Kurokawa D et al. (2010), Evolutionary origin of the Otx2 enhancer for it...

XB-ART-50013

Dev Biol 2010 Jun 01;3421:110-20. doi: 10.1016/j.ydbio.2010.03.013.

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Evolutionary origin of the Otx2 enhancer for its expression in visceral endoderm.

Kurokawa D , Ohmura T , Ogino H , Takeuchi M , Inoue A , Inoue F , Suda Y , Aizawa S .

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In the mouse, the Otx2 gene has been shown to play essential roles in the visceral endoderm during anterior-posterior axis formation and head induction. While these are primary processes in vertebrate embryogenesis, the visceral endoderm is a tissue unique to mammals. Two enhancers (VE and CM) have been previously found to direct Otx2 expression during early embryogenesis. This study demonstrates that in anterior visceral endoderm the CM enhancer does not have an activity by itself, but enhances the activity of the VE enhancer. These two enhancers also cooperate for the activities in anterior mesendoderm and cephalic mesenchyme. Comparative studies suggest that VE enhancer function was most likely established before the divergence of sarcopterygians into Actinistia, Dipnoi and tetrapods, while the nucleotide sequence corresponding to the VE enhancer was already present in the last common ancestor of bony fishes. The CM enhancer sequence and function would have been also established in ancestral sarcopterygians. The VE/CM enhancers and their gene cascades in the ancestral sarcopterygian head organizer would then have been co-opted by amphibian deep endoderm cells and mammalian visceral endoderm cells for the head development.

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Species referenced: Xenopus
Genes referenced: cer1 chrd dkk1 fgf8 foxa2 foxa4 lhx1 otx2 wnt3
GO keywords: endoderm development [+]

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	Fig. 1. Enhancer activity of the 51 bp VE and 49 bp CM sequences. (a–f) β-Gal expression directed by the α1 region of the mouse Otx2 gene in the AVE at E6.5 (a, b), in the AME at E7.5 (c, d) and in the CM at E8.5 (e) and E9.5 (f). We previously reported that panels (e) and (f) represent expression in cephalic neural crest cells at the migratory phase; the activity is lost when the cells settle (Matsuo et al., 1995, Kimura et al., 1997). (g–l) β-Gal expression in the AVE at E6.5 directed by the mouse Otx2 (− 884 to − 1) fragment (g), the (− 835 to − 1) fragment (h), the (− 594 to − 1) fragment (i), the (− 543 to − 1) fragment (j), the 1.8 kb α1 fragment that lacks the CM sequence (k) and the 1.8 kb α1 fragment that lacks the VE sequence (l). (m–s) β-Gal expression in the AME at E7.5 directed by the (− 884 to − 1) fragment (m), the (− 835 to − 1) fragment (n), the (− 594 to − 1) fragment (o), the (− 543 to − 1) fragment (p), the 1.8 kb α1 fragment that lacks the CM sequence (q), the 1.8 kb α1 fragment that lacks the VE sequence (r) and the 1.8 kb α1fragment that lacks the CM and VE sequences (s). (t–v) β-Gal expression in the CM at E8.5 directed by the 1.8 kb α1 region that lacks the CM sequence (t), the 1.8 kb α1region that lacks the VE sequence (u) and 1.8 kb α1 region that lacks the VE and CM sequences (v). (w) A schematic diagram of the constructs used for the above experiments. Panels (a), (e–l) and (t–v) show lateral whole mount views (anterior is to the left). Panels (c) and (m–s) show frontal whole mount views, while panels (b) and (d) show horizontal and sagittal sections, respectively. The number of β-Gal-positive embryos per total number of transgenic embryos generated is indicated in each panel. The arrowheads indicate weak activity in the AME (n, o, q, r) or CM (u). The arrows indicate weak activity in the heart (f, t) (Kimura et al., 1997). Abbreviations: AME, anterior mesendoderm; ANE, anterior neuroectoderm; AVE, anterior visceral endoderm; EP, epiblast.
	Fig. 2. Conservation of the VE and CM sequences among vertebrate Otx2 orthologs. (A) A vista plot of each jawed vertebrate Otx2 and lamprey OtxA α1 region aligned against the mouse α1 sequence. (B) The VE sequence in the vertebrate Otx2 genes. (C) The CM sequence in the sarcopterygian Otx2 genes. The numbers give the location of the sequences upstream of the Otx2 genes; the numbers in parenthesis show the percentage of the nucleotides identical to the mouse sequences. Sequences weakly conserved in skate do not overlap with the 51 bp VE sequence.
	Fig. 3. Enhancer activity of the α1 region of amniote Otx2 genes. β-Gal expression directed by the chicken α1 region (a–f), the mouse α1 region in which the VE sequence is replaced with the chicken VE sequence (g–i), the soft-shelled turtle α1 region (j–o) and the soft-shelled turtle α1 region that lacks the VE sequence(p–r) in the mouse AVE at E6.5 (a, g, j, k and p); the mouse AME at E7.5 (b, c, h, l, m and q); and the mouse CM at E8.5 (d, i, n and r), E8.75 (e) and at E9.5 (f and o). Panels (a), (b), (d), (f), (g–j), (l) and (n–r) show whole mount views. Panels (c) and (m) show sagittal sections, and panels (e) and (k) show horizontal sections. Panel (a), (d), (f), (g), (i), (j), (n–p) and (r) show lateral views and panels (b), (h), (l) and (q) show frontal views. Anterior is to the left in panels (a), (c), (d), (f), (g), (i), (j), (k), (m–p) and (r) and to the top in panels (b), (e), (h), (l) and (q). The arrowheads indicate faint expression in the AVE in panel (a), the distinct expression in the anterior neuroectoderm in panels (c) and (e), weak expression in the diencephalon in panel (f) and no expression in the anterior neuroectoderm in panel (h). The arrows indicate weak expression in the AME in panels (c) and (q) and in the CM in panel (r), the expression in the CM in panels (e) and (f), in the AME in panel (h) and in heart in panels (n) and (o), respectively. Abbreviation: CM, cephalic mesenchyme.
	Fig. 4. Enhancer activity of Xenopus and coelacanth Otx2 α1 regions. (A) The enhancer activity of the Xenopus α1 region. Panels (a–e) show the activity of the Xenopus α1 region. Panels (f–h) show the activity of the Xenopus α1 region lacking the VE sequence. Panels (i–k) show the activity of the mouse α1 region in which the VE sequence was replaced with the Xenopus VE sequence. Panels (a), (f) and (i) show β-Gal expression in the mouse AVE at E6.5. Panels (b), (c), (g) and (j) show β-Gal expression in the AME at E7.5. Panels (d, h and k) and (e) show β-Gal expression in the CM at E8.5 and E9.5, respectively. Panels (a), (b) and (d–k) show whole mount views, while panel (c) shows a sagittal section. Panels (a), (d–f), (h), (i) and (k) are lateral views and panels (b), (g) and (j) are frontal views. Anterior is to the left in panels (a), (c–f), (h), (i) and (k) and at the top in (b), (g) and (j). The arrowheads indicate weak expression in the AME in panel (g) and in the CM in panel (h). (B) The enhancer activity of the coelacanth α1 region. Panels (a–d) show the activity of the coelacanth α1 region. Panels (e) and (f) show the activity of the coelacanth α1 region that lacks the VE sequence. Panels (g–j) show the activity of the mouse α1 region in which the VE sequence was replaced with the coelacanth VE sequence. Panels (a) and (g) show β-Gal expression in the mouse AVE at E6.5. Panels (b), (e) and (h) show β-Gal expression in the AME at E7.5, and panels (c), (d) (f) (i) and (j) show β-Gal expression at E8.5. Panels (a–c) and (e–i) show whole mount views, and panels (d) and (j) show frontal sections at the level indicated by the line in panels (c) and (i), respectively. Panels (a), (c), (f) (g) and (i) show lateral views, and panels (b), (e) and (h) show frontal views. Anterior is to the left in panels (a), (c), (f), (g) and (i) and at the top in panels (b), (e) and (h). The arrowheads indicate weak expression in the AME in panel (b) and the distinct expression in the CM in panel (j). The arrows indicate the expression in the most anterior neuroectoderm in (c), (d) and (j).
	Fig. 5. Enhancer activity of the Xenopus, coelacanth and mouse Otx2 α1 regions in Xenopus and zebrafish embryos. (A) The enhancer activity of the Xenopus α1 region in Xenopus embryos. Panels (a), (c), (f), (i) and (k) show Egfp mRNA expression directed by the Xenopus α1 region. Panels (b), (d), (g), (j) and (l) show the endogenous Otx2 expression, and panels (e) and (h) show expression of Cerberus and Chordin. Panels (a–e) are from stage 9.5 (early gastrula) embryos. Panels (f–h) are from stage 10.5 (mid-gastrula) embryos, and panels (i–l) are from stage 14 (neurula). Panels (a), (b), (i) and (j) show whole mount dorsal views, and panels (c–h) show hemi-sections. Panels (d and e) and (g and h) are counterparts of the same embryos. Panels (k) and (l) are sagittal sections at the level indicated by the dotted lines in (i) and (j), respectively. Anterior is at the top. The arrowhead in panel (i) indicates ectopic activity in the notochord. (B) Enhancer activity of the coelacanth α1 region. Egfp mRNA expression directed by the coelacanth α1 region in stage 10.5 Xenopus (a) and shield-stage zebrafish (b) embryos. Panel (a) shows a hemi-section, and panel (b) shows lateral whole mount views. Anterior is at the top. (C) The enhancer activity of the mouse α1 region in zebrafish embryos. Egfp mRNA expression at the shield (a) and early somite stages (b). Panel (a) shows a lateral view, and panel (b) shows a dorsal whole mount view. Anterior is at the top. The arrows indicate the expression in the shield (a) and AME including the polster (b).
	Fig. 6. A comparison of the expression sites of the genes in the Otx2 gene cascade in the mouse visceral endoderm in Xenopus gastrula embryos. (A) Xlim-1 (B) FoxA4 (C) Dkk1 (D) Shisa. Each embryo was sagitally hemi-sectioned, and one half was hybridized for Otx2 and the other half for each gene.
	Supplementary Fig. S1. The nucleotide sequence of the α1 region of the mouse Otx2 gene. The 51 bp VE sequence is boxed with a solid line, and the 49 bp CM sequence is boxed with a dotted line. The A and B elements in the CM sequence and FOXA2 binding site in the VE sequence are indicated. #1 and #2 indicate the transcriptional start sites (Fossat et al., 2005, Acampora et al., 2009).
	Supplementary Fig. S2. Sequence conservation among the vertebrate Otx2 α1 regions. A vista plot of each vertebrate Otx2 α1 region aligned against the coelacanth (A) and Polypterus (B) α1 sequences. In Polypterus, dogfish, skate and lamprey, the Otx2 orthologs are expressed in their putative organizer tissues (Coolen et al., 2007, Suda et al., 2009). However, except for the VE domain, no conserved domain is present among the Polypterus, skate and coelacanth Otx2 α1 regions. Sequences weakly conserved in skate do not overlap with the 51 bp VE sequence.