Figure 4. The timing of Hedgehog induction of lymph hearts corresponds to the onset of en1 expression.

Cyclopamine or ethanol (as a solvent control) was added to embryos at the specified developmental stages; tadpoles were fixed at stage 42–45, and stained with 12/101 for presence of lymph heart musculature. The ethanol controls for all stages are combined in this graph, but χ² tests were performed between stage-matched cyclopamine and control samples for determination of statistical significance. Cyclopamine treatment has a less potent effect when added after stage 20 (4% wildtype at st.20 vs. 35% wildtype at st.22). Experimental and control samples are not significantly different when embryos are added to cyclopamine at stage 26, just prior to en1 expression in lymph heart myoblasts, or later in development (*p<0.01).