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Files in this Data Supplement:
Fig. S1. XRDH10 mRNA and exogeneous RA slightly upregulate Chordin expression at the blastula stage. Whole-mount in situ hybridization with antisense RNA against Chordin. (A) Uninjected embryo at stage 9 in dorsal view. (B) Embryo after microinjection of XRDH10 mRNA into the margin of each blastomere at the four-cell stage (total 4 ng). (C,D) Embryos treated from stage 7 onwards with 0.05% DMSO (dimethylsulfoxide) as a control (C) or with 5 µM RA in DMSO (D). Frequency of embryos with the indicated phenotypes was: A, 10/11; B, 11/11; C, 31/38; D, 47/47.
Fig. S2. XRDH10 mRNA and exogeneous RA have no effect on the organizer-specific expression of Noggin, Frzb, sFRP2 and Crescent at the early gastrula stage. (A-P) Vegetal view of embryos at stage 10.5. Embryos were uninjected (A,E,I,M), radially injected at the four-cell stage with 4 ng XRDH10 mRNA (B,F,J,N), or treated from stage 8 onwards with DMSO (C,G,K,O) or with 5 µM RA (D,H,L,P). Note that expression of Noggin, Frzb, sFRP2 and Crescent in the dorsal blastopore lip is not affected by any of the treatments. Frequency of embryos with the indicated phenotypes was: A, 16/16; B, 24/28; C, 53/53; D, 45/45; E, 12/12; F, 27/27; G, 12/12; H, 20/20; I, 16/16; J, 25/25; K, 24/24; L, 28/28; M, 15/15; N, 28/28; O, 62/62; P, 42/46.
Fig. S3. Downregulation of XRDH10 and XRALDH2 causes a posteriorwards shift of the hindbrain marker xCRABP. Morpholino oligonucleotides (MOs; each 2.6 pmol per embryo) were injected into the margin of a single blastomere at the two-cell stage. The non-targeted mRNA constructs XRDH10* and mRALDH2 (each 1 ng), and the lineage tracer nlacZ mRNA (100 pg), were co-injected. Embryos at the advanced neurula stage are shown in anterior-dorsal view (posterior to the top). The panels A′-F′ are magnifications of the embryos shown in A-F. Embryos were subjected to whole-mount in situ hybridization with an antisense RNA probe against xCRABP (cellular retinoic acid binding protein) that demarcates the presumptive hindbrain (square bracket). Nuclear Red-Gal staining (resulting from nlacZ mRNA expression) demarcates the injected right side. (A) An unspecific control-MO does not affect xCRABP expression. (B-D) XRDH10-MO, XRALDH2-MO, or a combination of both morpholinos, causes a posteriorwards shift of the xCRABP-expression domain. (E,F) The effects of XRDH10-MO and XRALDH2-MO are neutralized by XRDH10* and mRALDH2 mRNA, respectively. Frequency of embryos with the indicated phenotype was: A, 9/11; B, 10/11; C, 18/19; D, 12/12; E, 11/11; F, 17/19.
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