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First published online September 1, 2004
doi: 10.1242/10.1242/dev.01297

Development 131, 4511-4520 (2004)
Published by The Company of Biologists 2004
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vhnf1 integrates global RA patterning and local FGF signals to direct posterior hindbrain development in zebrafish

Rafael E. Hernandez1,2, Holly A. Rikhof1, Ruxandra Bachmann1,* and Cecilia B. Moens1,{dagger}

1 Howard Hughes Medical Institute and Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Seattle, WA 98109, USA
2 Medical Scientist Training Program and Molecular and Cellular Biology Program, University of Washington, Seattle, WA 98195, USA



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  		Fig. 1. vhnf1 acts downstream of RA to activate val. (A,B) vhnf1 (blue) is expressed posterior to the r4/5 boundary at about 10.7 hpf in control EtOH-treated embryos (A, krox20 expression in r3 and r5 is in red), and is expanded in its AP extent in the hindbrain of embryos treated with 10–7 M all-trans RA (B). (C,D) Treating embryos with 10–5 M AGN193109, a pan-RAR antagonist, from 4.5-11 hpf blocks vhnf1 expression and r5 specification (D) compared with DMSO-treated controls (C). (E,F) Wild-type embryos (E) exhibit robust vhnf1 expression by 9.5-10 hpf compared with their nls– siblings (F). (G-J) Embryos were treated with 2% DMSO or 10–5 M AGN193109, either alone or in combination with overexpression of vhnf1 (35 pg mRNA) as shown. (G) DMSO-treated controls show normal val expression (blue) in r5 and r6 at approximately 11.7 hpf and this expression is inhibited by treatment with AGN 193109 (H). Overexpression of vhnf1 causes a slight expansion of val expression in untreated embryos (I) and rescues val expression in AGN193109-treated embryos (J). A-F are shown as dorsal views with anterior to the left (A-D) or to the top (E,F).

 


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  		Fig. 2. vhnf1 and Fgfs cooperate to drive val expression. (A-C) vhnf1 requires Fgfs to drive val expression in the hindbrain. At 18 hpf val (blue) is normally expressed in r5 and r6 (A); krox20 expression in r3 and r5 and eng3 at the mid-hindbrain boundary (MHB) is in red. After injection of vhnf1 mRNA (50 pg), val expression expands anteriorly to approximately the level of r2 (B). By contrast, vhnf1 overexpression in fgf3–;fgf8– embryos does not drive val expression (C). (D-I) vhnf1 and fgfs cooperate to drive val expression. val (blue) is not normally expressed at 8.25 hpf (D), and injection of fgf3 mRNA (25 pg) alone is not sufficient to induce val (E). vhnf1 mRNA alone will induce a low level of val (F), while vhnf1 and fgf3 together induce val at a high level (G). Injection of noggin-gfp mRNA (20 pg) causes a dorsalization similar to fgf3 but when injected alone (H) or with vhnf1 mRNA (I) it does not induce val. (D'-I') Val protein is upregulated similarly to transcript levels at 8.25 hpf following mRNA injection (as in D-I), as detected by anti-Val immunoblot of lysed embryos. (J,K) Overexpression of caMek mRNA (20 pg) alone does not induce val expression (J) but like fgf3 can cooperate with vhnf1 to do so (K). (L,M) Robust upregulation of val downstream of fgf3 and vhnf1 requires val autoregulation. 12 hpf val+/+ and val+/– embryos expressing vhnf1 and fgf3 exhibit robust val expression (L), while little or no val is detected in val–/– embryos (M). A-C, are dorsal views with anterior to the left. D-M are optical cross sections near the dorsal midline.

 


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  		Fig. 3. Vhnf1 participates in a multistep process to repress r4 identity in r5 and r6. Wild-type (wt; left column), val– (middle column) and vhnf1– (right column). (A-C) krox20 (blue) in r3 and r5 of wt (A), with a small dorsal stripe in the r5 territory of val– embryos (B) compared with the variable dorsal and ventral expression in vhnf1– embryos (C). (D-L) Repression of hoxb1a (blue) in r5 and r6 requires multiple genes. hoxb1a is initially downregulated posterior to r4 (asterisks) at 10.7 hpf in wt (D), val– (E) and vhnf1– (F) embryos. By 11.7 hpf hoxb1a expression is largely restricted to r4 in wt (G) and val– (H) embryos but is upregulated posterior to r4 in vhnf1 mutants (I). At 18-20 hpf hoxb1a is clearly expanded in vhnf1 mutants (L) and mildly expanded in val mutants (K) compared with wt (J). (M-R) fgf3 (blue) expression is similar between wt (M), val– (N) and vhnf1– (O) embryos at 10.5 hpf. By 11.7 hpf fgf3 is normally only highly expressed in r4 (P), while in vhnf1 mutants (R) its expression expands posteriorly. (S-U) Expression of the RA-metabolizing enzyme cyp26b1 is restricted to r4 and r3 at 12 hpf in wt (S) val– (T) and vhnf1– (U) embryos. Expression of fgf8 is also limited to r4 and anterior by 12 hpf in wt (V) val– (W) and vhnf1– (X) embryos. (Y-AA) A single pair of Mauthner cells is present in r4 of wt (Y) and val– (Z) embryos, while supernumerary Mauthner cells are detected in 58% of vhnf1– embryos (AA; arrowhead). A-C are lateral views with anterior to the left and dorsal to the top. D-AA are dorsal views with anterior to the left. krox20 expression in r3 and r5 is in red (D-X). The mid-hindbrain boundary (MHB) is marked by pax2.1 (blue, J-L; red, M-O) or en3 (red, P-R).

 


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  		Fig. 4. vhnf1 and val mutant cells behave equivalently in genetic mosaics. (A) Donor-derived cells (brown) from a wild-type embryo contribute throughout the hindbrain of a wild-type host embryo. Krox20 expression in r3 and r5 is in blue. (B) vhnf1– cells are excluded from r5 and r6 of wild-type host embryos. (C) Wild-type cells form tight aggregates (arrowheads) in the r5-6 region of vhnf1– host embryos. These wild-type-derived cells express krox20 when in the r5 region. (D) vhnf1– cells were able to contribute throughout the hindbrain of val– host embryos, including the presumptive r5-6 region (bracket), and conversely (E) val– cells contributed to the entire hindbrain of vhnf1– host embryos. Cell behaviors in genetic mosaics are consistent with similar changes in Eph and ephrin expression in val and vhnf1 mutant embryos. (F,I) Wild-type (wt); (G,J) val–; (H,K) vhnf1–. (F-H) At 11.6 hpf, r5 and r6 expression of EphB4a (blue) is normally expressed in r2, r3, r5 and r6 of the hindbrain (F). The expression in r5 and r6 is strongly reduced in both val– (G) and vhnf1– (H) embryos. (I-K) ephrin-B2a (blue) is normally expressed in r1, r4 and r7 at 12 hpf (I). Its expression is upregulated posterior to r4 in val– (J) and vhnf1– (K) embryos. krox20 expression in r3 and r5 is in purple (F-H) or red (I-K). Embryos are shown in dorsal views with anterior to the left.

 


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  		Fig. 5. When overexpressed, vhnf1 requires val for its ability to repress both hoxb1a and ephrin-B2a in r4. Overexpression of vhnf1 by mRNA injection (25 pg) represses hoxb1a (blue) in r4 of wild-type (B) but not val mutant embryos (D). Similarly, injection of vhnf1 mRNA represses ephrin-B2a in r4 of wild-type (F) but not val mutant embryos (H). Dorsal views of 11.5-13 hpf embryos, anterior to the left, with krox20 in red.

 


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  		Fig. 6. A model for the functions of vhnf1 and val in directing hindbrain development. RA activates hoxb1b and hoxb1a expression to initiate an r4 development program, including the specification of Mauthner neurons. An unknown factor, `X', initially represses hoxb1a in r5 and r6, independent of vhnf1. RA activates vhnf1 expression, which reinforces repression of hoxb1a expression in r5 and r6, possibly in cooperation with an unknown co-factor, which may or may not be the same `X' above. Repression of hoxb1a by Vhnf1 blocks acquisition of r4 neuronal fates in r5 and r6, but Vhnf1 must act through Val to drive r5-6 neuronal development, including abducens cranial motor neurons (CMNs). Furthermore, Val is required for the acquisition of r5-6 cell-surface characteristics by both activating r5-6 EphB4a expression and repressing r4-like ephrin-B2a expression in r5 and r6. Finally, Val contributes to the maintenance of hoxb1a repression at later stages, possibly through activation of hox group 3 genes.

 

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© The Company of Biologists Ltd 2004