Fig. 3

CesA expression and regulation in different ascidian species. A–C In situ hybridization for CesA during embryonic development of Phallusia mammillata (A), Ascidia mentula (B), and Molgula appendiculata (C). In P. mammillata, the expression was first detected in the caudal part of late neurulae (Aii). Expression was found throughout the epidermis with weaker staining in the palp forming region (Aiv). A cross-section of the tail shows that the staining was limited to the epidermis and not found in internal tissues (Av). A similar pattern was found in A. mentula (B) and M. appendiculata (C) but with a much earlier onset of expression in the latter species (early gastrula: Ci). D In situ hybridization for CesA.b during embryonic development of P. mammillata. Transcripts were found throughout the epidermis from late tailbud stages (St. 24) with weaker expression in dorsal and ventral tail midlines (Diii–Dv). Embryos at the following stages are shown: gastrula (St. 10–11) (Ai,Bi,Ci), neurula (St. 14–16) (Aii,Bii,Cii), early/mid tailbud (St. 20–22) (Aiii-Av,Biii,Ciii,Di,Dii), and late tailbud (St. 24–25) (Avi,Biv,Civ,Diii-Dv). E–G Transcriptional regulation of Phmamm.CesA in the epidermis. A region of around 1 kb, Phmamm.CesA.up1, situated immediately upstream of CesA (E) was PCR-amplified and placed upstream of the LacZ reporter in inverted orientation. When tested in P. mammillata embryos (F), it was inactive at early tailbud stages (St. 19/20, no embryo stained in the epidermis, n = 185, a single experiment) and active in the epidermis at late tailbud stages (St. 23/24, 60% of the embryos with epidermis staining, n = 703 from 3 independent experiments). This region contains 2 putative Tfap2 binding sites (yellow bars in G). A synthetic version of Phmamm.CesA.up1, Phmamm.CesA.up, and 3 variants with Tfap2 mutations were placed in “correct” orientation upstream of LacZ, and their activity tested at mid-tailbud stages (St. 22) is summarized on the graph on the right in G (Phmamm.CesA.up, n = 296; Phmamm.CesA.up_mut1, n = 282; Phmamm.CesA.up_mut2, n = 363; Phmamm.CesA.up_mut12, n = 268; results from three independent experiments). H Swap assay of putative CesA promoters from three ascidian species. Each region was tested in both P. mammillata (Phmamm, blue) and C. intestinalis (Ciinte, orange), and the activity at mid/late tailbud stages in epidermis is displayed on the graph on the right (Phmamm.CesA.up1: in Phmamm n = 703 from 3 experiments, in Ciinte n = 348 from 2 experiments; Asment.CesA.up1: in Phmamm n = 899 from 4 experiments, in Ciinte n = 896 from 3 experiments; Moappe.CesA.up1: in Phmamm n = 767 from 3 experiments, in Ciinte n = 712 from 3 experiments). All pictures of embryos are lateral views with dorsal to the top and anterior to the left, except: animal views (Ai,Bi), vegetal view (Ci), neural plate views (Bii,Cii) with anterior to the left, frontal view (Aiv), dorsal view (Div), ventral view (Dv), and cross-section of the tail (Av). Individual data values can be found in Additional file 7: Table S1