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Figure 4.
tra2b Knockdown Reveals a Novel Inhibitory wnt11b Isoform
(A) RNA-seq read profile and Cufflinks assembled transcripts on the wnt11b locus show retention of intron 4. The top panel shows the JGI gene model for wnt11b, and the middle and bottom panels show read profiles and Cufflinks-assembled transcripts from control and morphants.
(B) Intron 4 retention results in a truncated protein (red, Wnt11b-short) resembling a dominant-negative ligand (yellow) that lacks 57 C-terminal residues compared with normal (black).
(C) wnt11b is expressed in the presomitic mesoderm/circumblastoporal region and somites. Embryos are shown in dorsal view with anterior to the left.
(D) wnt11b-short mimics wnt11b-dn in a pronephric tubule inhibition assay. Embryos were injected unilaterally into the prospective lateral mesoderm and examined by ISH for atp1a1, which marks the developing pronephros. Arrows point to the proximal pronephric tubules on the injected side.
(E) Summary of the pronephros tubule inhibition assay. Number of embryos scored: 144 (control), 72 (tra2bMO), 87 (wnt11b-dn), and 69 (wnt11b-short).
(F) qRT-PCR for induced cardiac gene expression (gata4) or axial mesoderm (t/bra) on animal caps injected with combinations of activin, wnt11b, wnt11b-dn, and wnt11b-short, showing that wnt11b-short acts similarly to wnt11b-dn and counters the effect of wnt11b. Bar plots show the mean of three independent experiments + SEM of normalized fold induction compared with activin-injected embryos.
(A) shows data from X. tropicalis and (C)–(F) show data from X. laevis. See also Figure S4. |
