In our laboratory, we investigate, at the cellular level, how the brain and nervous system develop from a single fertilized egg, using the ascidian and the freshwater snail. Here, we present our previous research achievements and outline prospects for our future studies.

Previous Research

1. Exploring Brain Development Through Ascidian Larvae

Ascidians are the closest marine invertebrate relatives of vertebrates, including humans (Fig. 1), and their developmental modes of the nervous system are the same as those of vertebrates. In addition, because the brain of an ascidian larva consists of only a few hundred cells, they offer an excellent model for studying, at the single-cell level, the developmental mechanisms of the brain that are shared with vertebrates. To understand these mechanisms, we investigate how cells behave during brain development and what genes play crucial roles in regulating the development of individual brain cells..

During my graduate studies, I (Oonuma) analyzed the transcription regulatory mechanisms controlling the expression of the Otx gene, which is essential for brain development (Oonuma et al., Dev Growth Differ, 2014; Oonuma et al., Zool Sci, 2014). In the course of this work, I discovered that the previously reported cell lineage of the brain—including the timing and number of cell divisions as well as cell fates—contained inaccuracies. This led me to conclude that a re-examination of the cell lineage was necessary to better understand brain development. However, removal of the chorion, a standard embryonic manipulation in the ascidian Ciona intestinalis type A, caused severe disruption of brain structure, and thus resolving this issue became our first priority. 

We therefore established a method for introducing exogenous genes into the Ciona eggs with  the chorion (egg envelope), thereby overcoming this technical limitation (Movie 2). In addition, by developing a single-cell labeling and live-tracking approach with using the photo-convertible fluorescent protein Kaede, we determined what cells give rise to particular neural and glial cell types in the brain (Fig. 2). Using these techniques, we elucidated the complete lineage of photoreceptor cells and dopaminergic neurons by investigating each mitotic division and its timing from the fertilized egg onward (Oonuma et al., Dev Biol, 2016; Oonuma and Kusakabe, Dev Biol, 2019; Oonuma and Kusakabe, Development, 2021). We further demonstrated that multiple neuronal classes exhibit left–right asymmetry during development and that brain-destined progenitors undergo dynamic migration during embryogenesis. (Oonuma and Kusakabe, Development, 2021).

We also demonstrated that the mitogen-activated protein kinase (MAPK) signaling pathway and the transcription factor Otx are essential for the development of dopaminergic cells (Oonuma and Kusakabe, Development, 2021). 

These methods we developed have been widely recognized and have underpinned domestic and international collaborations, with results reported in Science Advances and Nature (Akahoshi et al., Sci Adv, 2021; Todorov and Oonuma et al., Nature, 2024).

2. Biomphalaria glabrata as a new model for molluscan neurodevelopment

Although the nervous system of freshwater snails comprises on the order of 10,000 cells—far fewer than in vertebrates—it exhibits a complex architecture, including pronounced left–right differences in size and morphology (Fig. 3). Individual neurons are large, and neuronal populations that control rhythmic and complex behaviors such as feeding and locomotion have been identified. These features allow cellular level investigations of how the nervous system develops and how neural circuits control the behavior.

Among freshwater snails, we focus on Biomphalaria glabrata, a planorbid species. In addition to the features noted above, B. glabrata has high fecundity and a short life cycle, which facilitate husbandry and experimentation compared with the commonly used pond snail Lymnaea stagnalis.

Genetic engineering has become routine in developmental biology and is increasingly applied in neurophysiology and behavioral research. These approaches generally require the introduction of exogenous genes. However, the introduction of exogenous genes into the freshwater snail B. glabrata has been technically challenging. Embryos of freshwater snails are enclosed within egg capsules (Fig. 4). Removal of the egg capsule arrests embryogenesis, whereas introducing exogenous genes with the capsule intact is extremely difficult. Consequently, to investigate how the nervous system develops in B. glabrata using genetic engineering, it was necessary to establish (i) a method for introducing exogenous genes and (ii) a protocol for capsule-free embryo culture.

Therefore, by adapting the gene-introduction approach we had developed in the ascidian Ciona, I (Oonuma) established the microinjection technique for introducing exogenous genes into fertilized eggs of B. glabrata (Movie 3). I also established a capsule-free ex ovo culture protocol for B. glabrata. By combining these techniques with CRISPR/Cas9-mediated genome editing, I achieved the first targeted gene knockouts in B. glabrata. I have also demonstrated tissue-specific expression of GFP and transposon-mediated genomic insertion (knock-in). 

Collectively, these advances are opening the way for B. glabrata to serve as an emerging model for analyzing the development of the molluscan nervous system and its control of behavior.

Current Research

1. Elucidating how the brain develops in the Ciona larvae and the functions of each neuron in the larval brain

Our laboratory has developed the methods that currently provide the only experimental means to examine normal brain development in the Ciona larvae. By using the methods, we will identify multiple genes regulating left–right asymmetry and the development of each neuron, and to elucidate their relationships as gene regulatory networks.

In parallel, we are developing genetic approaches that selectively ablate target neurons. These tools will enable us to determine how individual neurons contribute to neural circuit function and larval behavior.

2. Elucidating neural development and behavior in snails

Although the molecular mechanisms underlying molluscan embryogenesis remain poorly understood, we have overcome key technical obstacles. Using B. glabrata, we will elucidate how the nervous system develops at cellular and molecular resolution. By monitoring neuronal activity with calcium imaging and selectively ablating targeted cells, we will also determine which neurons and ganglia control specific behaviors and how they do so.

We have generated mutants of the Th gene which encodes tyrosine hydroxylase—the rate-limiting enzyme in dopamine biosynthesis. Using this Th mutant snail, we will investigate the development of dopaminergic neurons and elucidate the molecular mechanisms linking dopamine-modulated neural circuits to behavior.