Amphioxus-human conserved noncoding sequences function as enhancers. (A–G) LacZ reporter gene expression driven by the conserved noncoding element (CNE) near amphioxus ZNF503/703 in mouse (A–C) and amphioxus (D–G). (H–L) Expression driven by the orthologous CNE near human ZNF503 in mouse (H–J) and amphioxus (K,L). (M) Expression of the endogenous amphioxus ZNF503/703 gene in the anterior central nervous system (n), somites (s), pharynx (p), and weakly in the notochord. (N–T) Expression of the orthologous CNE near ZNF703 in mouse (N–P) and amphioxus embryos (Q–T). B, I, and O show dorsal views of the mouse hindbrain; all other views are lateral, with anterior to the left. Mouse embryos shown at 9.5 d post conception. The mouse ZNF503 construct directs expression to the forebrain, midbrain, hindbrain, eye, branchial arches. Blue arrowheads show expression in the eye directed by the two human elements, but not the amphioxus element. The red arrowhead shows expression in a lateral domain that is directed by the amphioxus element, but not the human elements. Purple arrowheads indicate sites of expression observed for all three CNEs. The amphioxus ZNF503/703 CNE construct expresses at a high level in the amphioxus notochord (n) and somites(s) and at a lower level in the ectoderm (e) and central nervous system (nc). The embryo in D has an abnormal head, presumably due to injection trauma. Ectopic expression in necrotic cells in the gut lumen as in G, L, and S is common in amphioxus embryos injected with reporter constructs. Twenty-four-hour embryos (D,E,Q–T), 30 h larvae (K–M), 36 h larvae (F,G).
