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Fig. 8. XRDH10 contributes to CNS patterning and the posteriorizing effect of retinol. Morpholino oligonucleotides (MOs; each 2.6 pmol per embryo) were injected into the margin of one blastomere at the two-cell stage. The non-targeted mRNA constructs XRDH10* and mRALDH2 (each 1 ng) and the lineage tracer nlacZ mRNA were co-injected. Embryos are shown in dorsal view (anterior to the top). (A-F) Late gastrula embryos. XRDH10-MO, XRALDH2-MO, or a combination of both morpholinos, cause a reduction and posteriorward retraction of HoxD1expression, which is reverted by XRDH10* and mRALDH2 mRNA. (G-L) Neurula embryos showing expression of En2 (midbrain-hindbrain boundary), HoxB3 (hindbrain rhombomeres 5 and 6) and HoxC6 (anterior spinal cord). (M-R) Tailbud embryos depicting expression of Rx2A (eyes) and Krox20 (rhombomeres 3 and 5). (S) Effects of XRDH10 and XRALDH2 knockdown on hindbrain patterning. The posteriorward shift of Krox20 expression is shown in response to MO injections at the indicated doses. (T-W) Treatment with 100 µM retinol at stages 9-12 induces a robust anterior expansion of HoxD1 expression in late gastrula embryos (V). XRDH10-MO reverts the effect of retinol on the injected right-hand side (W). Frequency of embryos with the indicated phenotype was: A, 77/88; B, 55/105; C, 30/68; D, 54/88; E, 19/21; F, 23/25; G, 34/35; H, 17/31 (En2); H, 30/31 (HoxB3); H, 27/31 (HoxC6); I, 15/33 (En2); I, 31/33 (HoxB3); I, 32/33 (HoxC6); J, 6/9 (En2); J, 8/9 (HoxB3); J, 5/9 (HoxC6); K, 20/23; L, 39/39; M, 10/10; N, 35/73; O, 37/69; P, 38/60; Q, 10/10; R, 13/14; T, 9/9; U, 7/13; V, 9/9; W, 20/33 embryos. |
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Fig. 5. Overexpression of XRDH10 and XRALDH2 results in an anteriorward shift of neural markers, whereas XCYP26A1 has the opposite effect. Whole-mount in situ hybridization of embryos after microinjection of mRNA into the animal pole of one dorsal blastomere at the four-cell stage. The lineage tracer nlacZ (red nuclei) labels the injected right-hand side. (A-E) Late gastrula embryos in dorsal view (anterior to the top). HoxD1 demarcates the ectoderm and mesoderm in the trunk with an anterior expression boundary at the level of rhombomere 4 (horizontal line). (F-J) Early neurula embryos in dorsal view, showing Xlim1 expression in two lines of neural cells (arrow). (K-O) Early tailbud embryos in anterior view (posterior to the top) and schematic overviews demarcating Rx2A expression in the eyes and Krox20 expression in rhombomeres 3 and 5 of the hindbrain. (P-T) FoxG1 labels the telencephalon, and En2 the midbrain-hindbrain boundary. (U) Synergistic effects of XRDH10 and XRALDH2 on hindbrain patterning. The anteriorward shift of Krox20 expression is shown in response to mRNA injections at the indicated doses. Note that XRDH10 has little effect on its own, but strongly enhances the posteriorizing effect of XRALDH2. nlacZ mRNA was injected as a control. Injected RNA amounts were (where not otherwise noted): nlacZ (300 pg), XRDH10 (1 ng), XRALDH2 (1 ng) and XCYP26A1 (0.5 ng). ey, eye; rh, rhombomere; R2, XRALDH2; R10, XRDH10. The indicated changes in gene expression were observed in: B, 35/78; C, 43/59; D, 18/29; E, 9/9; G, 24/96; H, 45/95; I, 30/51; J, 13/13; L, 7/36; M, 22/33; N, 22/33; O, 15/15; Q, 6/56 (En2); R, 7/19 (En2); S, 8/20 (En2); T, 25/25 (En2) embryos. |
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Fig. 6. XRDH10 co-operates with retinol during head development. (A-D) Embryos were injected into the animal pole at the four-cell stage with the indicated mRNAs and treated with DMSO or retinol at stages 9-12. (A) DMSO-treated control embryo at tadpole stage. (B) Retinol (50 µM) induces microcephaly at the tadpole stage. (C) Injection of XRDH10 mRNA (1 ng into four blastomeres) and subsequent retinol treatment causes anencephaly. (D) XCYP26A1 mRNA (2.5 ng) partially restores eye and head structures in retinol and XRDH10-treated embryos. (E) Eye deficiencies induced by retinol and XRDH10, and dose-dependent rescue by XCYP26A1 mRNA in stage 40 embryos. (F) Control embryo at the tail bud stage after single injection of nlacZ mRNA. (G) Retinol (25 µM) leads to a slight reduction of the Rx2A-positive eye field (arrowheads). (H,I) In the retinol-treated embryos, XRDH10 mRNA (1 ng in one dorsal blastomere) causes a unilateral collapse of Rx2A expression (arrowhead in H), which is rescued by the co-injection of 2.5 ng XCYP26A1 mRNA (arrowhead in I). The indicated phenotypes were observed in: A, 24/24; B, 25/27; C, 28/45; D, 51/59; G, 13/15; H, 20/35; I, 10/13 embryos. |
