The role of vertebrate conserved non-coding elements in hindbrain development and evolution

Vertebrate conserved non-coding elements (CNEs) act as cis-regulatory modules of developmental genes. To assess their roles in coordinating gene expression during embryogenesis, CNEs were subjected to motif searches. Using reporter gene assays in zebrafish (Danio rerio) embryos, Pbx-Hox (TGATNNAT) motifs are demonstrated to be poor predictors of hindbrain enhancer activity. Hindbrain enhancer CNEs are distinguished from hindbrain negative CNEs accurately by virtue of co-occurring Pbx-Hox (TGA TDDA TKD) and Meis/Pknox (CTGTCA) motifs. The grammar of these motifs was investigated using a bioinformatic pipeline for the detection of multiple conserved motifs, revealing no patterns in their relative organisation aside from spatial co-occurrence. These motifs were then used to identify additional conserved hindbrain enhancers with high efficacy (89%). Substitutions targeted to either motif abrogate expression by the enhancer or generate ectopic reporter gene expression, suggesting that motif co-occurrence is required for efficient and segment-specific hindbrain activation. Pbx-Hox and Meis/Pknox motifs are enriched in gnathostome CNE sets but are not detected in invertebrate chordate CNEs. Furthermore the presence (or absence) of the hindbrain syntax correlates with the conservation (or lack thereof) of segment-restricted enhancer activity in orthologous CNEs from the sea lamprey. A library of zebrafish hindbrain regulatory elements is made available. The heterogeneity of function and the loose grammar of motifs are consistent with combinatorial factor binding; a model of CNEs as exceptionally well- conserved billboard enhancers is presented (inflexible billboard model). The implications of these data for models of the evolution of the vertebrate hindbrain are discussed. Several components of the hindbrain gene regulatory network are shared- derived characters of gnathostomes, suggesting the establishment and elaboration of the conserved regulatory code controlling hindbrain development on the vertebrate and gnathostome stems, respectively.