Overview of EGFP expression scoring process.

(A) Zebrafish with eGFP expression are scored using a (B) limited anatomy corresponding to (C) numerical values. (D) These are interpreted using the iPhone app Dragon Dictation. (E) A PERL script transforms text into numerical strings representing embryo expression in each anatomy. These data are analyzed to determine anatomical regions with significant expression for each CCE via a proportions test and a Wilcoxon rank-sum test (see Methods and Supplementary Data File S2).

Specific and Non-Specific CCE Activity.

(A) Examples of Specific CCE Activity. CCEs from the genes gria3b, rab11fip4a, prim1, and abca1a each drove robust expression in a finely localized anatomical region. Overall, 14 CCEs produced this type of specific expression (defined as expression in 4 or fewer anatomical regions). (B) This behavior contrasts with CCEs that drove robust but non-specific expression, such as CCE-ephb3a. 6 of the active CCEs drove nonspecific expression.

Comparison of CCE-lmo1 Stable and Transient Transgenic Expression.

(A) Stable transgenic F1 embryos from two independently generated lines displaying strong forebrain and hindbrain expression. Supplementary Figure S3 shows this behavior in a larger group of stable transgenic embryos. (B) Similarly, three transient transgenic embryos injected with CCE-lmo1 display analogous forebrain and hindbrain expression.

Representative images of CCE expression and host gene expression (mRNA in situ hybridization data from ZFIN) for 4 CCEs, showing overlap between CCE activity and host gene expression.

CCE-fez1 drives expression in multiple anatomies, with significant concurrent activity in forebrain, mid/hindbrain and eye. 4 representative embryos are shown.

CCE-sfrs3b, an alternatively spliced exon, is shown here to drive enhancer expression in the eye and brain, despite poison cassette activity of the exon.

Plasmid Design and Injection. Flanking Tol2 sequences integrate the control or experimental cassette into the zebrafish genome after injection with plasmid and transposase mRNA at the 1-cell stage.

A group of stable transgenic embryos (F1) derived from embryos injected with CCE-lmo1. Injected embryos were selected for forebrain and hindbrain expression and then crossed with wildtype zebrafish to yield the F1 generation.

Comparison of CCE-lmo1 expression to Zfin stages. CCE-lmo1 maintains strong similarity to the mRNA in situ hybridization of LMO1 throughout a large window of development (22–42 hpf).

CCE-islet expression in the heart.

CCE-ddx18 displays expression in the forebrain and midbrain, consistent with annotations in the ZFIN database. However, the diffuse expression patterns around the tectum and eye (particularly at ~22–24 hpf) make it difficult to visually determine whether there is agreement on a finer scale.

Images from the 20 significant CCEs and their corresponding anatomies. Images are labeled with (CCE-GeneName, ExonNumber), and the significant anatomy for each CCE is labeled. To view more images for each CCE, please visit: http://bioinformatics.bc.edu/chuanglab/C odingEnhancer

Images from the 20 significant CCEs and their corresponding anatomies. Images are labeled with (CCE-GeneName, ExonNumber), and the significant anatomy for each CCE is labeled. To view more images for each CCE, please visit: http://bioinformatics.bc.edu/chuanglab/C odingEnhancer

Images from the 20 significant CCEs and their corresponding anatomies. Images are labeled with (CCE-GeneName, ExonNumber), and the significant anatomy for each CCE is labeled. Note that CCE-ddx5 has voice-expression data but lacks an image of yolk expression. To view more images for each CCE, please visit: http://bioinformatics.bc.edu/chuanglab/C odingEnhancer