The functions in a genome are often conceptually divided into protein functions for coding DNA and regulatory functions for noncoding DNA. This division is based on the intuition that constraints associated with encoding a protein would prevent the evolution of noncoding functions in a coding region. However, the validity of this division has not been well-studied. One important class of regulatory functional elements in noncoding DNA is enhancers. These are DNA sequences classically found distal to gene promoters and associated with tissue- or temporally-specific transcriptional regulation of gene expression, especially for developmental genes 
. Here we investigate whether protein-coding DNA can contain enhancer functions similar to those found in noncoding DNA.
Prior computational and evolutionary studies at the motif level have shown that coding DNA can hold noncoding information. This ability to contain other functional information arises from the redundancy of synonymous codons. For example, Itzkovitz and Alon compared the human genetic code to alternative permuted codes, finding that the genetic code is nearly ideal for containing short functional motifs within protein-coding DNA 
. Using a sequence conservation approach, hundreds of unusually conserved nucleotide motifs have been found in coding sequences even after correcting for protein-level constraint 
. Additionally, multiple genome-wide transcription factor and histone modification studies have reported low-levels of protein binding within coding sequence 
. However, because a substantial fraction of protein-DNA binding is believed to be neutral, it has often assumed that such binding in coding regions is non-functional 
. In any case, assessments of functional motifs in coding sequence do not strongly test the ability of protein coding sequence to hold dual functions. This is because motifs are short in comparison to mRNA lengths.
Protein-coding sequences can be more critically tested by considering developmental enhancer activity. Developmental enhancers are typically much longer than individual TF-binding motifs and are often associated with strong sequence constraint. Highly conserved noncoding sequences have shown frequent enhancer activity in developmental expression assays 
. For example, three-fourths of noncoding sequences with >60% human-teleost conservation have shown enhancer activity in developmental assays 
. Therefore discovery of developmental enhancers in coding regions would indicate that long, highly constrained regulatory functions can evolve in coding regions despite the protein-coding constraint.
Relatively little is known about enhancers in coding sequence. Coding exon-controlled enhancer activity has been reported in a few cell line experiments, e.g. from the APOE, ADAMTS5 and BCL-2 genes 
, but whole embryo experiments would provide more definitive evidence of developmental activity. Such embryonic data is relatively sparse and has shown conflicting results. Tumpel et al observed that the second exon of Hoxa2
contains a consistent but weak developmental enhancer within a coding region, though reporter activity was found to be stronger when the coding region was combined with an adjacent noncoding sequence 
. Similarly, Lampe et al identified a coding enhancer in the first exon of Hoxa2 
. However, Woolfe et al tested three coding sequences (from Sox21, Pax6 and SHH in zebrafish developmental assay), but found little to no expression 
. The relative dearth of experimental data has made it unclear how prevalent coding developmental enhancers are.
To address this question, we investigated the enhancer functions of 31 coding sequences from a variety of developmental genes orthologous between human and zebrafish. We chose C
lements (hereafter CCEs) with strong conservation across vertebrate species for this study, as we expected these might be more likely to contain enhancers 
. Using whole-embryo experiments, we found that the coding sequence of many developmental genes contains enhancers that drive tissue-specific gene expression. Our results indicate that enhancers in coding regions and in noncoding regions have similar levels of activity, tissue-specificity and enhancer-associated histone modifications. Thus the protein-coding constraint does not exclude noncoding developmental regulatory information. Our work indicates that complex additional functions may be commonly harbored in protein-coding sequences of vertebrate genomes.