XB-IMG-3126
Xenbase Image ID: 3126
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Figure 7. WDR5 Is Essential for X. laevis Development(A) Dorsal (Aa), ventral (Ab), and lateral (Ac) view of stage 40 X. laevis tadpoles, developed from one-cell stage embryos injected with xWDR5 morpholino oligonucleotide to a final concentration of 10 μM. Axial deformities and abdominal lesions are indicated with open and closed arrowheads, respectively. Embryos coinjected with xWDR5 morpholino and GST-hWDR5 mRNA (Ad) or injected with GST-hWDR5 mRNA alone (Ae) do not significantly differ from control tadpoles (Af), indicating that phenotypes observed in (Aa)–(Ac) are the specific result of xWDR5 knockdown.(B) Effect of xWDR5 morpholino on xWDR5 expression and H3 K4 methylation. One-cell stage embryos control injected (lane 1), injected with xWDR5 morpholino (lane 2), or coinjected with xWDR5 morpholino and GST-hWDR5 mRNA (lane 3) were developed to the tailbud stage, and endoderm was excised to eliminate egg yolk. Embryos lacking endoderm were subsequently used for whole-cell extract preparation. Extracts were analyzed by immunoblotting with α-WDR5 (Ba), α-H3 K4(Me)3 (Bb), and α-tubulin (Bc) antibodies.(C) Quantitative analysis of axial deformities (“Axis”) and abdominal lesions (“Gut”) present in morpholino-injected embryos (“Morpholino”), embryos coinjected with morpholino and GST-hWDR5 mRNA (“Morph.+RNA”), or injected with GST-hWDR5 mRNA alone (“RNA”). Total number of counted embryos is indicated at the top of each column. Differences between morpholino-injected and RNA + morpholino-coinjected populations are highly statistically significant (p < 2 × 10−16) as determined by test for equal proportions and indicated at the top of the plot. Coinjection of GST-hWDR5 mRNA with morpholino rescues the phenotypes essentially to the penetrance observed in control sample (p = 0.35 for axial and p = 0.003 for the gut defects).(D) Feeding stage tadpoles derived from two-cell stage embryos were control injected (Da and Dc) or injected in one of the two blastomeres with 10 nl of 0.5 mM xWDR5 morpholino (Db and Dd). Morpholino-injected tadpoles exhibit axial deformation on the injected side, abnormal distribution of blood cells (blood is absent from heart while forming a hemorrhage in the tail), and failure of gut patterning.(E) Quantitative analysis of the penetrance of phenotypes presented in (D). Number of occurrences of axial defects (“Axis”), blood distribution abnormalities (“Blood”), and gut coiling defects (“Gut”) were noted in mock injected (“Control”) and morpholino injected (“Morpholino”) and plotted as proportions in total population counted. Total number of counted embryos is indicated at the top of each column. Observed differences are highly statistically significant (p ≤ 0.0001, test for equal proportions), and the p values are indicated at the top of the plot.(F) Whole-mount in situ detection of xHoxC8 transcript. Dorsal view of midtrunk region of stage 30 tadpoles derived from embryos control injected (Fa) or injected at two-cell stage in one of the blastomeres with xWDR5 morpholino (Fb–Fd) is shown. Purple stain indicates xHoxC8 expression, which is most prominent in neural tube. Arrows indicate misalignment of the xHoxC8 expression domains between left and right side of neural tube in morpholino-injected but not control embryos. All images are shown in the same orientation (see [Fa], upper right corner: A–P, anterior-posterior axis; L–R, left-right axis). Image published in: Wysocka J et al. (2005) Copyright © 2005. Image reproduced with permission of the Publisher, Elsevier B. V.
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