Enhancers are elements within our genome that control where and when genes are expressed throughout development. However, how the sequence of the enhancer regulates tissue-specific expression is not fully understood. We can investigate sequence by looking at the transcription factor binding motifs within enhancers. We want to better understand how combinations of motif syntax: the order, orientation, and spacing of motifs, interplays with motif affinity to regulate gene expression. We term the connections between syntax and affinity enhancer grammar. We use the neural plate Otx-a enhancer within Ciona intestinalis as a model to investigate if enhancer grammar is present, and to look for motif syntax and affinity trends giving different expression patterns. This enhancer activates when bound by GATA and ETS transcription factors. Our data shows that there is a grammar present within the Otx-a enhancer, as different grammatical variants give varying expression patterns. Our data suggests that changing the motif order to having ETS binding sites on both ends of the enhancer abolishes Otx gene expression in most developing embryos. Our data also shows a loss of expression caused by ETS motifs directly next to each other in combination with non-optimal spacing between high affinity GATA and ETS. We also found a grammatical variant with notochord expression, which may be due to two ETS sites close to a FoxA binding site. These findings help us better understand the grammar of the Otx-a enhancer and help us understand how enhancer sequence codes tissue-specific gene expression in development.