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doi: 10.1242/10.1242/dev.00572

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vhnf1 and Fgf signals synergize to specify rhombomere identity in the zebrafish hindbrain

¹ Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA
² Massachusetts Institute of Technology, Cambridge MA, USA

View larger version (110K): [in a new window]   Fig. 3. Fgf function is epistatic to vhnf1 function in the posterior hindbrain. (A-D) 18-somite stage embryos, dorsal view, anterior to the left. (A,B) hoxb1a expression is normally limited to the presumptive r4 domain (A) but is expanded to the posterior in vhnf1 mutant embryos (12/47 embryos) (B). (C,D) Morpholino oligos (MOs) directed against both fgf3 and fgf8 injected into embryos derived from vhnf1 heterozygous parents. Most (37/40) embryos show hoxb1a expression limited to the r4 domain (C). Expression of hoxB4 (purple) and krox20 (orange); all (56/56) embryos show no anterior expansion of hoxB4 expression (D). (E-G) One-somite-stage embryos, dorsal view, anterior to the top. (E,F) Injection of MOs directed against fgf8+fgf3 does not affect vhnf1 expression (E), although is sufficient to inhibit r5 krox20 expression in sibling embryos (F). (G) Clutches of embryos produced by vhnf1 heterozyogous parents show consistent expression of fgf8 in all embryos.

View larger version (70K): [in a new window]   Fig. 5. vhnf1 functions as a repressor of anterior identity independently of Fgf and val functions. (A-H) Overexpression of vhnf1 marked by co-injected lacZ (light blue stain). Panels labeled lacZ have only lacZ RNA injected. Dorsal view, anterior to the top. (A-F) hoxb1a expression (purple) and krox20 expression (orange) at six-somite stage. (A-C) vhnf1 does not affect hoxb1a expression at 100% epiboly (A) but represses hoxb1a expression (B). krox20 expression marks future r3 and r5 domains (orange). (C) When lacZ alone is injected, no repression of hoxb1a is observed. (D) Injection of vhnf1 into valentino (val) mutant embryos results in repression of hoxb1a expression at the six-somite stage and repression of the anterior (r3) krox20 expression. (E) Co-injection of fgf3 and fgf8-targeted morpholino oligos with vhnf1 RNA results in repression of hoxb1a expression. (F) Coinjection of fgf3 and fgf8 MOs with lacZ RNA has no effect on hoxb1a expression. (G,H) Expression of fgf8 RNA at tailbud stage. Injection of vhnf1 represses the anterior hindbrain expression of fgf8 (G), whereas injection of lacZ alone has no effect on fgf8 expression (H).

View larger version (76K): [in a new window]   Fig. 1. Loss of vhnf1 function results in transformation of posterior hindbrain to r4 identity. (A-F) 14-somite stage embryos. (A,B) hoxb1a gene expression (purple) is limited to the future r4 domain in a wild-type background and krox20 expression (orange) is present in presumptive r3 and r5 (A). hoxb1a transcripts are expanded throughout the posterior hindbrain in vhnf1 mutants, whereas r5-specific krox20 expression is reduced or absent in vhnf1 mutants (B). (C,D) hoxb3 expression (purple) is present in future r5 and r6 in the wild-type background (C), but is not expressed in vhnf1 mutants (D). (E,F) hoxB4 expression (orange) has an anterior boundary of expression at the future r6/r7 boundary and occurs throughout the anterior spinal column in wild-type (E); myod expression (posterior purple stain) identifies the mesoderm underlying the spinal column (s1=somite 1) and krox20 shows r3 and r5 (purple). In vhnf1 mutants, the anterior boundary of hoxB4 expression is indistinct and posteriorized (F). (G-J) One-somite-stage embryos. (G,H) valentino expression in presumptive r5 and r6 in wild-type embryos (G) is missing in vhnf1 mutants (H). (I,J) The r5 stripe of krox20 expression is present in wild-type embryos (I) but is severely reduced in vhnf1 mutants (J). (K,L) Reticulospinal neurons visualized in 48 hour embryos using anti-neurofilament (RMO44) antibody. Mauthner neurons (arrowheads) are limited to the single r4-derived pair in wild-type embryos (K) but appear in additional, posterior locations in vhnf1 mutants (red arrowheads) (L). (A-F) Dorsal view, anterior to the left. (G-L) Dorsal view, anterior to the top.

View larger version (95K): [in a new window]   Fig. 2. Early expression of vhnf1 in the posterior hindbrain. (A) Co-localization of hoxb1b (orange) and vhnf1 (purple). At 100% epiboly, the anterior boundary of vhnf1 expression is posterior to that of hoxb1b. (B) Co-localization of fgf8 (orange) and vhnf1 (purple). At 100% epiboly, fgf8 expression is apparent in the presumptive anterior hindbrain (arrowhead) and in the germ ring (arrow), and there are cells between the posterior boundary of fgf8 and the anterior boundary of vhnf1 that express neither gene. (C) Co-localization of fgf8 (purple) and hoxb1b (orange). At 100% epiboly, fgf8 and hoxb1b domains of expression are adjacent. (D) Co-localization of vhnf1 (orange) and krox20 (purple). At the one-somite stage, vhnf1 expression overlaps the posterior half of r5 krox20. (E,F) Co-localization of vhnf1 (purple) and hoxb1a (orange). At tailbud stage (E), the anterior boundary of hoxb1a expression lies anterior to that of vhnf1 but hoxb1a expression overlaps that of vhnf1 significantly throughout the posterior region. By the two-somite stage (F), hoxb1a expression has resolved to a single stripe and there is no overlap with vhnf1. (A-F) All embryos are dorsal view, anterior to the top. (G) Summary of expression data, anterior to the left.

View larger version (78K): [in a new window]   Fig. 4. The combination of vhnf1 expression and Fgf signal is sufficient to activate posterior hindbrain gene expression. (A) The experimental method consisted of injection of the indicated RNA into one cell at the two-cell stage, then implantation of a protein-coated bead after the shield stage. Embryos were aged to about the three-somite stage and fixed for analysis. (B,C) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead resulted in significant induction of krox20 expression both within the neural plate (B) and in lateral ectoderm (C). Arrowhead indicates the location of endogenous krox20 expression. (D) Injection of vhnf1 RNA and implantation of a BSA-coated bead does not induce krox20 expression. (E) The Fgf8-coated bead alone is not sufficient to induce ectopic krox20 expression. (F) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead induces valentino (val) expression. Arrowhead indicates the location of endogenous val expression. (G) Injection of vhnf1 RNA into a val mutant embryo (no endogenous r5 krox20 expression) and implantation of an Fgf8-coated bead does not induce krox20 expression. This dorsal view of the neural plate shows that r3 krox20 is repressed by vhnf1. Arrowhead indicates endogenous r3 krox20 on the uninjected side. (H) Injection of val RNA and implantation of a Fgf8-coated bead is not sufficient for induction of krox20 expression. (I) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead results in localized repression of hoxb1a expression. (J) Injection of vhnf1 RNA and implantation of a Fgf8-coated bead does not induce expression of the axial mesoderm marker no tail (ntl). (K) Summary of data. The combination of Fgf8+vhnf1 is sufficient to induce val and krox20 expression, and val function is required along with other Fgf+vhnf1-inducible factor(s) (X) for krox20 induction.

View larger version (79K): [in a new window]   Fig. 6. valentino (val) RNA is sufficient to recover some posterior hindbrain identity in vhnf1 mutants. (A-F) Injection of val+lacZ RNA or lacZ alone. Embryos are 12-somite stage. (A) Injection of val RNA into wild-type embryos has no effect on endogenous krox20 expression. (B) Injection of val RNA into vhnf1 embryos recovers r5 krox20 expression (arrow) within the injected cells in 7/8 mutant embryos. (C) Injection of lacZ RNA alone is not sufficient to recover krox20 expression. (D) Injection of val RNA into wild-type embryos at the 12-somite stage does not affect hoxb3 expression (arrow). (E) Injection of val RNA into vhnf1 mutant embryos recovers some hoxb3 expression in 5/14 embryos. (F) Injection of lacZ RNA alone does not rescue hoxb3 expression.

View larger version (26K): [in a new window]   Fig. 7. Model for the method by which vhnf1 functions to generate rhombomere-specific identity. (A) Summary of the data. rX represents the combined r5+r6 domain, a partially differentiated rhombomere that develops in val mutants. rX identity is distinct from rP, the unspecified tissue that remains when Fgf signals are reduced. (B) Model of the role of vhnf1 in generation of rhombomere identity. After hoxb1a, fgf8 and vhnf1 gene expression is established, vhnf1 functions to repress hoxb1a throughout the domain in which vhnf1 is expressed. In addition, Fgf signals from the anterior are received by posterior hindbrain cells, and the combination of Fgf signal transduction and vhnf1 function result in activation of val expression within the domain in which the overlap occurs. val function is then sufficient to activate krox20 expression, although this activation is limited to the presumptive r5 domain, suggesting that other factors limit this activation outside of r5 or promote it within r5.

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