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Fig. 4. XRDH10 induces retinoic acid signalling and differentially affects organizer gene expression. (A) Uninjected tadpole-stage embryo. (B) Animal injection of 4 ng XRDH10 mRNA at the four-cell stage induces a slight reduction of head structures and a shortening of the tail. (C) Treatment with 0.1 µM RA between stages 9 and 12 induces microcephaly and tail shortening. (D) Injection of 0.5 ng XCYP26A1 mRNA reverts the effect of XRDH10 mRNA and restores normal head and tail development. (E) Treatment with 4 µM citral at stages 9-12 abrogates the activity of XRDH10 mRNA. (F) RT-PCR analysis of animal caps explanted from stage 8 embryos and cultured until stage 12.5. Embryos were injected with 4 ng XRDH10 mRNA (lane 3) and animal caps treated with 5 µM RA (lane 4). Note that XRDH10 stimulates the transcription of all the RA target genes tested. (G-V) Whole-mount in situ hybridization of gastrula embryos in vegetal view. Insets depict lateral views. Embryos were injected in the margin of each blastomere at the four-cell stage with 1 ng XRDH10 mRNA (H,L,P,T) or treated from stage 8 onwards with DMSO as a control (I,M,Q,U) or 5 µM RA (J,N,R,V). Note that XRDH10 mRNA and RA expand the expression of Xlim1 and Chordin, but reduce the expression of Goosecoid and ADMP in the dorsal blastopore lip. Frequency of embryos with the indicated phenotypes was: B, 30/39; C, 25/25; D, 30/40; E, 29/39; G, 45/45; H, 19/39; I, 31/31; J, 41/41; K, 38/38; L, 29/43; M, 30/36; N, 16/29; O, 6/8; P, 4/6; Q, 22/28; R, 15/24; S, 14/14; T, 9/13; U, 64/69; V, 38/50. |
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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. 7. Knockdown of XRDH10 and XRALDH2 induces ventralization and influences mesodermal gene expression. Antisense morpholino oligonucleotides (MOs) were injected marginally at the two-cell stage (5.2 pmol per blastomere), followed by injection of non-targeted mRNA constructs (XRDH10* and mRALDH2) at the four-cell stage (1 ng per blastomere). (A,B) MOs target the translation initiation sites of two pseudoalleles of Xenopus laevis RDH10 and RALDH2. (C,D) Protein synthesis of XRDH10 and XRALDH2 is specifically inhibited by XRDH10-MO and XRALDH2-MO, but not by control-MO of random sequence. (E-J) Microinjection of XRDH10-MO and XRALDH2-MO leads to microcephaly and enlarged ventroposterior structures in tailbud embryos (E-G), and to reduced eye structures and shortened tails in tadpoles (H-J). Normal development is restored by XRDH10* and mRALDH2 mRNAs, respectively (insets). (K-S) Gastrula embryos in dorsal view. XRDH10- and XRALDH2-morphants have reduced Chordin expression (K-M) and expanded expression domains of Goosecoid (N-P) and ADMP (Q-S). (T-V) Neurula embryos in dorsal view (anterior to the top) after a single injection of MOs with the lineage tracer nlacZ mRNA (red nuclei). XRDH10-MO and XRALDH2-MO reduce Xlim1 expression in the pronephros (arrowhead). The indicated phenotypes were observed in: E, 101/117; F, 62/84 (inset, 73/98); G, 44/67 (inset, 80/84); H, 74/79; I, 39/50 (inset, 56/68); J, 18/48 (inset, 58/64); K, 33/33; L, 21/36 (inset, 19/27); M, 28/36 (inset, 20/28); N, 51/56; O, 28/38 (inset, 46/53); P, 37/49 (inset, 34/44); Q, 52/52; R, 14/26 (inset, 51/62); S, 51/62 (inset, 23/32); T, 11/15; U, 14/20 (inset, 28/32); V, 16/24 (inset, 50/51) embryos. |
