Ishioka et al., 2011 - Retinoic acid-dependent establishment of positional information in the hindbrain was conserved during vertebrate evolution. Developmental Biology   350(1):154-168 Full text @ Dev. Biol.

Fig. 4 Regulatory function of the hoxb1b upstream DNA during somitogenesis. (A–F) Expression of hoxb1b mRNA from 80% epiboly to the 20-somite stage (20-s). (G–J) Expression of 5′hoxGFP mRNA in Tg embryos from the bud stage to 30 hpf. (K–N) Expression of 5′hoxGFP (K), hoxb1b (L), hoxb5b (M), and hoxb6b (N) was compared with krox20 expression in r3 and r5 by in situ hybridization. (O–S) Transient (O) and stable (P-S) expression of 5′hoxGFP was visualized by fluorescence from 4-somite stage through 28 hpf. (A–C, E–N, S) Dorsal views with anterior to the top (A–C, E–I, K–N) or to the left (J, S). (D, O–R) Lateral views with anterior to the left and dorsal to the top. The anterior expression boundaries of hoxb1b and 5′hoxGFP are shown with open arrowheads, whereas those of hoxb5b and hoxb6b are shown with black arrowheads. Transgene expression in the posterior pharyngeal arches, diencephalon, MHB, and otic vesicles is shown with black/white thin arrows, yellow thin arrows, open circles, and asterisks, respectively. Scale, 200 μm.

Fig. 5 Early expression of hoxb1b in the posterior neural plate is driven by the downstream DNA. (A–D) Expression of 3′5′hoxGFP was visualized by WMISH using DIG-labeled probe in injected embryos (transient expression, A) or in Tg embryos (B–D). (E–P) Expression of 3′5′hoxGFP was detected as fluorescence under a stereomicroscope (E–L) or by confocal microscopy (M–P) in injected embryos (E) or in Tg embryos (F–P). (M-O) Expression of krox20 in r3 and r5 (M) was detected in a Tg embryo expressing 3′5′hoxGFP (N). Their merged image (O) shows that the anterior expression boundary of 3′5′hoxGFP coincides with the r3/r4 boundary. (A–D, H, K, M–O) Dorsal views with anterior to the top (A–D, H, M–O) or to the left (K). (E–G, I, J, L, P) Lateral views with anterior to the left. Anterior expression boundaries of 3′5′hoxGFP are shown with open arrowheads. The expression in the posterior hindbrain (F), pharyngeal arches, notochord, MHB otic vesicles, and diencephalon (J, L) are shown with a curve, white thin arrows, open thick arrows, open circles, asterisks, and yellow thin arrows, respectively. Scale, 200 μm.

Fig. 6 Downstream region is responsible for the RA-mediated regulation of hoxb1b. (A–H) Wild-type embryos (A, B, E, F) or 3′5′hoxGFP Tg embryos (C, D, G, H) were untreated (A, C, E, G), treated with RA at the shield stage (B, D), or treated with DEAB at the shield stage (F, H), and cultured until 80% epiboly, when the expression of hoxb1b (A, B, E, F) or 3′5′hoxGFP (egfp) (C, D, G, H) was examined by WMISH. Numbers of embryos showing the expression pattern represented by the photos vs. numbers of stained embryos are shown at the bottom-right. (I–L) 3′5′hoxGFP Tg embryos (I, J) or 5′hoxGFP Tg embryos (K, L) were untreated (I, K) or treated (J, L) with RA at the shield stage and allowed to develop to the 8-somite stage, when GFP expression was observed. (M–P) 3′5′hoxGFP Tg embryos were untreated (M) or treated with RA during the specified periods (N–P) and observed for fluorescence at the 8-somite stage. (Q–T) 3′5′hoxGFP Tg embryos were untreated (Q, S) or treated (R, T) with DEAB for 3 h and examined for fluorescence at the specified stages. RA-induced reporter expression in the polster was also observed for the RARE from RARβ-2 (Perz-Edwards et al., 2001), suggesting that this ectopic expression is inherent in the function of the RARE. Anterior expression boundaries of the transgene are marked with open arrowheads. (A–H, Q–T) Dorsal views with anterior to the top. (I–P) Lateral views with anterior to the left. p, polster; scale bars, 200 μm.

Fig. 7 Downstream region mediates the Wnt and Fgf signal in hoxb1b regulation. Wild-type embryos (A, B, E, F, I, J) or 3′5′hoxGFP Tg embryos (C, D, G, H, K, L) were treated with LiCl as specified (A–D) or injected with mRNA for the specified genes (E–H, 150 pg/embryo; I–L, 75 pg/embryo) (E–L), and examined for 3′5′hoxGFP mRNA expression by WMISH. Anterior expression boundaries of the transgene are marked with open arrowheads. Numbers of embryos showing the expression pattern represented by the photos vs. numbers of stained embryos are shown at the bottom-right. Scale, 200 μm.

Fig. 8 Localization of the regulatory activities in the downstream region of hoxb1b. The downstream 4.6-kb DNA (A, B) or its subfragments (D–L) were co-injected with 5′hoxGFP DNA (D–J) or hsp-GFP (A, B, K, L) into embryos, which were observed at 80% epiboly (A), bud stage (L), or 5–8 somite stages (B–K). When 5′hoxGFP (C) or hsp-GFP (not shown) was injected alone, little expression was observed at this stage. Anterior expression boundaries of the transgene are marked with open arrowheads (B) The ectopic expression sometimes seen in the mesendoderm is marked with an asterisk.

Fig. 10 RARE sequences in the downstream region mediate the RA signal in the regulation of hoxb1b. (A) In F1-GFP and F4-GFP, F1 and F4 DNA were placed downstream to the egfp gene, respectively. The RAREs in these downstream subregions were deleted from the constructs by inverse PCR, giving rise to F1ΔR-GFP and F4ΔR-GFP. (B–I) Transient expression of GFP fluorescence in injected embryos at the specified stages. (D, E, H, I) The percentages of embryos showing expression in the posterior neural pate and the numbers of scored embryos (n) are shown at the bottom-right. The blastoderm margin, anterior boundary of reporter expression, and anterior end of the head are shown with dashed lines (B, F), open arrowheads (C, G), and open arrows (C, G), respectively. Scale, 200 μm.

Fig. 11 Electrophoretic mobility shift assay showing specific binding of RAR/RXR with the two RAREs in the downstream region of hoxb1b. (A) The DIG-labeled oligonucleotides containing 3′-RARE1 and 3′-RARE3 (D1R and D4R, respectively) were incubated with zebrafish RAR/RXR in the absence or presence of 200-molar excess of competitors (D1R, D4R, and RfR), and run on a 5%-polyacrylamide gel. (B) DIG-labeled D1R and D4R probes were incubated with RAR/RXR in the absence or presence of 200-molar excess cognate oligonucleotides (D1R, D4R) or their mutated versions (D1m, D4m). (C) Sequences of the oligonucleotides used as probes or competitors. The RARE sequences are marked with underlines, and base substitutions are shown in lower case.

Acknowledgments:
ZFIN wishes to thank the journal Developmental Biology for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

Reprinted from Developmental Biology, 350(1), Ishioka, A., Jindo, T., Kawanabe, T., Hatta, K., Parvin, M.S., Nikaido, M., Kuroyanagi, Y., Takeda, H., and Yamasu, K., Retinoic acid-dependent establishment of positional information in the hindbrain was conserved during vertebrate evolution, 154-168, Copyright (2011) with permission from Elsevier. Full text @ Dev. Biol.