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

Noriyuki Satoh,Koki Nishitsuji,Jun Inoue,Hitoshi Tominaga,Masato Kiyomoto,Kanako Hisata

doi:10.3389/fcell.2021.696875

Abstract

Among chordate taxa, the cephalochordates diverged earlier than urochordates and vertebrates; thus, they retain unique, primitive developmental features. In particular, the amphioxus notochord has muscle-like properties, a feature not seen in urochordates or vertebrates. Amphioxus contains two Brachyury genes, Bra1 and Bra2. Bra2 is reportedly expressed in the blastopore, notochord, somites, and tail bud, in contrast to a low level of Bra1 expression only in notochord. To distinguish the expression profiles of the two Brachyury genes at the single-cell level, we carried out single-cell RNA-seq (scRNA-seq) analysis using the amphioxus, Branchiostoma japonicum. This scRNA-seq analysis classified B. japonicum embryonic cells into 15 clusters at developmental stages from midgastrula to early swimming larva. Brachyury was expressed in cells of clusters 4, 5, 8, and 9. We first confirmed that cluster 8 comprises cells that form somites since this cluster specifically expresses four myogenic factor genes. Cluster 9 contains a larger number of cells with high levels of Bra2 expression and a smaller number of cells with Bra1 expression. Simultaneous expression in cluster 9 of tool-kit genes, including FoxA, Goosecoid, and hedgehog, showed that this cluster comprises cells that form the notochord. Expression of Bra2, but not Bra1, in cells of clusters 4 and 5 at the gastrula stage together with expression of Wnt1 and Caudal indicates that clusters 4 and 5 comprise cells of the blastopore, which contiguously form the tail bud. In addition, Hox1, Hox3, and Hox4 were highly expressed in Bra2-expressing clusters 4, 5, 8, and 9 in a temporally coordinated manner, suggesting roles of anterior Hox genes in specification of mesodermal organs, including somites, notochord, and tail bud. This scRNA-seq analysis therefore highlights differences between the two Brachyury genes in relation to embryonic regions in which they are expressed and their levels of expression. Bra2 is the ancestral Brachyury in amphioxus, since expression in the blastopore is shared with other deuterostomes. On the other hand, Bra1 is a duplicate copy and likely evolved a supplementary function in notochord and somite formation in the Branchiostoma lineage.

Highlights

The origin and evolution of chordates are two of the most intriguing evo-devo research subjects of metazoans (Holland et al, 2015; Lowe et al, 2015; Satoh, 2016; Gee, 2018)
ScRNA-seq of Branchiostoma Embryonic Cells the four earlier stages (Figures 4A,B). These results indicate that cells of cluster 8 comprise the developing somites, consistent with results of previous studies showing that amphioxus myogenic factor (MF) genes are expressed exclusively in embryonic cells that give rise to paraxial muscle or somites at gastrula and neurula stages (Schubert et al, 2003; Urano et al, 2003; Aase-Remedios et al, 2020)
Detailed identification and characterization of muscle structural genes in the developing cephalochordate notochord using scRNA-seq data are the subject of future research, here we examined four genes, gene for troponin I2 (TNNI2; LOC109469528) (Supplementary Figure 4D), for troponin T3 (TNNT3; LOC109481859) (Supplementary Figure 4E), for possible notochord-specific actin (LOC109482101) (Supplementary Figure 4F), and for cysteine- and glycine-rich protein 2 (CSRP2) (LOC109481702) (Supplementary Figure 4G)

Summary

Introduction

The origin and evolution of chordates are two of the most intriguing evo-devo research subjects of metazoans (Holland et al, 2015; Lowe et al, 2015; Satoh, 2016; Gee, 2018). The cephalochordate notochord is formed at roughly the neurulation stage by “pouching-off ” from the dorsal region of the archenteron, like somites, which are pinched off from both the left and right sides of the archenteron (Conklin, 1932; Hirakow and Kajita, 1994; Figure 1). This contrasts with urochordate and vertebrate notochords, which are formed by convergent extensions of precursor cells that are bilaterally positioned in the early embryo (Munro and Odell, 2002). Vacuolation within notochord cells provides both stiffness and an increase in cell volume in ascidians and vertebrates (Jiang and Smith, 2007)

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Results

Conclusion