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

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.

SUBMITTER: Satoh N

PROVIDER: S-EPMC8321703 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: A common feature of chemosensory systems is the involvement of G protein-coupled receptors (GPCRs) in the detection of environmental stimuli. Several lineages of GPCRs are involved in vertebrate olfaction, including trace amine-associated receptors, type 1 and 2 vomeronasal receptors and odorant receptors (ORs). Gene duplication and gene loss in different vertebrate lineages have lead to an enormous amount of variation in OR gene repertoire among species; some fish have fewer than 100 OR genes, while some mammals possess more than 1000. Fascinating features of the vertebrate olfactory system include allelic exclusion, where each olfactory neuron expresses only a single OR gene, and axonal guidance where neurons expressing the same receptor project axons to common glomerulae. By identifying homologous ORs in vertebrate and in non-vertebrate chordates, we hope to expose ancestral features of the chordate olfactory system that will help us to better understand the evolution of the receptors themselves and of the cellular components of the olfactory system. RESULTS: We have identified 50 full-length and 11 partial ORs in Branchiostoma floridae. No ORs were identified in Ciona intestinalis. Phylogenetic analysis places the B. floridae OR genes in a monophyletic clade with the vertebrate ORs. The majority of OR genes in amphioxus are intronless and many are also tandemly arrayed in the genome. By exposing conserved amino acid motifs and testing the ability of those motifs to discriminate between ORs and non-OR GPCRs, we identified three OR-specific amino acid motifs common in cephalochordate, fish and mammalian and ORs. CONCLUSION: Here, we show that amphioxus has orthologs of vertebrate ORs. This conclusion demonstrates that the receptors, and perhaps other components of vertebrate olfaction, evolved at least 550 million years ago. We have also identified highly conserved amino acid motifs that may be important for maintaining receptor conformation or regulating receptor activity. We anticipate that the identification of vertebrate OR orthologs in amphioxus will lead to an improved understanding of OR gene family evolution, OR gene function, and the mechanisms that control cell-specific expression, axonal guidance, signal transduction and signal integration.

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

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