Enhancer activities of amphioxus Brachyury genes in embryos of the ascidian, Ciona intestinalis

Abstract

The notochord and somites are distinctive chordate structures. The T-box transcription factor gene, Brachyury, is expressed in notochord and plays a pivotal role in its formation. In the cephalochordate, Branchiostoma floridae, Brachyury is duplicated into BfBra1 and BfBra2, which are expressed in the somite-formation region as well. In a series of experiments to elucidate the regulatory machinery of chordate Brachyury expression, we carried out a lacZ reporter assay of BfBra in embryos of the urochordate, Ciona intestinalis. Vista analyses suggest the presence of conserved non-coding sequences, not only in the 5'-upstream, but also in the 3'-downstream and in introns of BfBra. We found that: (1) 5'-upstream sequences of both BfBra1 and BfBra2 promote lacZ expression in muscle cells, (2) 3'-downstream sequences have enhancer activity that promotes lacZ expression in notochord cells, and (3) introns of BfBra2 and BfBra1 exhibit lacZ expression preferentially in muscle and notochord cells. These results suggest shared cephalochordate Brachyury enhancer machinery that also works in urochordates. We discuss the results in relation to evolutionary modification of Brachyury expression in formation of chordate-specific organs characteristic of each lineage.

A Preliminary Single-Cell RNA-Seq Analysis of Embryonic Cells That Express Brachyury in the Amphioxus, Branchiostoma japonicum

“…The present scRNA-seq study showed that Bra2 is expressed at higher levels and in broader embryonic regions than Bra1 (Figure 6). Since Bra2, but not Bra1, is expressed in cells of the blastopore (Yuan et al, 2020; this study) and since Brachyury expression in the blastopore or archenteron invagination region is a feature shared by most metazoans, Bra2 is the ancestral Brachyury and Bra1 is a duplicated copy, with more divergent properties, as proposed by previous studies (Tominaga et al, 2018;Yuan et al, 2020). Bra2 was strongly expressed first in the archenteron invagination region and functions in gastrulation, then in invagination-like movements of cells of the upper archenteron to form the notochord and that of cells of bilateral sides of the archenteron to form somites, and contiguously from the blastopore, in tail bud formation at the posterior-most region of amphioxus embryos.…”

“…They showed that the zygotic expression level of Bra2 is much higher than that of Bra1, and that Bra2 is highly expressed in the blastopore, tail bud, and notochord, while Bra1 is weakly transcribed only in the notochord. In addition, a heterogenic transplantation assay of cis-regulatory sequences of Bra1 and Bra2 into the Ciona embryonic system demonstrated that the 5' upstream sequence of Bra2 contains higher enhancer activity in both the notochord and somites, compared to that of the 5' upstream sequence of Bra1 in the notochord (Tominaga et al, 2018). These results suggest that both Bra1 and Bra2 are Brachyury orthologs and that Bra1 has diverged more.…”

A single motor neuron determines the rhythm of early motor behavior inCiona

“…The urochordate ascidian is the closest relative of vertebrate ( 13 ), and one of the main basal chordates in addition to the cephalochordate (amphioxus) and jawless vertebrate (lamprey). Thus, Ciona have shared enhancer machinery ( 47 , 48 ) and gene expression patterns ( 49 ) with amphioxus and lamprey. Ciona also has similar characteristics as motor circuits with amphioxus and lamprey.…”