Mr. Nakazawa and Yamazawa equally contributed to this study.
Formation of the digestive tract in Ciona intestinalis includes two distinct morphogenic processes between its anterior and posterior parts
Version of Record online: 29 JUL 2013
Copyright © 2013 Wiley Periodicals, Inc.
Volume 242, Issue 10, pages 1172–1183, October 2013
How to Cite
Nakazawa, K., Yamazawa, T., Moriyama, Y., Ogura, Y., Kawai, N., Sasakura, Y. and Saiga, H. (2013), Formation of the digestive tract in Ciona intestinalis includes two distinct morphogenic processes between its anterior and posterior parts. Dev. Dyn., 242: 1172–1183. doi: 10.1002/dvdy.24009
- Issue online: 20 SEP 2013
- Version of Record online: 29 JUL 2013
- Accepted manuscript online: 29 JUN 2013 12:00AM EST
- Manuscript Accepted: 17 MAY 2013
- Manuscript Revised: 2 MAY 2013
- Manuscript Received: 12 JAN 2013
- JSPS. Grant Numbers: 18370088, 22570207
- Digestive tract morphogenesis;
- Ciona intestinalis;
- Endodermal strand;
Background: In the ascidian Ciona intestinalis, the digestive tract, an essential system for animals, develops during metamorphosis from the two primordial tissues, the endoderm and endodermal strand, located in the larval trunk and tail, respectively. However, it has been largely unknown how the digestive tract develops from these primordial tissues. We examined the metamorphosing larvae for the tubular formation of the digestive tract, focusing on the epithelial organization of the endoderm, by combined confocal microscopy and computational rendering. Results: The tubular structure of the esophagus to the stomach was formed through the folding and closure of the endodermal epithelia in the central-to-right posterior trunk. By contrast, the intestine was formed in the left posterior trunk through the accumulation and rearrangement of the cells originated from the endodermal strand. This was confirmed by the cell-tracing experiment using Kaede expression construct driven in the endodermal strand. Thus, the tubular formation of the digestive tract in C. intestinalis includes distinct morphogenetic processes and cell lineages between its anterior and posterior parts. Conclusion: This study provides the first detailed description of the digestive tract morphogenesis in C. intestinalis and serves as an important basis toward thorough understanding of its digestive tract development. Developmental Dynamics, 242:1172–1183, 2013. © 2013 Wiley Periodicals, Inc.
The digestive tract is an essential system for animals, and it develops from the endoderm. In that development, several processes such as endoderm formation, endoderm patterning, duct formation, and organ specification must be included. To date, molecular analyses of each of these processes as well as the morphogenetic descriptions of the digestive tract in chordates have been reported largely on vertebrate embryos (Zorn and Wells, 2009). In vertebrate embryos, however, it is rather difficult to analyze thoroughly the process of their digestive tract development due to the anticipated complexities. By contrast, such studies may be feasible in primitive chordate ascidians with simple body plans of their larvae and adults.
Ascidians belonging to the subphylum Urochordata are regarded to be the closest extant relative of vertebrates (Delsuc et al., 2006; Putnam et al., 2008). Ascidian eggs develop into tadpole larvae, which share the prototypical morphogenesis and body plan of chordates characterized by the formation of a hollow dorsal neural tube, notochord and paraxial mesoderm (Satoh, 1994, 2003). Nevertheless, larvae in most ascidian species, including Ciona intestinalis, do not possess the digestive tract. Instead, the larva possesses the digestive tract primordia in two regions: one in the trunk (the head of the tadpole-shaped ascidian larva) and the other in the tail. They are designated the endoderm and the endodermal strand, respectively. Formation of the digestive tract takes place during the metamorphosis, when the tail is absorbed into the trunk (Satoh, 1994). The endoderm is the tissue, consisting of approximately 500 undifferentiated endodermal cells, and is placed in the ventral trunk containing a luminal space designated “the endodermal cavity” located in the middle of the tissue (reference therein, Satoh, 1994). It has been shown with the metamorphosing larva of Halocynthia roretzi that the anterior endoderm forms the branchial basket consisting of the branchial sac, endostyle, peripharyngeal band, dorsal tubercle, and peribranchial epithelium, and the posterior endoderm develops into the digestive tract, consisting of the esophagus, stomach, and intestine (Hirano and Nishida, 2000). However, the detailed information regarding the ways the digestive tract is formed and/or which digestive organ develops from which part(s) of the posterior endoderm is unknown. This holds true with the endodermal strand that is the tissue, running ventrally along the length of the notochord in the tail (Satoh, 1994). It is unknown how the endodermal strand is involved in the digestive tract formation.
In the ascidian, C. intestinalis, juveniles have a relatively large branchial basket and a rather simple digestive tract, consisting of four digestive organs, the esophagus, stomach, intestine, and the pyloric gland being the only accessory gland (Chiba et al., 2004). Although the digestive tract of C. intestinalis is relatively simple, our previous study revealed that Hox genes, Hox10, Hox12, and Hox13, are expressed in the posterior part of the digestive tract with distinct expression domains aligned along the anterior–posterior axis (Ikuta et al., 2004). This is reminiscent of the developing hindgut in chicken embryo, where Hox10, Hox11, and Hox13 genes are expressed and their individual expression domains are aligned along the A–P axis in the gut's endoderm (Yokouchi et al., 1995). These observations suggest that some molecular mechanism of regionalization of the digestive tract may be shared between the ascidians and vertebrates. Furthermore, several of transgenic lines, which harbor green fluorescent fluid gene expressed in the specific region within the digestive tract of the juvenile, has been isolated in C. intestinalis (Yoshida and Sasakura, 2012). Thus, C. intestinalis, together with its abundant genomic as well as cell lineage information, is an attractive model for studying the digestive tract development and its diversity in chordates.
In this study, we have first described the morphogenetic process of the digestive tract, because it has been largely unknown how the tubular structure of the digestive tract develops either in C. intestinalis or in other ascidian species. For this, the metamorphosing larvae and juveniles were fixed and stained with phalloidin, and optical sections were obtained sequentially using a confocal laser-scanning microscope. Focusing on the epithelial structure, the formation of the tubular structure of the digestive tract was traced four-dimensionally. We found that the endodermal cells located posterior central to right of the trunk width, facing the endodermal cavity, became epithelialized and formed the esophagus and stomach by folding and closure of the endodermal epithelia. By contrast, the intestine was formed in the central to left of the trunk width through accumulation and rearrangement of the cuboidal cells largely of the endodermal strand origin. This was confirmed by a novel cell lineage-tracing experiment, using a Kaede expression construct. The present study provides the first detailed description of the tubular structure formation in the digestive tract development in C. intestinalis.
Computational Rendering Reconstruction of Outlines of the Digestive Tract in the Juvenile After Body Axis Rotation
To describe tubular structure formation processes of the digestive tract, we used confocal microscopy with phalloidin staining to recognize the epithelial tissue, the most conspicuous structure of the digestive tract, and rendering procedure to construct three-dimensional (3D) models out of the epithelial tissue images of developing digestive tract.
As the first step, we examined the inner structure of C. intestinalis juveniles at stage 37, the late body axis rotation stage, since Chiba et al. have reported that the digestive organs are recognizable at this stage (Chiba et al., 2004). As shown in Figure 1, a tubular epithelial structure was clearly recognizable within the juvenile body (Fig. 1A–F). From the left to central of the juvenile body width, the intestine was located spanning along the body axis. The posterior end (the anus) of the intestine was located close to the left atrial siphon primordium (Fig. 1A,B), and the other end was opened to the large lumen of the stomach, occupying central to right of the juvenile body width (Fig. 1C–E). The stomach was, in turn, connected to the esophagus on the right side (Fig. 1D–F). The opening of the esophagus to the branchial basket was seen above the bulge of the stomach (asterisk in Fig. 1D), thus the esophageal duct was found to be located in the central to right of the juvenile body width. The digestive tract, spanning from the esophagus to the end of the intestine, is schematically shown in Figure 1G. Additionally, the pyloric gland, an organ branching from the posterior stomach and lying along the intestine, was identifiable (Fig. 1B,C). On each cross-sectional image of the digestive tract, epithelium was colored manually on computational images, and with the images of colored epithelia, a 3D model of the digestive tract was constructed using DeltaViewer software (see the Experimental Procedures section). The reconstructed 3D model clearly showed the outlines of the digestive tract of a metamorphosing juvenile at stage 37 (Fig. 1H–J), suggesting this rendering method to be useful.
Reconstruction of Outlines of the Digestive Tract in the Juvenile During Body Axis Rotation
Next, we examined the larvae during the body axis rotation (stages 35 to 37) for the development of the digestive tract epithelia (Fig. 2A–D), and 3D models were constructed (Fig. 2E–L). The images showed that the digestive tract at stage 35 had a very similar architecture, but consisted of a simple smaller tube in comparison to that at stage 37 (Fig. 2E,I,H,L). In other words, we suggest that it can be seen that the simple small tube is enlarged during body axis rotation stages, and forms the digestive organs (Fig. 2E–L). This formation is accompanied by the distinct morphological changes, such as expansion of the stomach and elongation of the esophagus and the intestine. The pyloric gland becomes recognizable on the outer surface of the intestine at early stage 36 (Fig. 2J) and is elongated along with the intestine, keeping the end of the gland at the central intestine (Fig. 2K,L).
A series of sections of the 3D model at the levels shown in Figure 2M revealed a tubular lumen of the digestive tract at stage 35 (Fig. 2N), which starts dorsally (b in Fig. 2N), goes rightward, turns toward the ventral (a in Fig. 2N), then goes leftward and finally turns toward the dorsal (f in Fig. 2N). The sections of the 3D model also indicate the presence of a branching lumen, corresponding to the pyloric gland (d, e in Fig. 2N).
Considering the above observations, it is suggested that the esophagus, stomach, and intestine are formed in the right, center, and left regions of the posterior trunk, respectively, before the onset of body axis rotation. Therefore, in the following sections, the three regions of the metamorphosing larvae were separately examined to determine how and when the tubular lumen of the digestive tract formation takes place.
Duct Formation of the Prospective Stomach in the Central Posterior Trunk of the Metamorphosing Larvae
In the larva at early stage 27, immediately after hatching, we examined sagittal sections (Fig. 3). This revealed that only a short stretch of epithelium was present in the dorsal endoderm, while ventral endodermal cells were not epithelialized yet (Fig. 3A). By contrast, in larvae at late stage 27 onward, both ventral and dorsal endodermal cells facing the endodermal cavity were completely epithelialized (Fig. 3B–F). Three-dimensional models rendered with the epithelia clearly showed duct-forming processes in the central region (Fig. 3G–K). At late stage 27, the epithelium facing the posterior endodermal cavity constituted a V-shaped furrow along the left–right (L-R) axis (Fig. 3G). At stage 28, the anterior–dorsal and anterior–ventral edges of the V-shaped epithelial furrow came close to each other (blue arrows in Fig. 3H) and eventually fused by stage 29 (Fig. 3I), which resulted in a small luminal space (red lining in Fig. 3I). At stage 30 onward, the cells surrounding the luminal space became columnar (Fig. 3E) and the luminal space-surrounding cells were completely separated from the endodermal cavity epithelium (Fig. 3J,K). Thus, a duct running along the L-R axis was formed ventrally through folding and closure of the epithelia in the posterior central trunk by stage 32 (Fig. 3K).
In addition, the 3D models viewed from the opposite side (Fig. 3M–O) unveiled that the lumen of the duct became divided into two as development proceeded. One duct located ventrally would form the main digestive tract and the other located dorsally would form the duct of pyloric gland.
Tubular Lumen Formation of the Prospective Pharynx and Esophagus in the Right Posterior Trunk of the Metamorphosing Larvae
Tubular lumen formation in the right posterior trunk of the larvae at stages 29, 30, and 32 was examined at three parasagittal levels (a, b, and c in Fig. 4A) by constructing 3D models out of the endodermal epithelia (Fig. 4B–I). In the sections at the level a, it was revealed that the tubular lumen formation became evident in the ventral endodermal epithelium at stage 29, and additionally, another tubular lumen formation took place dorsally around stage 30 (Fig. 4B–D). The former tubular lumen formation was very similar to that described in the previous section. The latter tubular lumen formation was evident at the level b (Fig. 4E,F) but it was incomplete at the level a, leaving the ventral side of the lumen open to the endodermal cavity (Fig. 4D). At the level c, it was revealed that the luminal space formation was initiated in a way reminiscent of that in the central region. However, this resulted in an elongated luminal space along the D–V axis (Fig. 4G–I). The luminal space eventually connected the two tubular lumens running dorsally and ventrally along the L-R axis (Fig. 4I,J). The dorsally running tubular lumen opened to the endodermal cavity at the immediate right to the notochord (Fig. 4D,J). We suggest the tubular lumen and the opening correspond to the esophagus and its anterior end connecting to the pharynx, and the prospective esophagus was connected to the prospective stomach located ventrally (Fig. 4I,J). Furthermore, the distorted tubular lumen of the prospective stomach was divided into two tubular lumens, continuing to the prospective intestine ventrally and to the pyloric gland dorsally (Fig. 4J, see also Fig. 3O). Thus, the anterior prospective digestive tract, from the pharynx to the stomach, was formed through folding and closure of the endodermal epithelia by stage 32 (Fig. 4J).
Prospective Intestine Formation in the Left Posterior Trunk of the Metamorphosing Larvae
In the next stage, posterior left trunk in larvae at stages 27 to 32 was examined for the development of the intestine. Because the intestine in the juvenile connects the stomach and the opening to the left atrial siphon, first, parasagittal sections at the level immediate right to the atrial siphon primordium were examined (Fig. 5).
In larvae at stage 27, epithelium-like cells were observed in the region posterior right to the atrial siphon primordium, where the prospective intestine develops (Fig. 5A). However, epithelium-like cells were missing from the position at stage 28 (Fig. 5B). By contrast, in larvae at stage 29, a cluster of cells with epithelium-like appearance consisting of large cuboidal cells was emerging in the region posterior right to the atrial siphon primordium and dorsal to the prospective stomach, or the duct to be connected to the prospective stomach (Fig. 5C). This cluster changed its contour shape as the development proceeded (Fig. 5D–F). In larvae at stage 30, a lumen was formed, being surrounded by most, but not all, of the cuboidal cells (upper red lining in Fig. 5D) on the dorsal side of the duct connected to the prospective stomach (Fig. 5D). In larvae at stage 32, a tubular lumen running along the D–V axis was observed (Fig. 5F). The lumen is expected to be of the prospective intestine (Fig. 5F). The anus did not open to the atrial siphon until juvenile stage (data not shown). Thus, the findings out of the parasagittal sections suggest that the intestine forms through accumulation and rearrangement of the large cuboidal cells.
Cells Forming the Prospective Intestine Originate From the Endodermal Strand
To explore the origin of the cuboidal cells, horizontal sections at the level ventral to the notochord were examined in larvae at stages 27 to 32 (Fig. 5A–F,G–L). In the larva at stage 32, the duct of the prospective intestine was located on the left side of the prospective stomach (Fig. 5L). By contrast, in the larva at stage 31, the duct was not so evident but a group of large cuboidal cells was recognizable in the prospective intestine region (Fig. 5K). In the larvae at stages 29 and 30, the large cuboidal cells were aligned in a row spanning from the prospective intestine forming region to the more posterior central region (Fig. 5I,J). In the larvae at stages 27 and 28, the prospective intestine cells were hardly identifiable among cells of similar morphology, and neither were the large cuboidal cells in a row (Fig. 5G,H). In the sagittal section of the larva at stage 29, a stretch of cells with somewhat similar morphology to the cuboidal cells was observed aligned along the ventral to the anterior notochord (Fig. 5N). These cells are aligned as continuation of the endodermal strand cells that are located as a file of squamous cells immediate ventral side of the notochord in the tail (Fig. 5N). These cells and/or the endodermal strand cells were missing from the posterior to anterior as development proceeded (Fig. 5N,O) and disappeared by stage 31 (Fig. 5P).
Based on these observations, we hypothesized that the cells of the endodermal strand move toward anterior into the left posterior trunk of the larva, where they form the intestine. To test this hypothesis, we labeled and traced the endodermal strand cells using the larvae introduced with pZip-Kaede expression construct and those of the transgenic line harboring the pZip-Kaede expression construct (see the Experimental Procedures section), which drives the expression of the Kaede protein in a part of the sensory vesicle, posterior endoderm and in approximately anterior two-thirds of the endodermal strand at larva stage (Fig. 6A, see the Discussion section).
A larva at stage 27 introduced with the pZip-Kaede expression construct, emitting green but not red fluorescence of the Kaede protein in the endodermal strand (Fig. 6A, data not shown), was irradiated with ultraviolet (UV) light only at anterior two-thirds of the tail. Immediately after UV irradiation, it was confirmed that the larva emitted the red fluorescence of the converted Kaede protein solely in the endodermal strand (Fig. 6B,C; red fluorescence is represented by magenta color). Such larvae were allowed to develop and were examined for the red fluorescence at various stages. In larvae at stage 30, well before the tail absorption, red fluorescence was located in the left posterior trunk, but not in the tail (Fig. 6D,E). It should be noted that left-sided localization of the red fluorescence was always the case, so far as the larvae of the stable pZip-Kaede transgenic line developed within the chorion were concerned (n = 21). In the larvae introduced with pZip-Kaede by electroporation, the left-sided fluorescence localization was approximately one-quarter (n = 11), probably due to the removal of the chorion as has been reported with the left-sided expression of Ci-Pitx and Ci-Nodal (Yoshida and Saiga, 2008, 2011). When the larvae metamorphosed into juveniles, red fluorescence was detected in the intestine (Fig. 6G) and some other parts, including the periesophageal body and the tail residue (Fig. 6G′, see the Discussion section). These results indicated that the endodermal strand cells move from the tail into the left posterior trunk, which occurs well before the tail absorption, and they contribute to form most part of the intestine.
In the present study, we have described for the first time the morphogenetic processes of the digestive tract in the ascidian, C. intestinalis on the basis of confocal microscopic observations. It has been clearly shown that anterior and posterior parts of the digestive tract are formed through distinct processes from the posterior endoderm and the endodermal strand, respectively. The endodermal cells destined to the intestine move out of the tail much earlier before its absorption, which is also first described in the present study.
Summary of the Digestive Tract Morphogenesis in C. intestinalis
The digestive tract morphogenesis in C. intestinalis is summarized in Figure 7. The anterior digestive tract to form the esophagus and stomach develops through epithelialization, followed by folding and closure of the epithelia, in the two regions of dorsal and ventral in the central to right posterior endoderm facing the endodermal cavity; the dorsal region gives rise to the esophagus and the ventral region to the stomach. These morphogenetic events occur during stages 27 to 32 (Figs. 3, 4). By contrast, the intestine is formed in the left posterior trunk in a different manner. Cells to constitute the intestine originate from the endodermal strand in the tail; the cells in the endodermal strand move anteriorly into the left posterior trunk of a larva by stage 30, which occurs earlier than the absorption of the tail. During stages 30 to 32, the intestine is formed by accumulation and rearrangement of the cells originated in the endodermal strand (Figs. 5, 6). The duct of the pyloric gland is formed around stage 30 through bifurcation of the lumen left to the stomach (Fig. 3). Thus, the basic tubular structure of the digestive tract is formed by stage 32, followed by the differentiation of the distinct digestive organs that becomes evident during the body axis rotation stages. The digestive tract subdivisions are complete by stage 37.
Some Considerations on the Strategy to Describe Digestive Tract Morphogenesis
The strategy we took in the present study is a simple method combining confocal microscopy and computational rendering, focusing on the epithelial organization of the endoderm, a characteristic of the digestive tract. Feasibility of the method depends on the fact that epithelial organization of the digestive tract endoderm is easy to be identified by its morphology. At present, no antibodies that specifically stain endodermal epithelium in the larval trunk are available and genes specifically expressed in the endodermal epithelium of the developing digestive tract have been unknown in C. intestinalis. Considering these circumstances, the method applied can be assessed as reasonable.
Cells Labeled by pZip-Kaede Expression Construct
For tracing the endodermal strand cells, we used the pZip-Kaede expression construct. This allows labeling of the endodermal strand cells that express Kaede protein by local UV radiation at the tail.
In the larvae harboring the pZip-Kaede expression construct, approximately anterior two-thirds of the entire length of the endodermal strand emitted green fluorescence, when examined immediately after hatching. It has been reported that germ cell precursors derived from the B8.12 blastomere pair at the gastrula are localized in the distal region of the endodermal strand, expressing CiVH, the protein of the vasa gene homolog in C. intestinalis (Shirae-Kurabayashi et al., 2006). This region is likely corresponding to the region without Kaede fluorescence, and it has been shown that the germ cell precursors are carried into the trunk at the time of the tail absorption (Shirae-Kurabayashi et al., 2006). By contrast, the endodermal strand cells destined to the intestine move into the left posterior trunk much earlier than the onset of tail absorption, which is first described in the present study. Therefore, it is highly likely that the germ cell precursors are not included among the cells labeled by pZip-Kaede expression construct.
Anterior Two-thirds of the Endodermal Strand May Contain Some Distinct Cell Fates
Although the present study has clearly shown that the cells in the anterior two-thirds of the endodermal strand in the larva contribute to the intestine, red Kaede fluorescence was observed in other parts than the intestine. In some of the juveniles developed from the eggs introduced with the pZip-Kaede expression construct by injection or electroporation, red fluorescence was observed in a small vesicle structure, which was apparently distinct from the digestive tract tube and was located near the esophagus and the tail residue (Figs. 6G′–I′). Considering its morphology and location, the vesicle structure is presumed to be the periesophageal body, which has been reported by Okada and Yamamoto as the structure located just below the ventral margin of the esophageal opening, and filled with round vesicles and Golgi complexes, possibly with an endocrine function (Okada and Yamamoto, 1999). It is, however, unclear whether the cells constituting periesophageal body are of the endodermal strand origin. Additionally, it has also been reported by Shirae-Kurabayashi et al. that CiVH expressing cells derived from the B8.11 blastomere pair, the sister of the B8.12 blastomere, are localized in the anterior half of the endodermal strand, and that they coalesce into the intestinal wall but not the gonad after metamorphosis (Shirae-Kurabayashi et al., 2006). Nevertheless, the fate of those cells may be distinct from the neighboring endodermal cells as speculated by Takamura et al. (Takamura et al., 2002). Thus, the anterior two-thirds endodermal strand may contain some distinct cell fates, though a large part of the constituting cells are destined to develop into intestine epithelium.
To understand how the digestive tract is formed is a key step toward understanding of the thorough process of the digestive tract development in C. intestinalis. Hence the present study will serve as an important basis for the future studies on morphogenetic as well as molecular mechanisms of the digestive tract formation, their diversity and evolution in ascidians and/or chordates.
Adult Ciona intestinalis were obtained at the Onagawa Field Research Center of Tohoku University and provided by the Maizuru Fisheries Research Station of Kyoto University and Misaki Marine Biological Station of University of Tokyo through the National Bio-Resource Project (NBRP) of the MEXT, Japan. Eggs and sperm were obtained surgically from the gonoducts. After insemination, fertilized eggs were washed and cultured in a Petri dish filled with artificial seawater or filtered seawater at room temperature (18–22°C). Developmental staging of larvae was done according to FABA2 (http://chordate.bpni.bio.keio.ac.jp/faba2/2.2/top.html). Juveniles were harvested after detaching from the Petri dish using a tungsten needle.
Phalloidin staining was carried out as described previously with some modifications (Christiaen et al., 2005). Larvae and juveniles were fixed with 3.7% formaldehyde in Ca2+/Mg2+ free seawater for 15 min. Fixed samples were permeabilized with the phosphate-buffered saline containing 0.4% Triton X-100 and 0.2% Tween 20 (PBTT1) for 10 min. To quench autofluorescence of aldehyde, samples were treated with phosphate-buffered saline containing 50 mM NH4Cl, 0.2% Triton X-100 and 0.1% Tween 20 (PBTT2) for 15 min. Then, samples were washed in PBTT2 for 5 min twice and were stained for 15 min with Alexa Flour 488 or 546 phalloidin (Invitrogen) diluted to 1:100 in PBTT2. Samples were further washed in PBST for 5 min twice and were immersed in VECTASHIELD with DAPI (4′,6-diamidine-2-phenylidole-dihydrochloride;Vector Laboratories). All manipulations were done at the room temperature. Stained samples were kept at 4°C in dark until microscopy.
Confocal Laser Scanning Microscopic Analysis
Stained larvae or juveniles were mounted in the center of a two-tiered sticky toroidal-shaped seal attached on the top of a slide glass. A cover slip was placed over the seal. Samples stained with phalloidin were visualized under a confocal laser scanning microscope (LSM5) with the LSM software, ZEN 2008 (Zeiss). An X20 objective lens was used. Image data were acquired in a z-stack mode (section thickness, 1.028 μm; pinhole, 75 μm; filters, LP 505; laser, 488 nm), (section thickness, 1.114 μm; pinhole, 81 μm; filters, BP 560–615; laser, 543 nm).
Confocal microscopic image data were analyzed using ImageJ software (provided by NIH). Three-dimensional constructions of epithelial tissues were done according to the procedure described on the DeltaViewer provided by Wada through HP: (http://delta.math.sci.osaka-u.ac.jp/DeltaViewer/index-j.html) (Wada, M., Osaka University, Osaka, Japan).
pZip-Kaede Expression Construct and Its Transgenic Line
Ci-Zip is a zinc transporter gene of C. intestinalis, expressing in the endodermal strand and a part of central nervous system (CNS) in tail-bud stage (aniseedV3_13279). To generate an expression construct that drives a given gene in the endodermal strand, an enhancer of Ci-Zip was amplified by polymerase chain reaction using primers (5′-TTTCTGCAGTGCTGACTCGGATGTATG-3′ and 5′-TTGGATCCTTGTGGAAAAGCATCAA-3′) as described by Shi et al. (2009). The endodermal strand enhancer was connected to the Ci-fkh basal promoter, which includes the upstream 148 bp from its initiation codon (Harafuji et al., 2002), and was inserted into pSP-Kaede (Hozumi et al., 2010) to generate the expression construct, pSPCiZipCifkhK (hereafter denoted as pZip-Kaede). pZip-Kaede drives expression of Kaede in the endodermal strand and a part of CNS from the tail-bud stage onward.
For generation of a transgenic line, a stretch of the nucleotide sequence including the endodermal strand enhancer, Ci-fkh basal promoter and Kaede coding region of pCiZipCifkhK was subcloned into pMiDestF (Sasakura et al., 2008) using a Gateway system kit (Invitrogen), and the plasmid, pMiCiZipCifkhK, was obtained. The transgenic line, Tg[MiCiZipCifkhK]1, was created by electroporation of the wild-type eggs with 60 μg of pMiCiZipCifkhK and 80 μg of Minos transposase mRNA. Selection of electroporated animals was performed as described by Matsuoka et al. (Matsuoka et al., 2005).
Electroporation of the Expression Construct
Electroporation of reporter constructs into fertilized eggs was carried out using a protocol adapted from a previous report (Corbo et al., 1997). For electroporation, a Gene Pulser Xcell electroporator (Bio-Rad) and electroporation cuvettes of 0.2 cm in width were used. Fertilized eggs were treated for 5 min in the dechorionation solution (sea water containing 1% of sodium thioglycolate, 0.05% actinase E, and 0.04N NaOH), thoroughly washed in fresh seawater twice and further washed in 0.693 M mannitol in 10% seawater twice. One hundred fifty microliters of egg suspension was mixed with 250 μl of DNA in mannitol (21:4 mixture of 0.77 M mannitol and 1 mg/ml plasmid DNA in TE buffer) to give a total volume of 400 μl and the plasmid DNA concentration of 100 μg/ml. The egg suspension was immediately transferred to a cuvette and exposed to an electric charge; 10 ms square pulse at 30 V. The eggs were returned to fresh seawater and reared at 18°C.
Exposure of a Larva Harboring pZip-Kaede to UV Radiation and Observation of the Larva and the Resulting Juvenile
A swimming larva developed from the egg introduced with pZip-Kaede construct was transferred to a drop of seawater mounted in the center of a four-tiered sticky toroidal seal attached on the top of a slide glass. To anesthetize the larva, it was resuspended in 20 μl of 0.2% MS-222 (tricaine mesylate) seawater. Then, the tail of the larva was irradiated with UV light using an Olympus BX60 microscope. The larva was returned to fresh seawater in a Petri dish and cultured at 18°C to the adhesion period or juvenile stage. For photographing, the juvenile was transferred into 7–10 μl of half-saturated L(-)-menthol in seawater mounted in the center of the four-tiered sticky toroidal seal attached on the top of a slide glass. Images were taken using the BX60 microscope equipped with an Olympus DP70 camera and processed by Photoshop (Version 7.0, Adobe Systems).
We thank the National Bio-Resource Project, MEXT, and members of the Education and Research Center of Marine Bioresources of Tohoku University, Maizuru Fishery Research Station of Kyoto University and Misaki Biological Marine Station, the University of Tokyo for providing us with adult Ciona intestinalis. Special thanks are due to the late Ms. K. Hirayama at Kyoto University for her devoted working for the NBRP. We thank Drs. K. Fukuda, N. Takatori, K. Yoshida, and T. Ikuta for discussions and Mr. H. Nezu for his work at the initial stage of this study. Thanks are also due to Ms. E. Zerinska for critical reading of the manuscript. TY was supported by Japan Science Society fellowship. H.S. was funded by Grants-in-Aid for Scientific Research (B) and (C) from JSPS (18370088, 22570207) and partly by JAMBIO.
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