Steventon B et al. (2012), Mutual repression between Gbx2 and Otx2 in sens...

XB-ART-45517

Dev Biol 2012 Jul 01;3671:55-65. doi: 10.1016/j.ydbio.2012.04.025.

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Mutual repression between Gbx2 and Otx2 in sensory placodes reveals a general mechanism for ectodermal patterning.

Steventon B , Mayor R , Streit A .

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In the vertebrate head, central and peripheral components of the sensory nervous system have different embryonic origins, the neural plate and sensory placodes. This raises the question of how they develop in register to form functional sense organs and sensory circuits. Here we show that mutual repression between the homeobox transcription factors Gbx2 and Otx2 patterns the placode territory by influencing regional identity and by segregating inner ear and trigeminal progenitors. Activation of Otx2 targets is necessary for anterior olfactory, lens and trigeminal character, while Gbx2 function is required for the formation of the posterior otic placode. Thus, like in the neural plate antagonistic interaction between Otx2 and Gbx2 establishes positional information thus providing a general mechanism for rostro-caudal patterning of the ectoderm. Our findings support the idea that the Otx/Gbx boundary has an ancient evolutionary origin to which different modules were recruited to specify cells of different fates.

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Species referenced: Xenopus
Genes referenced: dmrta1 en1 eya1 eya2 fdx1 foxe3 gbx2.1 gbx2.2 mst1 otx2 pax2 pax3 pax6 pax8 pdf runx3 tbx2
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	Fig. 1. Otx2 and Gbx2 form a boundary within the PPR. (A) Expression of Eya2 marks the PPR (bracket). (B)–(D) Double ISH for Otx2 (red) and Gbx2 (blue) at HH7. Lines indicate the level of sections shown in (C) and (D). (E)–(J) Parasagittal sections of stage HH5 (E)–(G) and HH7 (H)–(J) chick embryos after Gbx2 ISH (black (E), (F), (H), (I)) and Otx2 immunostaining (green; (F), (G), (I), (J)); DAPI labels nuclei ((G), (J); magenta). Arrows in (H)–(J) indicate anterior limit of Gbx2. (K) and (L) Double ISH in Xenopus for Otx2 ((K), (L); turquoise) and Gbx2 ((L); blue) at stage 12, dorsal to right anterior to the top. Red brackets indicate overlapping expression. (M) Diagram summarising expression of Otx2 and Gbx2 at stage 12 in Xenopus; red bracket: PPR. (N) Eya1 at stage 13 labels the PPR (bracket). (O) and (P) Double ISH for Otx2 ((O), (P); turquoise) and Gbx2 ((P); blue) at stage 13, dorsal to the right. Black brackets indicate the PPR. (Q) Diagram summarising neural plate and PPR expression of Otx2 and Gbx2 at stage 13 in Xenopus
	ig. 2. The Otx2/Gbx2 boundary separates otic and trigeminal precursors. (A) Diagram showing HH 7 stage chick embryo: the distance from the center of Hensen's node to the anterior tip of prechordal plate (hn-pc) and from the midline to the edge of the neural plate (ml-np) were set to 100%, respectively. The position of DiI label was measured and expressed as percentage of each distance. (A′). The position of the Gbx2/Otx2 boundary was measured using the same landmarks. In total 9 embryos were measured with the boundary on average at 35±7%; most anterior position measured: 42%, most posterior position: 24.5%. (B) Diagram combining labels from this study and published fate maps ( [Streit, 2002] and [Xu et al., 2008]); gray: labels contributing to the trigeminal placode; blue: labels contributing to the otic placode. Circles: labels from published fate maps; squares: labels with dual fate from the current study; stars: labels from the current study. 35% indicates the average position of the Otx2/Gbx2 boundary (dotted line)±standard deviation (small arrow); note: mixed trigeminal and otic fates mostly locate near this boundary. (C) HH7 embryo with DiI labeled cells posterior to the average position of the Otx2/Gbx2 boundary (white line). (D) and (E) At HH12 their descendants contribute to the otic placode as shown in whole mount (D) and in transverse sections (E) and (F). HH7 embryo with DiI label anterior to the average position of the Otx2/Gbx2 boundary (black line). (G) and (H) At HH11 their descendants overlap with Pax3 protein (green) in the trigeminal placode. In total, 21 labels were placed into the Otx2+ and 12 into the Gbx2+ domain. (I) Diagram showing the experimental design: blastomeres were injected at the 64-cell stage in Xenopus and their position scored at stage 14. Arrows show the orientation of all embryos. (J)–(L) Neighboring blastomeres were injected with nGFP and nRFP and grown until stage 14. Descendants from injected cells are intermingled as indicated by red and green outlines in L (100%, n=10). (M)–(O) When injected with nGFP/Otx2 and nRFP/Gbx2 descendants from adjacent blastomeres do not mix (boundary in 79% of embryos, n=14). Red and green outlines in O show the distribution of cells.
	Fig. 3. Otx2 and Gbx2 mutually repress each other in the PPR. (A) and (B) Injection of Otx2 mRNA into the D1 blastomere of 8-cell stage embryos (A) shifts En-1 posteriorly on the injected side (50%. n=28; FDX: turquoise), while injection into the A3 blastomere of a 32-cell stage embryo has little effect ((B); 5% affected, n=36). (C) and (D) Injection of Gbx2 mRNA into the D1 blastomere at 8-cell stage (C) shifts En-1 anteriorly on the injected side (68%; n=64; FDX: turquoise), while injection into the A3 blastomere at 32-cell stage has no effect ((D); 0% affected, n=31). Dorsal view, anterior to the top. (E) and (F) Injection of Otx2 (E; 68% affected, n=31) or Otx2-EnR ((F); 77% affected, n=17) into A3 at the 32-cell stage inhibits Gbx2 in the PPR. Compare bracket in the injected (FDX: turquoise) and uninjected side. (G) and (H) Injection of Gbx2 into A3 at the 32-cell stage leads to Otx2 repression (68%; n=26); compare brackets (G) on the injected ((H): FDX, turquoise) and uninjected side. (I) and (J) Co-injection of splice and translation blocking Gbx2 morpholinos into A3 at 32-cell stage leads to Otx2 expansion (73%; n=33); compare brackets (I) on the injected ((J) FDX, turquoise) and uninjected side. (K) and (L) Injection of Gbx2-EnR into A3 at the 32-cell stage leads to a loss of Otx2 in 62% of embryos (n=52); compare brackets (K) on the injected ((L) FDX, turquoise) and uninjected side. (E)–(L) Frontal view, dorsal to the top.
	Fig. 4. Gbx2 is required for otic specification. (A)–(C) Injection of splice and translation blocking Gbx2 morpholinos inhibits otic Pax8 (A; 54%, n=28) and otic Pax2 ((B); 55%, n=131; Splice MO: 66% affected, n=29; ATG MO: 49% affected, n=79). There is no effect on Eya1 (0% affected, n=30. (C): blue). (D)–(F) At stage 25, Pax2 expression is reduced and the otic vesicle is small (asterisk in transverse section F) after injection of splice and translation blocking Gbx2 morpholinos ((E) 59% affected, n=66; splice MO: 44% affected, n=25) when compared to the uninjected side (D). (G)–(I) Injection of inducible Gbx2-EnR-GR: activation at stage 10 reduces otic Pax8 (arrow) at stage 13 ((H); 49%, n=43) compared to uninjected side (G) and Pax2 at stage 16 ((I); arrow; 46% n=18). (J)–(L) Activation of inducible Gbx2-EnR-GR at stage 10 (J) reduces Pax8; no change is observed in absence of DEX ((K); 0% affected, n=17) or when DEX is added at stage 14 ((L) 0% affected, n=110). (M)–(O) Gbx2 mRNA does not expand Pax8 ((M); 0% affected, n=35), Pax2 ((N); 0% affected, n=22), and Eya1 ((O) 0% affected, n=22). (P), (Q) Overexpression of Gbx2 reduces Dmrt-4 ((P) 66%, n=29) and FoxE3 ((Q) 92%, n=13). Small panels in (P) and (Q) show higher magnification of the control (top) and injected (bottom) side. All embryos were injected into the A3 blastomere at the 32-cell stage; inserts in (A), (B), (G)–(N) high magnification of the otic region. In (C) and (O) turquoise staining reveals FDX. Crosses indicate the orientation of embryos; a: anterior, l: left, p: posterior, r: right, d: dorsal, v: ventral.
	Fig. 5. Activation of Otx2 target genes is required for trigeminal placode specification. (A), (B) Otx2-EnR inhibits Runx3 at stage 23 ((A); 59%, n=26; arrowhead: trigeminal placode on uninjected side). This is rescued by co-injection of Otx2 ((B) inhibition reduced to 17%, n=36). Inserts show higher magnification of the trigeminal region. (C), (D) At stage 28 the profundal and trigeminal placodes can be distinguished; both are reduced after Otx2-EnR injection (78%, n=18). Compare control (C) and injected side (D). (E), (F) Injection of Gbx2 splice and translation blocking morpholinos ((F) 0% affected, n=25) does not affect Runx3; compare uninjected (E) and injected side (F). (G), (H). Injection of Gbx2-EnR does not affect Runx3 ((H); 3% affected, n=31); compare control (G) and injected side (H). (I)–(K) Activation of Otx2-EnR-GR at stage 10 inhibits Pax3 the profundal placode ((J) 49%, n=33); no change is observed without DEX ((I) 5% affected, n=61; PR: profundal placode) or when added at stage 14 ((K) 5% affected, n=42). Magnifications show profundal region (dotted outline) on the uninjected (top) and injected side (bottom; FDX: turquoise). All embryos were injected into the A3 blastomere at the 32-cell stage.
	Fig. 6. Activation of Otx2 target genes is required at early and late stages of lens placode formation. (A), (B) Otx2-EnR inhibits Pax6 in the lens at stage 17 (50%, n=26). This is rescued by co-injection of Otx2 ((B); inhibition reduced to 15%, n=20). (C)–(F) Activation of Otx2-EnR-GR at stage 10 reduces lens-specific Pax6 at stage 18 ((D) 52%, n=25); without DEX Pax6 expression is normal ((C) 0% affected, n=23). FoxE3 at stage 25 is normal without DEX ((E) 0% affected, n=30), while addition of DEX at stage 18 leads to reduction ((F) 64%, n=22). (G) Otx2-E1A activates Otx2 targets but does not affect lens Pax6 (0% affected, n=25). All embryos were injected into the A3 blastomere at the 32-cell stage and are shown in frontal view with dorsal to the top. High magnifications of the lens region are shown below each panel; dotted lines demarcate placodal Pax6. Turquoise staining reveals the lineage tracer FDX.
	Fig. 7. Activation of Otx2 target genes is required at early and late stages of olfactory placode formation. (A), (B) Otx2-EnR inhibits the olfactory placode marker Dmrt4 at stage 21 (59%, n=29). This is rescued by co-injection of Otx2 ((B) inhibition reduced to 12%, n=33). (C–F) Otx2-EnR-GR injections: in the absence of DEX Dmrt4 expression is normal ((C) 5% affected, n=20); when DEX is added at stage 10 Dmrt4 expression is lost at stage 18 ((D) 59%, n=44). At stage 25 Dmrt4 is normal in absence of DEX ((E) 8% affected, n=26), while activation at stage 18 strongly reduces Dmrt4 ((F); 63%, n=24). (G) Otx2-E1A has no effect on Dmrt4 (0% affected, n=14). (H)–(I) Otx2 mRNA reduces Pax2 ((H) 72%, n=25), but does not change Eya1 ((I) 0% affected, n=44). All embryos were injected into the A3 blastomere at the 32-cell stage and are shown in frontal view with dorsal to the top. High magnifications of the olfactory region are shown in small panels; dotted lines demarcate placodal Dmrt4. Turquoise staining reveals the lineage tracer FDX.
	Fig. S1. ( NO caption given on website or in pdf as of 6/28/2012) cjz

References [+] :

Acampora, Forebrain and midbrain regions are deleted in Otx2-/- mutants due to a defective anterior neuroectoderm specification during gastrulation. 1995, Pubmed