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First published online 20 August 2003
doi: 10.1242/dev.00614

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The Mix family homeodomain gene bonnie and clyde functions with other components of the Nodal signaling pathway to regulate neural patterning in zebrafish

¹ Department of Biochemistry and Biophysics, Programs in Developmental Biology, Genetics and Human Genetics, University of California, San Francisco, San Francisco, CA 94143-0448, USA
² Abteilung für Entwicklungsbiologie, Biologie I, Universität Freiburg, Hauptstrasse 1, D-79104 Freiburg, Germany

View larger version (103K): [in a new window]   Fig. 1. bon mutant embryos exhibit anterior neural defects. (A,B) Lateral views (anterior to the left) of wild-type and bon–/– embryos at 28 hpf. Compared with wild-type siblings, bon–/– embryos show characteristic pericardial edema (arrowhead), as well as slightly smaller forebrain (brackets) and smaller eyes (arrows). (C,D) Dorsal views (anterior to the top) of otx2 expression in the presumptive forebrain and midbrain regions of wild-type and bon–/– embryos at the tailbud stage. The otx2 expression domain is smaller in bon–/– embryos. (E,F) Dorsal views (anterior to the top) of emx1 and her5 expression in wild-type and bon–/– embryos at the 1-somite stage. emx1 expression marks the anterior edge of the neural plate and her5 expression marks the midbrain-hindbrain boundary (MHB). The distance between the anterior edge of emx1 expression and the posterior tip of her5 expression (brackets) is reduced by about 10% in bon–/– embryos as compared with wild-type siblings. These anterior neural plate phenotypes (shown in D and F) segregated completely with the bon mutation, as assessed by genotyping.

View larger version (78K): [in a new window]   Fig. 2. bon is required in the axial mesoderm for neural patterning. Restricted injections of bon MO into a single cell at the 32-cell stage result in tissue specific knockdown of Bon function. Restriction of bon MO was determined by antibody staining for the fluorescein moiety conjugated to the MO (A-C) or by localization of co-injected 10 kDa rhodamine-dextran (D-F). (A-C) Lateral views (dorsal to the right) of otx2 expression in bon MO-injected embryos at the tailbud stage. Embryos with restriction to the axial mesoderm (n=25; A), lateral mesoderm (n=3; B) and ventral mesoderm (n=10; C) are shown. Arrowheads point to the localization of the bon MOs, whereas arrows mark the area of otx2 expression. Only embryos with bon MOs in the axial mesoderm showed a reduction of the otx2-expression domain (A). (D-F'') Lateral views of bon MO-injected embryos at 80% epiboly (D-F) and 28 hpf (D'-F''). The same embryos were followed and examined at 80% epiboly (D-F), 28 hpf for bon-MO restriction (D'-F') and morphological defects in head formation (D''-F''). (D,D',D'') Embryos with bon MOs in axial mesoderm, derivatives of which populate the notochord (white arrowhead) and head mesenchyme (white arrow), exhibited anterior defects, with a reduction in eye size (black arrow) being most prominent (n=27). Embryos with bon MO in non-axial tissues, such as ventral mesoderm (n=5; E) and neural ectoderm (n=2; F), exhibited no defects in neural development (E'',F''). Head size was determined on individual embryos by measuring the distance from the MHB to the tip of the telencephalon at 28 hpf. This distance was 272±8 µm in embryos with axial mesoderm restriction of the bon MO (n=27), and 300±12 µm in wild-type embryos or those with neuroectoderm or ventral mesoderm morpholino restriction (n=7).

View larger version (118K): [in a new window]   Fig. 3. bon mutant embryos exhibit defects in anterior axial mesoderm gene expression. Whole-mount in situ hybridization analyses at the shield stage (A), and at 50% (D,G) and 90% (B,C,E,F,H,I) epiboly, showing dorsal views (A-C; anterior to the top), animal pole views (D-F; D, dorsal to the right; E,F, anterior to the top) and lateral views (G-I; dorsal to the right). (A) At the shield stage, wild-type and bon–/– embryos show indistinguishable gsc expression. (B,C) At 90% epiboly, the gsc expression domain is reduced in bon–/– embryos as compared with wildtype. (D,G) At 50% epiboly, wild-type and bon–/– embryos show indistinguishable bmp4 expression. Arrowheads point to the dorsal bmp4 expression domain. (H,I) At 90% epiboly, wild-type and bon–/– embryos show a wild-type pattern of ventrolateral bmp4 expression, but (E,F) the anterior axial mesoderm bmp4 expression domain is dramatically reduced in bon–/– embryos (arrows). These phenotypes segregated completely with the bon mutation, as assessed by genotyping.

View larger version (117K): [in a new window]   Fig. 4. bon interacts with sqt to regulate neural patterning. Nomarski images at 30 hpf (A-D) and whole-mount in situ hybridization analyses at 1-somite (E-L) and tailbud stages (M-P), showing lateral (A-H; A-D, anterior to the left; E-H, dorsal to the right) and animal pole views (I-P; anterior to the top). Compared with wild-type siblings (A), bon–/– embryos (B) have severe pericardial edema (arrowhead) and smaller forebrain structures (arrow), sqt–/– and some bon+/–;sqt+/– embryos (C) are cyclopic, and bon–/–;sqt–/– embryos (D) have severe pericardial edema (arrowhead) and lack anterior structures (arrow). (E-L) Whole-mount in situ hybridization analyses with emx1 and krox20 at the 1-somite stage. At the 1-somite stage, emx1 marks the anterior boundary of the neural plate and krox20 rhombomeres 3 and 5 (r3 and r5). In bon–/– embryos (F,J), the distance between the anterior neural ridge (emx1) and the r5/r6 boundary is reduced (brackets), and the distance between r3 and r5 is also reduced. The lateral borders of the emx1-expression domain (asterisks) are also shifted medially in bon–/– embryos (J). In sqt–/– or bon+/–;sqt+/– (G) and bon–/–;sqt–/– (H) embryos, the reduction in the distance between the anterior edge of emx1 expression and the r5/r6 boundary (brackets) is more pronounced. In addition, instead of outlining the neural plate, emx1 expression spreads medially throughout the entire area of the anterior ventral neural plate in sqt–/– or bon+/–;sqt+/– (K) and bon–/–;sqt–/– (L) embryos. This expansion does not appear to be an expansion of anterior neural fates as otx2-expression domains are reduced in sqt–/– or bon+/–;sqt+/– (O) and bon–/–;sqt–/– (P) embryos, when compared with either wild-type (M) or bon–/– embryos (N). These neural patterning defects segregated completely with the respective bon, sqt and bon;sqt mutations, as assessed by genotyping.

View larger version (103K): [in a new window]   Fig. 5. bon and sqt function in parallel to regulate mesendodermal gene expression. Whole-mount in situ hybridization analyses of dkk1 (A-H) and ntl (I-L) expression, showing animal pole (A-D,I-L; dorsal to the right) and dorsal views (E-H; anterior to the top). At 50% epiboly, dkk1 expression is seen in all marginal blastomeres in wild-type embryos (A). In bon–/– embryos, dkk1 expression exhibits a slight dorsal gap (B). In sqt–/– or bon+/–;sqt+/– embryos, this dorsal gap appears more extensive (C). In bon–/–;sqt–/– embryos, dkk1 expression is seen only in the ventral half of the margin (D). At 70% epiboly, dkk1 is expressed in cells of the PCP in wild-type embryos (E). In bon–/– embryos, the dkk1-expressing cells appear to coalesce aberrantly (F). In sqt–/– or bon+/–;sqt+/– embryos, dkk1 expression in the PCP is dramatically reduced (G). In bon–/–;sqt–/– embryos, dkk1 expression appears to be completely absent (H). At 50% epiboly in wild-type and bon–/– embryos, ntl is expressed around the margin of the embryo (I,J). In sqt–/– embryos, ntl expression appears reduced around the entire margin (K). In bon–/–;sqt–/– embryos, ntl expression appears reduced around the margin and is absent from the dorsal side (L). The dkk1 and ntl expression defects segregated completely with the respective bon, sqt and bon;sqt mutations, as assessed by genotyping.

View larger version (103K): [in a new window]   Fig. 6. bon interacts with sur (foxh1) to regulate mesendodermal gene expression and neural patterning. Nomarski images at 28 hpf (A,B) and whole-mount in situ hybridization analyses at 50% (C,D,K,L) and 70% epiboly (G,H), and at the tailbud stage (E,F,I,J). A,B and E,F are lateral views (A,B, anterior to the left; E,F, dorsal to the right); C,D and I-L are animal pole views (C,D,K,L, dorsal to the right; I,J, anterior to the top); and G,H are dorsal views (anterior to the top). Compared with wild-type siblings (A), bon–/–;sur–/– embryos (B) have severe pericardial edema (arrowhead) and lack anterior structures (arrow). (E,F,I,J) Whole-mount in situ hybridization analyses with emx1 and krox20 at the tailbud stage. At the tailbud stage, the distance between the anterior neural ridge (emx1) and the r5/r6 boundary (brackets) is dramatically reduced in bon–/–;sur–/– embryos (F), similar to that observed in bon–/–;sqt–/– embryos (Fig. 4h). In addition, emx1 expression is also expanded medially in bon–/–;sur–/– embryos (J). At 50% epiboly, dkk1 expression is seen in all marginal blastomeres in wild-type embryos (C), whereas in bon–/–;sur–/– embryos it exhibits a dorsal gap (D). At 70% epiboly, dkk1 is clearly expressed in cells of the PCP of wild-type embryos (G), whereas in bon–/–;sur–/– embryos it is dramatically reduced (H). At 50% epiboly, ntl is expressed around the margin of the embryo (K), whereas in bon–/–;sur–/– embryos it is absent from the dorsal side (L). These neural patterning and mesendodermal gene expression defects segregated completely with the bon;sur mutations, as assessed by genotyping.

View larger version (18K): [in a new window]   Fig. 7. A model for the genetic network of Nodal signaling. Combining our results with biochemical (Germain et al., 2000) and molecular epistasis data (Alexander et al., 1999), a model emerges in which the Nodal signal provided by Sqt is transduced by a Smad2/Smad4 complex. In endoderm formation, Bon functions downstream of Nodal signaling. The identity of the transcriptional mediator (Y) of Nodal signaling regulating bon expression is not known. The genetic interactions between bon;sqt and bon;sur indicate that Bon also functions in parallel to Sqt and Sur (Foxh1) to regulate mesendodermal target genes, such as dkk1 and ntl. These genes in turn regulate neural patterning. The more than additive defects seen in bon–/–;sqt–/– and bon–/–;sur–/– embryos, which are not seen in MZsqt–/– embryos, suggest that an additional, as yet unidentified, factor (X) may be involved in this network, regulating Bon function at least. Whether factor X regulates Bon function through Smad activation remains to be determined.

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