Fig. 4

Gh6 expression and regulation in different ascidian species. A–D In situ hybridization for Gh6 during embryonic development of Ciona intestinalis (A), Phallusia mammillata (B), Ascidia mentula (C), and Molgula appendiculata (D). For the three phlebobranch species, Gh6 was first detected at early tailbud stages in the epidermis at the tip of the tail. Then expression started anteriorly in the palp region and in the tail epidermis at late tailbud stages. Tail epidermis expression was detected in the four medio-lateral rows of cells (Bv,Bvi,Bviii). Expression in the palp region was likely absent from the future protruding papillae (Bvii). By contrast, Moappe.Gh6 was expressed broadly in the epidermis starting at neurula stages (Dii–Div). Embryos at the following stages are shown: gastrula (St. 10–11) (Bi,Ci,Di), neurula (St. 14–16) (Ai,Bii,Cii,Dii), early/mid tailbud (St. 20–22) (Aii,Aiii,Biii,Ciii,Diii), and late tailbud (St. 24–25) (Aiv,Biv–Bviii,Civ,Div). E Transcriptional regulation of Phmamm.Gh6 in the epidermis. A 3.6-kb region starting at the beginning of the scaffold 1127 immediately upstream of Gh6, Phmamm.Gh6.up1, was PCR-amplified, placed upstream of the LacZ reporter, and tested in vivo. It was active only in a part of the Phmamm.Gh6 expression domain, the tail epidermal medio-lateral cells at late tailbud stages (St. 19–22: 3% of embryos with staining in this expression domain, n = 401; St. 24–25: 75% of embryos with staining in this expression domain, n = 521; results from four experiments). Phmamm.Gh6.up2, a 1.5-kb region embedded in Phmamm.Gh6.up1 containing conserved segments, had a similar activity (St. 19–22: 0% of embryos with this expression domain, n = 670 from three experiments; St. 24–25: 56% of embryos with this expression domain, n = 556 from four experiments). F Transcriptional regulation of Cirobu.Gh6 in the epidermis. Cirobu.Gh6.up1, a 2.7-kb region that almost corresponds to the entire upstream intergenic region, fully recapitulated Gh6 expression with tail tip activity (arrows) detected in early tailbuds, and palp (arrows) and tail epidermis activity in late tailbuds (St. 19–22: 45% of embryos with expression in endogenous territories, n = 300; St. 24–25: 73% of embryos with expression in endogenous territories, n = 300; results from two experiments). G Phmamm.Gh6.up1 was not active in C. intestinalis embryos (n = 367 from two experiments). H Cirobu.Gh6.up1 was active in Gh6 expression domains in P. mammillata embryos (St. 21–22: 36% of embryos with expression in endogenous territories, n = 448 from three experiments; St. 23–24: 72% of embryos with expression in endogenous territories, n = 210 from two experiments). All pictures of embryos are lateral views with dorsal to the top and anterior to the left, except: animal views (Bi,Ci), vegetal view (Di), neural plate views (Ai,Cii,Dii) with anterior to the left, frontal view (Bvii), dorsal view (Bv), ventral view (Bvi), and cross-section of the tail (Bviii). Individual data values can be found in Additional file 7: Table S1