Fig. 5. Two phases of RA signalling in the mouse cardiac field. A-E (left-side views) and F-K (frontal views). A-E and G-I are mouse Raldh2 (orange) and mouse Tbx5 (purple) double in situ hybridizations. (A) Late bud (LB) stage. Cardiac precursors express mouse Tbx5 and occupy an anterior position. Mouse Raldh2 is expressed in mesoderm posterior to the node (*). Separation between cardiac precursors and RALDH2 is maximal. (B) LB stage. Raldh2 expression expands in the anterior lateral mesoderm. Mouse Tbx5 expression increases forming a stripe in the lateral mesoderm oriented in a posterior-direction towards the advancing RALDH2 caudorostral wave. (C) Early headfold stage (EHF). The gap separating cardiac precursors from RALDH2 is decreased. (D) EHF stage. mouse Raldh2 expression advances to contact the most posterior cardiac precursors. (E) Late headfold stage (LHF). RALDH2 penetrates the cardiac field and overlaps posterior cardiac precursors (white arrowhead). (F) LHF stage. Double mouse Tbx-5 in situ hybridization/lacZ staining in RA-indicator embryos. Mouse Raldh2 expression takes RA signalling to posterior cardiac precursors. In this embryo RA signalling overlaps the posterior third of the cardiac field (bracket). (G-I) Somite stages. Embryos display increasing overlap between Raldh2 expression and cardiac precursors (black arrows). (J,K) RALDH2 immunohistochemistry. Arches of RALDH2 expression joined at the midline in sino-atrial tissue below the anterior intestinal portal (AIP) (star) in embryos respectively displaying looped and unlooped hearts.

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