Motivation

Modern vertebrates have a sophisticated nervous system which have mesmerized researchers for centuries. However, despite ongoing efforts, the evolution of many vertebrate neural cell types and differences in central nervous system (CNS) development between invertebrates and vertebrates remains poorly understood.

To understand vertebrate evolution, many evolutionary biologists have turned to sister lineages, one of which is the cephalochordate lineage. One of the most commonly studied modern-day living cephalochordate species is amphioxus, a type of small, marine-living fish-like animal that shares many characteristics with vertebrates. Amphioxus has been studied for over a century, and many researchers have given special attention to its central nervous system (CNS). The amphioxus CNS is composed of a neural tube along the back of the animal with an enlarged anterior region (referred to the 'cerebral vesicle'). However, despite significant progress, many questions remain: most amphioxus CNS cell types are poorly described, developmental trajectories and their regulation are unknown and there is debate around proposed vertebrate novelties such as the brain and neuromesodermal progenitors (NMPs).

Key Findings

Amphioxus (Branchiostoma floridae) embryo samples from seven developmental stages spanning blastula to late neurula were sequenced, obtaining over 320k cells and achieving a median gene count per cell of over 1600.

By generating this high-quality single-cell dataset covering most key developmental stages across amphioxus embryogenesis, we were able to identify neural cell clusters including a group of neurohypophysis-like neurons and hypothalamus-like neurons. Through experimental validation, our results provide strong support for the presence of hypothalamus- and neurohypophysis-like cells in the anterior CNS of the last chordate ancestor, which may have the potential to form a hypothalamus-like neural network given their close proximity.

By projecting cells from different developmental stages into the same two-dimensional map based on transcriptome similarity, we were able to deduce precursor-descendant cell relationships and draw a predicted map of amphioxus development at the single-cell level. By comparing these trajectories for amphioxus to predicted trajectories of five other animals (mouse, zebrafish, tunicate, sea urchin, and sea anemone), we show three developmental origins for the vertebrate nervous system: an anterior FoxQ2-dependent mechanism that is deeply conserved in invertebrates, a less-conserved route leading to more posterior neurons in the vertebrate spinal cord and a mechanism for specifying NMPs that is restricted to chordates.

By using gene knockout techniques, we were also able to test our proposed developmental trajectory by targeting transcription factor genes predicted to be crucial for cell differentiation and CNS development in amphioxus. We show that the amphioxus cerebral vesicle can be separated into two regions, an anterior region that is dependent on FoxQ2a expression, and a posterior region that can still form without FoxQ2a presence. Furthermore, we identify a group of NMP cells in the amphioxus tailbud that co-express vertebrate NMP markers SoxB1c and Bra2, and we observe ‘overspill’ of neural gene expression from posterior neural and neuromesoderm cell clusters to general tailbud clusters in Brachyury double knock-out embryos, supporting possible Brachyury transcription factor-mediated suppression of neural gene expression in the amphioxus, falling in line with Brachyury suppression of proneural gene expression in vertebrates.

We have generated a rich, in-depth single-cell level dataset for amphioxus that has allowed us to construct a predicted developmental trajectory for all main body structures in the amphioxus embryo, and we demonstrate through using gene knockout the validity of our key transcription factor predictions for CNS cell development. Many more exciting hypotheses deriving from our analyses remain to be explored.