Cross-species regulatory activity of the C. robusta and D. rerio Hox5 and Otx CNEs. (A) Representation of the C. robusta Hox5 CNE expression in the most anterior part of the tail neural tube at larva stage. (B) The D. rerio Hoxa5a CNE shows well defined expression profile in the anterior part of the Ciona neural tube in a pattern reminiscent that of the Ciona Hox5 CNE (black arrowheads). (C) Representation of the C. robusta Otx CNE-B expression in the sensory vesicle(black arrow). (D) The D. rerio Otx CNE-B shows expression in the sensory vesicle (black arrow) and extends posteriorly along the visceral ganglion (black arrowhead). (E) C. robusta Otx CNE-B is active in a D. rerio transgenic embryo at 72 hpf. GFP signal (green) is visible in Te (white arrowhead), RGC (white arrow) and MB (orange arrowhead). As control, RFP expression (red) in muscles under the control of cardiac actin promoter. Diffuse GFP signal in the yolk is unspecific (asterisk). (F) Schematic representation of the D. rerio Otx1 gene endogenous expression at 72 hpf. Otx1 is expressed in RGCs, Te, D, MB and HB. Abbreviations: D, diencephalon; HB, hindbrain; MB, midbrain; RGC, retinal ganglion cells; Te, telencephalon. A-E Ciona embryos at larva stage. B, dorsal view; A,C-F, lateral view, anterior is on the left. 

Comparison of the regulatory activity of tunicate-specific and chordate-specific Hox5 and Otx T-CNE/CNE. (A-C) Comparable expression of M. occulta Hox5 T-CNE, C. robusta chordate-specific Hox5 CNE and D. rerio Hoxa5a CNE in the most anterior part of the tail neural tube (black arrowheads). MoccHox5 T-CNE shows a more restricted expression in a subdomain of CrHox5 and DrHox5CNEs at larva stage. (D-F) The tunicate-specific M. occulta Otx T-CNE (D), and the chordate-specific C. robusta Otx CNE-A (E) and CNE-B (F) show a similar expression profile in the ventral part of the sensory vesicle (black arrows). (G,H) The tunicate-specific OdOtx T-CNE reproduces almost the same expression profile of the chordate-specific DrOtx CNE-B in the posterior sensory vesicle and in the neural axons along the visceral ganglion. Black/white arrowheads and arrows indicate the corresponding conserved territories of expression. Asterisks indicate ectopic expression in the mesenchyme. A-F,H: lateral view; G, dorsal view. Anterior is on the left.

Representation of the tunicate-specific CNEs showing no signal or not consistent staining in the mesenchyme. (A, B, E)The Hox5 T-CNEs from B. schlosseri and O. dioica and the Otx T-CNE from C. robusta show extended expression in the head mesenchyme. (C, F) MoccIpf1 T-CNE and BsOtx T-CNE are expressed in the mesenchymal pockets of the head. (D) The C. robusta Hox5and Ipf1 T-CNEs do not show any signal in transgenic larvae.