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Fig. 2. Morpholino-based studies of sizzled function.
(A) 1 cell embryos were injected with RNA encoding either Sizzled-HA or SizzledD92N-HA (200 pgs/embryo) together with 200 pgs/embryo GFP RNA either alone or together with 7 ngs/embryo sizzled morpholino; immunoblot analysis of stage 11 embryos indicated that GFP accumulation was unaffected by morpholino injection, while Sizzled-HA protein levels were reduced, but not completely eliminated. Injection of the sizzled morpholino into 1 cell of 2 cell embryos, together with RNA encoding lacZ as a lineage marker, revealed that compared to control embryos (B), there was a decrease in xbra in situ hybridization staining (C) that could be rescued by the co-injection of sizzled RNA (D). Similarly, the expression of the endodermal marker endodermin (E) was increased in sizzled morphant embryos (F), and this increase was reversed by sizzled RNA injection (G). These effects extended into later stages. Staining with the monoclonal antibody 4A6, which labels the pronephros, revealed a decrease in staining on the injected (H*) compared to the uninjected sides (H) of stage 30 embryos. Similarly, the expression of myoD ((I)-control, (J)-sizzled morpholino) (* indicates injection artifact) and the organization of somatic myotomal muscle, visualized by whole-mount staining with an antibody against tropomyosin, ((K), (M)-control, (L)-sizzled morpholino-injected side, (N)-section of whole-mount stained, injected embryo) were disrupted in sizzled morpholino injected regions of the embryo (* indicates injected side in part N). |
