XB-IMG-75044
Xenbase Image ID: 75044
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Fig. 9. Morpholino-mediated knockdown of Crescent. Four-cell-stage embryos were injected into the dorsal equatorial region with MOs as indicated on the left side of panels. (A) Efficacy of cresMO to block the splicing of crescent transcript. (a) RT-PCR showing aberrant splicing in MO-injected embryos. RT−, RT minus. (b) Normal and cryptic splice donor sites in exon 1 (indicated by arrowheads). (B) Morphological appearances of injected embryos at the tailbud stage (stage 33/34). Compared to normal embryos (a), the morphants showed short body axis and small head with small eyes (b), whereas these phenotypes were weakened by co-injection with crescent mRNA (c). Anterior is to left. (C) Anterior development and neurulation are affected at the neurula stage. The expression of Rx2A (a–c,g) at stage 17, or that of Dlx3 (d–f,d′–f′,h) and Xhairy2a (i–k) at stage 15 was examined. Anterior view; dorsal is upwards in (a–c). Dorsal view, anterior is upwards in (d–f,i–k). Cross sections at the position indicated by dashed lines in (d–f) are shown in (d′–f′), respectively. DAPI staining revealed that wider neural plate and notochord. Notochord was indicated by white dots. (g,h) Width of Rx2A domain (indicated in (a–c) and the width per length ratio of the Dlx3-negative region (neural plate) were measured. The vertical axes in (g) and (h) indicate arbitrary units and the width per length ratio, respectively. The width and length of the Dlx3-negative region were defined as indicated in the bottom panel (h). Because similar results were obtained from at least two independent experiments, a representative experiment is shown. The number of samples (no) was indicated below the bar graph. Arrows indicate floor plate expression (i–k). (D) cresMO does not affect on the expression of the mesoderm and neural markers. The expression of Xbra (a,b), XmyoDa (c,d), and chordin at stage 11 (e,f), goosecoid at stage 10.5 (g,h), and sox2 at stage 14 (i,j) were examined. nβ-gal mRNA was coinjected as a lineage tracer (C)–(E), but distributions of nβ-Gal activity does not necessarily correspond to those of MOs because MOs could be more diffusible than mRNAs. crescent mRNA was coinjected for rescue experiments (B) and (C). Amounts of MOs (ng/embryo), or mRNAs (pg/embryo): standard MO (stdMO), 200 (Ca,d,i; D) or 400 (A); cresMO, 200 (A; B; Cb,c,e,f,j,k; E) or 400 (A); 5 mm MO, 400 (A): crescent mRNA, 5–10; nβ-gal mRNA, 30. Numbers in each panel indicate the frequency of the represented phenotype from 1 (D), 2 (B, Ck), or 3 (Ci,j) experiments. Image published in: Shibata M et al. (2005) Copyright © 2005. Image reproduced with permission of the Publisher, Elsevier B. V.
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