Functional Study On Conserved Non-coding Elements Around Pax Genes In Deuterostomes

Decades of study in developmental genetics has revealed that the embryonic patterning of all metazoans is controlled by a common set of regulatory genes,such as Pax,BMP,Wnt,Nodal and FGF.These genes sometimes show common expression patterns and genetic interactions,suggesting the existence of similar or even conserved regulatory circuits.Over thousands of conserved regulatory sequences(also called conserved non-coding elements,CNEs)have been identified around these patterning genes within an animal group like vertebrates,flies and nematode,but surprisingly no such sequence were found among all metazoans.Aiming to resolve this discrepancy,here we focused on the Pax family genes which show many similar expression patterns among all examined deuterostomes.By carefully aligning the Pax gene sequences from vertebrates,Urochordata Ciona intestinalis,Cephalochordata Branchiostoma belcheri and Echinodermata Strongylocentrotus purpuratus,we found an unexpected large number of CNEs which are conserved in all studied species including the much derived Urochordata tunicate.Further functional assays to three of these CNEs from Pax2/5/8 gene in amphioxus and zebrafish embryos showed that each orthologous CNE from mouse,tunicate,amphioxus or sea urchin could drive LacZ reporter gene to express in a similar pattern in amphioxus or zebrafish embryos.When comparing their expression patterns between amphioxus and zebrafish we found all three CNEs have co-optioned novel regulatory activities in vertebrate brain development,probably through recruiting new transcription factors(TFs)on them.In summary,our study demonstrated for the first time that the transcriptional-developmental genes were regulated by common sets of cis-regulatory elements among deuterotomes or even among all metazoans.We predicted that more CNEs would be identified between diverse animal groups in future studies.Additionally,our work also suggested that by binding to novel TFs,old CNEs could confer novel regulatory activities to their target genes and thus expand the expression profiles of the target genes.This finding provided an alternative explanation for the evolution of vertebrate gene regulatory complexity,and thus gave new insights into the origin of vertebrates.In addition,we analysed and discussed the development of high efficiency Actin promoters and a stable genome editing method in amphioxus in the Chapter 2 and 4.