Fig. 2

Genomic structure, plasmid constructs, and results of the transient transgene expression assay. (A) EcoRI restriction map of the genomic region flanking the zebrafish isl1 gene. The lines under the map indicate the regulatory regions of the isl1 gene. ICP, the isl1 gene core promoter; CM, an enhancer for the cranial motor neurons; SS and SSX, enhancers for the primary sensory neurons and the fin motor neurons. The thick line, isl1, indicates the transcribed unit of the isl1 gene. Note that the isl1 gene is composed of six exons divided by five introns. (B) Maps of plasmids used in this study. Each enhancer fragment was inserted immediately upstream of the promoter sequences. PLAP, human placental alkaline phosphatase gene. (C and D) Each plasmid construct shown here was injected into one-cell stage zebrafish embryos to examine its enhancer activity. The number on the right side of the name of each construct indicates the number of embryos exhibiting expression of GFP in cranial motor neurons (C) or primary sensory neurons (D) among all injected embryos. X, XhoI; P, PstI; B, BstXI. (C) Note that nucleotide conversion in TAAT to GGGG (termed zCREST1m, asterisk in Fig. 5A) completely inactivated its enhancer activity. (E) Dorsal view of a 48-hpf embryo injected with the zCREST1-hsp70:GFP plasmid and expressing GFP in the majority of cranial motor neurons IV, trochear motor neurons. (F and G) Lateral views of 32-hpf embryos injected with the zCREST2-hsp70:GFP plasmid and expressing GFP in the trigeminal ganglion neurons (F) and Rohon-Beard neurons (G).