Developmental Biology
Neuroscience
Evolutionary Biology

Development and evolution of photoreceptors
Cell-fate specification and cell differentiation in the central nervous system
Regulation of CNS-specific gene expression
Evolution of cis-regulatory systems and development
Molecular evolution of gene families

My primary interest is to understand how the interaction between transcription factors and cis-regulatory systems has evolved and controls gene expression during development. As a model system to study gene regulation in development, I have been using a group of primitive chordates, ascidians. Within a day after fertilization, the ascidian egg develops into a tadpole larva, which consists of 2,600 cells. The ascidian tadpole larva exhibits the hallmarks of a chordate, including a motile tail containing a notochord, a dorsal nerve cord, and striated muscle cells. There are several advantages for developmental research in ascidians. We have developed an efficient method for introduction and expression of foreign genes in ascidian embryos. Functions of a given gene can be efficiently inhibited by using antisense morpholino oligos in ascidian embryos and larvae. Ascidians have a small genome size and their embryos have low cell numbers, contain only a few different tissue types, develop rapidly, and have a well-known cell lineage. The genome size is about 160 Mbp/haploid containing approximately 15,500 genes. In December 2002, the draft genome sequence of the ascidian Ciona intestinalis was released. Ascidians are also favorable for evolutionary research because of their phylogenetic position near the vertebrates. The existence of closely-related species of ascidians with different modes of development enables us to study evolutionary changes in developmental mechanisms which occurred recently. We are using C. intestinalis, a species used by researchers throughout the world.

My research focuses on development of the central nervous system (CNS) in the ascidian larva. The CNS of the ascidian larva shows organization similar to that of the vertebrates. The CNS of the ascidian tadpole contains only about 330 cells; among them, less than 100 cells are neurons. Most anterior part of the CNS is a brain vesicle with two sensory organs called the otolith and ocellus. The CNS extends via the visceral ganglion containing motor neurons into the tail nerve cord, which consists of glial eppendymal cells. In contrast to organs such as muscle, epidermis, and endoderm, in which cell fates are autonomously determined by maternally inherited cytoplasmic factors, successive cell-cell interactions play central roles in cell-fate specification of the nervous system in ascidian embryos. The CNS of C. intestinalis provides researchers with a simple and primitive model to analyze the developmental and functional complexities of the vertebrate CNS.

Systematic analysis of cis-regulatory regions of ascidian genes

Interactions between transcription factors and cis-regulatory sequences are one of the main mechanisms controlling development. Therefore, to fully understand genetic programs underlying development of organisms, structure and function of cis-regulatory regions of many developmentally-regulated genes should be elucidated. Recently, genome sequence data have become available for several model organisms. However, in contrast to protein coding sequences, many of which can be predicted by bioinformatics, cis-regulatory sequences are still very difficult to be found in the genomic sequence in silico. Therefore, even after completion of the whole genome sequencing, experimental analysis would be a main approach to study function of cis-regulatory sequences. Ascidians have several advantages for such analysis. First, they have a small genome size and the genome sequence data is available; second, transgenes can be easily introduced into many embryos at the same time by electroporation; third, relatively short upstream regions, usually within 3 kb from the transcription start site, are sufficient for proper expression patterns of a reporter gene introduced into embryos.

We are conducting a systematic analysis of cis-regulatory regions of the CNS-specific genes. To efficiently isolate upstream regions of genes, we optimized PCR-based genomic walking methods. We intend to analyze function of cis-regulatory regions of all the CNS-specific genes identified. Comparative and bioinformatic analyses of many genes will facilitate identification and characterization of cis-regulatory sequences. This study will provide important information to understand the genetic program controlling the ascidian CNS development.

Development and evolution of chordate photoreceptors

Vertebrates have evolved two types of eyes; the lateral eyes (paired eyes) and the median eyes (pineal or parietal eyes). The urochordate ascidian larva has an eye-spot (ocellus) in its brain. Putative photoreceptor organs have also been reported in adult ascidians. Understanding evolutionary relationships between the ascidian photoreceptors and the vertebrate eyes is a key to uncover the origin and evolution of the vertebrate eyes. In C. intestinalis, we have characterized and examined expression patterns of homologues of genes involved in function or development of the vertebrate eyes. The results suggest that ascidians have photoreceptor systems more similar to those of vertebrates than to those of other invertebrates. The larval ocellus expresses a vertebrate-type opsin gene and the surrounding brain cells express visual cycle genes similar to those found in the retinal pigment epithelium of vertebrates. A number of genes related to eye function and development are also expressed in part of the primordial pharynx and atrial primordia, suggesting that adult photoreceptors develop in these regions, possibly oral and atrial siphons. Based on comparisons of the developmental origins, gene expression patterns, and functions of eyes between vertebrates and ascidians, we propose a hypothesis that the larval ocellus and the adult anterior photoreceptors (of the oral siphon) are homologous to the vertebrate median eye and lateral eyes, respectively. The last common ancestor of urochordates and vertebrates may have possessed distinct precursors of the lateral eyes and the median eye of vertebrates.

Molecular bases of diversity of larval forms in ascidians

Closely related species with different modes of development are attractive systems to investigate the evolution of developmental mechanisms. In ascidians, several types of evolutionary modification of the larval development have been reported. We are studying development of the nervous systems in ascidian species that exhibit evolutionary modification of larval forms.

Most ascidian species show indirect development in which the embryo develops into a tadpole larva. The tadpole (or urodele) larva consists of a head, containing a brain with a neural sensory organ(s), and a tail, containing a notochord and flanking bands of striated muscle cells. Anural development is an alternate mode of development in which the embryo develops into a tailless (or anural) larva. Anural embryos lack typical urodele features, including the brain sensory organ, notochord, and differentiated muscle cells. Fewer than 20 ascidian species have been described with anural development, and most of these species are classified in the family Molgulidae. Urodele development is thought to be ancestral in ascidians. Since anural embryos have lost characteristics of chordates (dorsal CNS, notochord, and paraxial striated muscle) during recent evolution, studies on the development and evolution of anural embryos would provide important clues to understand developmental mechanisms of the chordate body plan.

Previous studies with anural embryos have mostly addressed their attention to development of the notochord and the tail muscle cells. The nervous system should also be greatly modified in anural embryos. For example, the anural embryo lacks the brain sensory organ with a pigment cell. We have obtained a number of neural marker genes in the ascidian C. intestinalis. We are looking at differences of structure and development between urodele and anural species by using neural marker genes and antibodies. This study would give insights into evolutionary mechanisms of a radical change of development as well as developmental mechanisms of the ascidian nervous systems.

Another interesting modification in the larval nervous system in ascidians is found in the diversity of the brain sensory organs. The larval brain of Ciona and Halocynthia contains two sensory organs, the otolith (gravity-sense organ) and the ocellus (photoreceptor organ). In some ascidian species, such as those in the genus Molgula, however, the brain contains only the otolith. On the other hand, other species, including Botryllus schlosseri, have a single sensory organ, the photolith, responding to both gravity and light. We intend to compare development of the brain sensory organ in these species with that of Ciona. This study will contribute to our understanding how developmental pathways can be modified during evolution.