Akane Sasaki

Development of Ciona intestinalis Juveniles (Through 2nd Ascidian Stage)

Abstract Following the reading of its draft genome sequence and the collection of a large quantity of cDNA information, Ciona intestinalis is now becoming a model organism for whole-genome analyses of the expression and function of developmentally relevant genes. Although most studies have focused on larval structures, the development of the adult form is also very interesting in relation to tissues and organs of vertebrate body. Here we conducted detailed observations of the development of tissues and organs in Ciona intestinalis larva and juveniles until so-called the 2nd ascidian stage. These observations included examination of the oral siphon, tentacle, oral pigments and atrial pigments, atrial siphon, ganglion and neural gland, longitudinal muscle, stigmata, transverse bar and languet, longitudinal bar and papilla, heart, digestive organ, gonad, endostyle, and stalk and villi. The findings from these observations make a new staging system for juvenile development possible. Based on the development of the internal organs, we propose here nine stages (stage 0 ∼ stage 8) starting with swimming larvae and proceeding through juveniles until the 2nd ascidian stage. These descriptions and staging system provide a basis for studying cellular and molecular mechanisms underlying the development of adult organs and tissues of this basal chordate.

Gene expression profiles in young adult Ciona intestinalis

Abstract. Comparison of 12,230 expressed sequence tags (ESTs) of 3′ ends of cDNA clones derived from young adults of Ciona intestinalis allowed us to categorize them into 976 independent clusters. When the 5′-end sequences of 10,400 ESTs of the 976 clusters were compared with the sequences in databases, 406 of the clusters showed significant matches (P < E-15) with reported proteins with defined functions, while 117 showed matches with putative proteins for which there is not enough information to categorize their function, and 453 had no significant sequence similarities to known proteins. The 406 clusters with sequence similarity to proteins with defined functions consisted of 304 clusters related to proteins with functions common to many kinds of cells, 73 related to proteins associated with cell-cell communication and 29 related to transcription factors. Spatial expression of all of the 976 clusters was examined by a newly improved whole-mount in situ hybridization method. A total of 430 clusters did not show distinct in situ hybridization signals, while 122 clusters showed ubiquitous distribution of signals, and 253 clusters showed signals in multiple tissues. The remaining 171 clusters showed signals specific to a certain organ or tissue: 16 showed epidermis-specific expression, 3 were specific to the neural complex, 1 to heart, 6 to body-wall muscle, 94 to pharyngeal gill, 3 to esophagus, 26 to stomach, 1 to intestine and 21 to endostyle. Many of these organ-specific genes encode proteins with no sequence similarity to known proteins. The present analysis thus highlights characteristic gene expression profiles of Ciona young adults and provides not only molecular markers for organs and tissues but also transcriptomic information useful for further genomic analyses of this model organism.

Novel Endostyle-Specific Genes in the Ascidian Ciona intestinalis

Abstract The endostyle is a pharyngeal organ of Urochordata, Cephalochordata and larval Cyclostomata. This organ secretes mucus-proteins for internal filter feeding, a feeding system that must have developed in the common ancestor of these subphyla. Therefore, the endostyle is a key structure to understanding the origin and evolution of chordates. A previous study of the overall gene expression in Ciona intestinalis young adults yielded several candidates for ascidian endostyle-specific genes. In the present study, we determined in detail the expression profiles of six novel endostyle-specific genes. Ci-VWFL1 and CiVWFL2 encode related proteins similar to vertebrate von Willebrand factor, and were continuously expressed in zones 4 and 2 of the developing endostyle, respectively. The expression of Ci-Ends8 was observed in the entire region of zone 6 in young adults; however, the expression of this gene was restricted to the dorsal- and ventral-regions of zone 6 in the adult endostyle. The expression of Ci-Ends9 and Ci-Ends10 was observed in zones 6 and 4 in young adults, respectively, and was downregulated in the adult endostyle. Ci-Ends11 showed an expression pattern similar to that of Ciona TTF-1, which encodes a thyroid-related transcription factor. The predicted amino acid sequence of Ci-Ends10 showed similarity to Trip230, and that of Ci-Ends11 resembled Ptp4E. These molecules might be useful for further analysis of the development, function and evolution of the endostyle.

Regeneration in the Hemichordate Ptychodera flava

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worms body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.

A cDNA Resource for Gene Expression Studies of a Hemichordate, Ptychodera flava

Recent investigations into the evolution of deuterostomes and the origin of chordates have paid considerable attention to hemichordates (acorn worms), as hemichordates and echinoderms are the closest chordate relatives. The present study prepared cDNA libraries from Ptychodera flava, to study expression and function of genes involved in development of the hemichordate body plan. Expressed sequence tag (EST) analyses of nine cDNA libraries yielded 18,832 cloned genes expressed in eggs, 18,739 in blastulae, 18,539 in gastrulae, 18,811 in larvae, 18,978 in juveniles, 11,802 in adult proboscis, 17,259 in stomochord, 11,886 in gills, and 11,580 in liver, respectively. A set of 34,159 uni-gene clones of P. flava was obtained. This cDNA resource will be valuable for studying temporal and spatial expression of acorn worm genes during development.

Effects of 5-aza-2′-deoxycytidine on the Gene Expression Profile During Embryogenesis of the Ascidian Ciona intestinalis: A Microarray Analysis

Abstract DNA methylation is an important epigenetic factor that participates in silencing genes. Genomic approaches to studying DNA methylation promise to be particularly fruitful, since DNA methylation is involved in global control of gene expression in many organisms. With its draft genome completed and a large quantity of available cDNA data, Ciona intestinalis is newly emerging as an invaluable model organism for investigating genome-wide gene expression and function. Here we examine the effects of 5-aza-2′-deoxycytidine (5-aza-CdR), a chemical that blocks CpG methylation, on the gene expression profile of early C. intestinalis embryos, using oligonucleotide-based microarray analysis. Embryos treated with 5-aza-CdR show delayed gastrulation and are developmentally arrested at the neurula stage. They subsequently lose cellular adhesion and finally die. Apoptosis was not detected in these embryos by TUNEL staining at 12 h, indicating that the defects observed did not result from 5-aza-CdR-induced apoptosis. Gene expression profiles of 12-h-old 5-aza-CdR-treated embryos compared to wild-type revealed 91 upregulated genes and 168 downreg-ulated genes. Although nearly half of these encoded proteins with unknown functions, several encoded cell-signaling molecules and transcription factors. In addition, genes associated with the stress response and cell defense were upregulated, whereas genes involved in cell adhesion were downregulated.

Regeneration in the Hemichordate

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worms body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.

Regeneration in the HemichordatePtychodera flava

When the body of P. flava is severed, the animal has the ability to regenerate its missing anterior or posterior as appropriate. We have focused on anterior regeneration when the head and branchial regions are severed from the body of the worm. After transection, the body wall contracts and heals closed in 2 to 3 days. By the third day a small blastema is evident at the point of closure. The blastema grows rapidly and begins the process of differentiating into a head with a proboscis and collar. At 5 days the blastema has increased greatly in size and differentiated into a central bulb, the forming proboscis, and two lateral crescents, the forming collar. Between 5 and 7 days a mouth opens ventral to the differentiating blastema. Over the next few days the lateral crescents extend to encircle the proboscis and mouth, making a fully formed collar. By 10 to 12 days a new head, sized to fit the worms body, has grown attached to the severed site. At about this time the animal regains apparently normal burrowing behavior. After the head is formed, a second blastema-like area appears between the new head and the old body and a new branchial region is inserted by regeneration from this blastema over the next 2 to 3 weeks. The regenerating tissues are unpigmented and whitish such that in-situ hybridization can be used to study the expression of genes during the formation of new tissues.