Fig. 7. Summary of expression of endodermal markers in amphioxus at the mid-neurula (20 hours) and early larval (30 hours) stages for (A) controls, (B) RA-treated embryos and (C) BMS009-treated embryos. The thickness of the lines is proportional to the relative level of expression. Changes of expression in the mouth and gill slit primordia are not shown. The position of the pigment spot in the nerve cord at the level of somite 5 is not affected by levels of RA signaling. Markers of the posterior foregut/midgut endoderm (AmphiRAR, AmphiHox1 and AmphiWnt3) are in light blue, those with expression restricted to the pharynx from the early neurula (AmphiPax1/9, AmphiPitx and AmphiNotch) are in purple, markers with expression becoming restricted to the pharynx at the early larval stage (AmphiNodal and AmphiOtx) are in orange, and those expressed throughout the length of the endoderm at all stages (AmphiIslet, AmphiFoxA2 and AmphiHh) are in brown. These groupings in controls (A) are predictive of the changes in expression in response to RA (B) and the RA antagonist BMS009 (C). In controls (A) AmphiRAR, AmphiHox1 and AmphiWnt3 are most strongly expressed in the middle third of the endoderm. RA (B) shifts their expression anteriorly. BMS009 (C) downregulates AmphiRAR and AmphiHox1 and by 30 hours shifts posteriorly their anterior limits of expression and expands posteriorly the domain of AmphiWnt3. In controls (A), expression of genes in the second group (AmphiPax1/9, AmphiPitx and AmphiNotch) is restricted to the pharynx at both stages. RA (B) shifts the posterior limits of their expression anteriorly and by 30 hours downregulates expression of AmphiPitx and AmphiNotch. BMS009 (C) has the opposite effect, expanding the domains of all three posteriorly. In controls (A), expression of genes in the third group (AmphiNodal and AmphiOtx) becomes restricted to the pharynx by 30 hours and is not affected by RA (B) until 30 hours, when expression is truncated posteriorly and/or downregulated. BMS009 (C) keeps expression of both genes high throughout much of the length of the endoderm. In controls (A), genes in the fourth group (AmphiIslet, AmphiFoxA2 and AmphiHh) are expressed throughout most of the length of the endoderm at both stages. The posterior limits of their expression are not markedly affected by either RA (B) or BMS009 (C).