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Fig. 1. Double WISH reveals the spatial relationship between Rarγ2 and posterior Hox, PSM and CNH genes. (A-M) Rarγ2 is stained with BM Purple and the other genes are stained with Fast Red. Rarγ2 is caudal to Myod and Tbx6 (A-D), but synexpressed with Msgn1 (F,G) in neurula stage Xenopus embryos. (E,H) Rarγ2 is synexpressed with the caudal domain (CD) of Hoxc10 but not with neural tube (NT) or lateral plate mesoderm (LPM) of Hoxc10 in tailbud stage embryos. Rarγ2 overlaps with S–III domains of Ripply2 (I,J) and Thyl2 (L,M) expression, but not with more anterior somitomeres (S–II, S–I, S0). (K) Rarγ2 overlaps with xNot expression in neurula stage embryos. Dorsal and lateral views shown with anterior to the left, except in K (caudal view with dorsal at top).
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Fig. 2. Posterior Hox and PSM markers are reduced by RARγ-selective agonist and expanded by RARγ-selective inverse agonist. (A-F) WISH from embryos treated post-gastrulation (stage 12.5) with 10 nM 4647, 0.5 µM 5099 or vehicle (0.1% ethanol). Dashed red line represents half the embryo axis. 4647 diminishes and 5099 expands the expression of (A) Hoxd10 (4647, 16/16; 5099, 17/17 embryos), (B) Hoxc10 (4647, 14/14; 5099, 21/21), (C) Hoxc13 (4647, 12/12; 5099, 16/16), (D) Tbx6 (4647, 11/12; 5099, 17/17), (E) Msgn1 (4647, 15/15; 5099, 14/14), and (F) Rarγ2 (4647, 15/15; 5099, 9/9) relative to control vehicle. Embryos shown in lateral or dorsal view at tailbud stage, anterior to left.
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Fig. 3. PSM markers are modulated by RARγ-selective agonist and inverse agonist. (A-R) WISH from embryos treated post-gastrulation (stage 12.5) with 10 nM 4647, 0.5 µM 5099 or vehicle (0.1% ethanol). (A-D) Control expression of Msgn1, Tbx6, Thyl2 and Ripply2. (E) Msgn1 expression diminished by 4647 treatment (17/17 embryos). (F) Tbx6 expression expanded by 4647 treatment (22/22). (G,H) Somitomere domains of Thyl2 (19/19) and Ripply2 (17/17) are thicker and posteriorly expanded. (I,J) Msgn1 (17/17) and Tbx6 (13/13) expression expanded by 5099 treatment. (K,L) Somitomere domains of Thyl2 (15/17) and Ripply2 (26/26) are fewer and thinner. Embryos are shown in dorsal view at neurula stage, anterior to left. (M-R) Caudal views of Msgn1 and Tbx6.
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Fig. 4. RARγ2 knockdown alters expression of posterior Hox and PSM markers. (A-J′) Embryos were injected unilaterally at the 2- or 4-cell stage with 7.5 ng Rarγ2.1 MO+7.5 ng Rarγ2.2 MO. Injected side is indicated by magenta β-gal lineage tracer. Rarγ2.1/2.2 MO decreases expression of (A) Hoxc10 (18/18 embryos), (B) Hoxd10 (12/12), (C) Hoxa11 (9/9) and (D) Hoxc13 (16/16) in tailbud stage embryos. Rarγ2.1/2.2 MO decreases lateral, but expands midline, expression (green lines) of (E) Msgn1 (10/13) and (F) Tbx6 (8/11), knocking down and shifting expression rostrally of (G) Thyl2 (13/15) and (H) Ripply2 (13/14) in tailbud stage embryos. Rarγ2.1/2.2 MO decreases lateral, but expands midline, expression (green lines) of (I) Msgn1 (35/36) and (J) Tbx6 (20/20) in neurula stage embryos. Embryos shown in dorsal view with anterior on left. (I′,J′) Caudal views of I and J.
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Fig. 5. Rarγ2 mRNA rescues posterior Hox and PSM expression in Rarγ2 MO embryos. (A-H) Embryos injected unilaterally at 2- or 4-cell stage. Injected side is indicated by magenta β-gal lineage tracer. (A,E) 5 ng Rarγ2.1 MO+5 ng Rarγ2.2 MO+control (mCherry) mRNA diminishes Hoxc10 and Msgn1 expression, curving the embryo axis in 100% of embryos (Hoxc10, 23/23; Msgn1, 13/13). (B,C,F,G) Co-injection of Rarγ2 MO and 1 ng Rarα2 mRNA or 1 ng Rarβ2 does not rescue the phenotype; however, (D,H) 1 ng Rarγ2 mRNA partially rescues axial curvature and Hoxc10 (18/23) and Msgn1 (23/35) expression. Tailbud embryos shown in dorsal view with anterior to left.
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Fig. 6. c-SMRT overexpression knocks down posterior Hox, PSM and CNH markers. Embryos injected unilaterally at 2- or 4-cell stage with 4 ng c-smrt mRNA or control (mCherry) mRNA. Injected side indicated by magenta β-gal lineage tracer. (A,C,E,G,I,K) Control expression of Hoxc10, Hoxd10, Hoxc13, Msgn1, Tbx6 and xNot. (B,D,F,H,J,L) c-smrt overexpression shortens the axis on injected side in 70% of embryos. (B) c-smrt mRNA results in lateral knockdown (13/23 embryos), neural knockdown (7/23) or neural rostral shift (7/23) in Hoxc10 expression. (D) c-smrt mRNA produces neural and lateral knockdown (15/19) or lateral knockdown alone (4/19) of Hoxd10 expression. (F,H,J) c-smrt mRNA knocks down expression of Hoxc13 (14/18), Msgn1 (12/14) and Tbx6 (15/15). Tailbud embryos shown with anterior to left. (H′,J′) Caudal views of H and J. (L) c-smrt mRNA knocks down xNot (12/15) expression in neurula stage embryos (caudal view, dorsal to top).
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Fig. 7. VP16-RARγ2 overexpression knocks down posterior Hox and PSM marker expression. Embryos injected unilaterally at 2- or 4-cell stage with 0.3 ng VP16-Rarγ2 mRNA or control (mCherry) mRNA. Injected side is indicated by magenta β-gal lineage tracer. Control expression of Hoxc10, Hoxd10, Msgn1 and Tbx6 is shown in Fig. 6A,C,G,I. (A-D) VP16-Rarγ2 overexpression shortens the axis on injected side in 100% of embryos. (A,B) VP16-Rarγ2 mRNA results in neural/midline rostral shift and lateral knockdown in Hoxc10 (9/13 embryos) and Hoxd10 (7/13) expression. Neural/midline knockdown is also observed (Hoxc10, 4/13; Hoxd10, 7/13). (C,D) VP16-Rarγ2 mRNA rostrally shifts and/or knocks down Msgn1 (12/12) and Tbx6 (13/13) expression. Tailbud embryos shown with anterior to left. (A′-D′) Caudal views of A-D.
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Fig. 8. Overexpression of DN-Rarγ2 mRNA expands expression of PSM and CNH markers, shifting or knocking down somitomere markers Thyl2 and Ripply2. (A-J) Embryos injected unilaterally at 2- or 4-cell stage. Injected side is indicated by magenta β-gal lineage tracer. (A,C,E,G,I) Control (mCherry) mRNA does not alter expression of Tbx6, Msgn1, Thyl2, Ripply2 or xNot. (B,D,F) 2 ng DN-Rarγ2 mRNA expands expression of Msgn1 (8/11) and Tbx6 (15/23) (green lines) and rostrally shifts xNot (8/10). (H,J) DN-Rarγ2 overexpression produces multiple phenotypes of Thyl2 and Ripply2 expression, as characterized and scored in K. Neurula embryos shown in dorsal view with anterior to left.
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Fig. 9. DN-Rarγ2 mRNA rescues posterior Hox expression in Rarγ2 MO embryos. (A-D) Embryos injected unilaterally at 2- or 4-cell stage. Injected side is indicated by magenta β-gal lineage tracer. (A) 2.5 ng Rarγ2.1 MO+2.5 ng Rarγ2.2 MO or (B) 5 ng Rarγ2.1 MO+5 ng Rarγ2.2 MO diminishes Hoxc10 expression and curves the embryo axis. (C,D) 2 ng DN-Rarγ2 mRNA partially rescues this effect and expands neural expression of Hoxc10. Tailbud embryos shown in dorsal view with anterior to left. (E) Detailed scoring of the rescue experiment.
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Fig. 10. RARγ functions as both transcriptional activator and repressor during somitogenesis and axial elongation. RARγ is activated by RA near the determination wavefront where PSM differentiates into somitomeres, then mature somites. The progenitor pool within the PSM and CNH domains, which is maintained by Rarγ repression, feeds into the wavefront until exhausted, as somitogenesis proceeds faster than progenitors are replenished (Gomez and Pourquie, 2009). As PSM and CNH domains diminish, the distance between RA/wavefront (blue line) and the posterior tip of the embryo (green line) becomes shorter. RA is able to enter the posterior, activating Rarγ, switching its function from repressor promoting growth to activator terminating growth. RXR, retinoid X receptor.
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