G) but not in bon (F) and MZsur;bon (H) embryos. (I-L) bik is induced in bon (J) and MZsur;bon and MZsur;bon/+ (J and not shown) but not in MZsur (K) mutant embryos.

 


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  		Fig. 9. Regulation of Smad2 phosphorylation by bon and sur. Western-blot analysis of wild-type, MZsur, bonMO and MZsur;bonMO embryos at dome and shield stage. At dome stage, MZsur and MZsur;bonMO embryos have lower phosphorylated Smad2 levels but bonMO embryos are not affected. Phosphorylated-Smad2 levels recover by shield stage. Detection of actin serves as loading control.

 




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