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Keywords:

  • adhesive organ development;
  • ascidian;
  • papilla;
  • Pitx ;
  • transcription regulatory mechanism

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Pitx genes play important roles in a variety of developmental processes in vertebrates. In an ascidian species, Halocynthia roretzi, Hr-Pitx, the only Pitx gene of this species, has been reported to be expressed in the left epidermis at the tailbud stage. In the present study, first, we have shown that Hr-Pitx is also expressed in the papilla-forming region at the neurula to tailbud stages, and then we addressed transcription regulatory mechanisms for the expression of Hr-Pitx in the papilla-forming region. We have identified the genomic region ranging from 850 to 1211 bp upstream from the translation start site of the Hr-Pitx gene as an enhancer region that drives the transcription of Hr-Pitx in the papilla-forming region. Within the enhancer region, putative transcriptional factor binding sites for Otx as well as Fox were shown to be required for its activity. Finally, we carried out knocking down experiments of Hr-Otx function using an antisense morpholino oligonucleotide, in which the knocking down of Hr-Otx function resulted in reduction of the enhancer activity and loss of the expression of Hr-Pitx in the papilla-forming region. In Xenopus laevis, it has been reported that Pitx genes are expressed downstream of Otx function during development of the cement gland, an adhesive organ of its larva. Taken together, it is suggested that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Spatially and temporally coordinated gene expression is essential for the development of animal body plan and it is primarily controlled by the regulatory genes encoding sequence-specific transcription factors and components of signaling pathways. Regulatory relationships among transcription factors through binding to their target cis-regulatory modules in other regulatory genes constitute gene regulatory networks (GRNs), which determine the course of animal development (Levine & Davidson 2005).

Ascidians (Urochordata) are the closest living relatives of vertebrates (Delsuc et al. 2006), thus, exhibit the characteristic body plan of chordates with the hollow dorsal neural tube, and the notochord and paraxial mesoderm in the tadpole larva stage. It has been proposed that the comparison of the architecture of developmental GRNs between ascidians and vertebrates may provide insights into the origin of the chordate body plan (Lemaire et al. 2008). Therefore, the analysis of the regulatory linkage surrounding transcription factors in the development of ascidians should be important in better understanding the developmental mechanism of chordates and its evolution.

Pitx genes encode Paired-like homeodomain transcription factors and are widely distributed among animal species ranging from Drosophila to vertebrates (Gage et al. 1999). In vertebrates, three groups of Pitx genes, Pitx1, Pitx2 and Pitx3, are known to exhibit distinct expression patterns and play important roles in the development of a variety of tissues and organs (Lamonerie et al. 1996; Lanctot et al. 1997, 1999; Szeto et al. 1999). Additionally, three transcriptional isoforms, Pitx2a, Pitx2b and Pitx2c are known for Pitx2. Also, it has been well documented that Pitx2c is expressed on the left side of the lateral plate mesoderm, which is under control of the left-sided Nodal signaling and that Pitx2c plays an essential role in the establishment of left-right asymmetry (Logan et al. 1998; Piedra et al. 1998; Ryan et al. 1998; Yoshioka et al. 2008; Kitamura et al. 1999). In ascidians, a single Pitx gene has been identified in Ciona intestinalis and H. roretzi (Boorman & Shimeld 2002; Christiaen et al. 2002; Morokuma et al. 2002). In both species, left-sided expression in the early tailbud stage being under control of Nodal has been reported (Morokuma et al. 2002; Yoshida & Saiga 1997). In C. intestinalis, two transcriptional isoforms, Ci-Pitxa/b and Ci-Pitxc, which have been designated by analogy to the vertebrates’ Pitx2a/b and Pitx2c, are known (Christiaen et al. 2005). Ci-Pitxa/b is expressed in the anterior neural boundary at the tailbud stage and in the stomodeum of the larva (Boorman & Shimeld 2002; Christiaen et al. 2002, 2005). Ci-Pitxc is expressed in the left epidermis of the tailbud embryos and photoreceptor cells of the larvae (Boorman & Shimeld 2002; Christiaen et al. 2005). These transcriptional expression patterns of ascidian Pitx gene in the development are reminiscent of those known for vertebrate three Pitx genes.

In some vertebrates, an adhesive organ is formed during the development and in its formation, involvement of Pitx genes has been suggested. In Xenopus laevis larvae, the cement gland, the adhesive organ of this species, is formed at the ventral rostral end and it has been reported that Pitx1 and Pitx2c are expressed in the cement gland formation (Campione et al. 1999; Hollemann & Pieler 1999; Schweickert et al. 2001b). It has also been reported that the ectopic expression of Pitx1 induces an ectopic cement gland (Chang et al. 2001) and that Pitx genes act downstream of Otx2 in the cement gland formation (Schweickert et al. 2001a; Dickinson & Sive 2007). In the teleost fish, Astyanax mexicanus, the casquette, which is formed on the head of its larvae, is regarded to be homologous to the cement gland in Xenopus (Pottin et al. 2010). In A. mexicanus, Pitx1 and Pitx2 are identified and both of them are expressed in the casquette (Pottin et al. 2010). Pottin and colleagues have proposed that the cement gland-like structure that expresses Pitx as well as other factors in its development may be an ancestral characteristic of chordate larvae (Pottin et al. 2010; Retaux & Pottin 2011). In ascidians, an adhesive organ designated papillae, consisting of three cone-shaped protrusions, are formed at the rostral end of the larvae. Although the cell lineage and the morphological description have been well accumulated (Katz 1983; Nishida 1987), much remains unknown about functional relationships among factors expressed in its development. In the present study, we have demonstrated that Hr-Pitx, the Pitx gene of H. roretzi, is expressed in the papilla-forming region from the neurula through tailbud stages and examined transcription regulatory mechanisms for the expression of Hr-Pitx in the papilla-forming region. We have identified an enhancer responsible for the regulatory mechanism, in which putative binding sites of Otx and Fox transcriptional factors are necessary for its activity. In consistence, knockdown of Hr-Otx lead to the reduction of the enhancer activity and loss of the expression of Hr-Pitx in the papilla-forming region. These results suggest that the expression of Pitx in the papilla forming region is lying downstream of Otx gene function, and we suggest that the expression regulatory mechanism of Pitx, involving Otx as the upstream gene, in the developing adhesive organ is conserved between ascidians and vertebrates.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Ascidians

Adult ascidians, H. roretzi, were obtained from the International Coastal Research Center of the Ocean Research Institute, University of Tokyo, Iwate, Japan, and Research Center for Marine Biology, Asamushi, Graduate School of Life Science, Tohoku University, Aomori, Japan. Naturally spawned eggs were fertilized with suspension of non-self-sperm, and these eggs were cultured in filtered sea water at 13°C.

Isolation of Hr-Pitx gene

Previously, we carried out reverse transcription–polymerase chain reaction (RT–PCR) using degenerated primers to isolate PRD type homeobox sequences and poly(A) RNAs from tailbud stage embryos, and among PCR fragments, we identified a partial homeobox sequence of Hr-Pitx. Using the partial homeobox sequence of Hr-Pitx, a H. roretzi genomic library constructed using λ DASH as the vector was screened, which resulted in isolation of a clone containing Hr-Pitx and its flanking sequences. Messenger RNA and genomic nucleotide sequences of Hr-Pitx and its 5’ upstream have been deposited in DDBJ databank under the accession numbers AB731221 and AB731222, respectively.

Whole-mount in situ hybridization

Whole-mount in situ hybridization (WISH) and fluorescence two color WISH were carried out using digoxigenin and/or fluorescein labeled RNA probes as described by Wada et al. (1996) and by Ikuta & Saiga (2007), respectively.

Preparation of reporter constructs

All reporter constructs were prepared by inserting genomic DNA fragments out of 5′-upstream region of Hr-Pitx into the multicloning site of pPD1.27 vector (Fire et al. 1990) or its variant, pPD46.21 vector, both of which harbor the lacZ gene with a nuclear localization signal. The us7488 construct was prepared by inserting PCR products into the PstI/BamHI sites within the pPD1.27 vector. Other constructs, us2028, us1211, us849, us1134 and us1034, were prepared by inserting PCR products into SalI/BamHI sites within the pPD46.21 vector. To make constructs of Δ927–856 and Δ1028–856, upstream region 927–856 and 1028–856 bp from the translation start site were deleted from the us1211 construct using PCR. Putative binding sites of Fox and Otx factors were identified using the TFSEARCH program (http://mbs.cbrc.jp/research/db/TFSEARCH.html), which is owed to the TRANSFAC databases (Heinemeyer et al. 1998). To make mFox and mOtx, mutations at three and two nucleotide residues were introduced into the putative Fox and Otx binding sequences, respectively, in the construct of us1211 using PCR with mutagenic primers. Nucleotide sequences of wild type and mutated Otx and Fox binding sites were as follows; Otx; 5′-TAATCY-3′, mOtx; 5′-TCATAY-3′, Fox; 5′-TRTTTRTT-3′ and mFox; 5′-GRGGTRTT-3′. Underlined residues indicate mutated nucleotides. Y and R indicate C or T and A or G, respectively.

Microinjection of reporter constructs, histochemical detection of β-galactosidase activity and knocking down of Hr-Otx

Microinjection of reporter constructs into fertilized eggs and histochemical detection of β-galactosidase activity were carried out as described previously by Oda-Ishii and Saiga (2003). Knocking down of Hr-Otx was performed as described previously (Wada et al. 1999), using antisense morpholino oligonucleotide against Hr-Otx, which was originally designated HrothMO1 by Wada et al. (2004).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Expression of Hr-Pitx during embryogenesis

Spatial and temporal expression of Hr-Pitx during embryogenesis was examined by WISH. Expression of Hr-Pitx was first detectable by WISH at the mid neurula stage. Hr-Pitx was expressed in eight cells of the anterior trunk epidermis, which were aligned in an arc shape (Fig. 1a). In the late neurula stage, the number of cells expressing Hr-Pitx increased from 8 to 16 (Fig. 1b). In the early tailbud stage, Hr-Pitx expression in the anterior trunk was downregulated except for three spots where papillae would form in the larva (Fig. 1c). Expression of Hr-Pitx in the papilla-forming region was maintained up to the mid tailbud stage (Fig. 1d) and disappeared by the mid-to-late tailbud stage (Fig. 1g). In the early tailbud embryos, as described previously (Morokuma et al. 2002), Hr-Pitx was expressed on the left-side of the epidermis (Fig. 1e). This asymmetric expression continued until the mid tailbud stage (Fig. 1f, g), and became downregulated and disappeared during the late tailbud stage (data not shown). Asymmetric expression of Hr-Pitx is also observed at the mid-to-late tailbud stage in photoreceptor lineage cells, which are located on the right side of posterior sensory vesicle (Fig. 1g). This expression continued up to the swimming larva stage and was detected in photoreceptor cells, which are located on the right-ventral side of the ocellus pigment cell (Figs. 1h and S1). In addition, Hr-Pitx was expressed in the stomodeum region and on the left side of the anterior sensory vesicle of the larvae (Figs. 1h and S1).

image

Figure 1. Expression of Hr-Pitx during the development of Halocynthia roretzi. Expression of Hr-Pitx detected by whole-mount in situ hybridization (WISH) at the mid (a) and late (b) neurula stages, initial-to-early (c, e), early (d, f) and mid-to-late (g) tailbud stages, and the swimming larva stage (h). (a–d) Frontal view, dorsal to the top. (e–h) Dorsal view, anterior to the left. Open arrowheads in (g, h) indicate the expression of Hr-Pitx in photoreceptor cell lineages. The arrow and the closed arrowhead in (h) indicate the Hr-Pitx expression in the stomodeum region and on the left side of the sensory vesicle, respectively. (a–g) Embryos are shown with the same magnification. Scale bars indicate 100 μm.

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A regulatory region of Hr-Pitx expression in the papilla-forming region is located in the upstream 1211 bp region

To reveal the transcription regulatory mechanism of Hr-Pitx, we carried out a reporter analysis on upstream region of Hr-Pitx. First, we generated the reporter construct of us7488 harboring 7488 base pair (bp) upstream region from the translation start site of Hr-Pitx and injected the construct into fertilized eggs (Fig. 2a). The eggs were allowed to develop to the tailbud stage and then fixed for the histochemical detection of β-galactosidase activity. Expression of lacZ was detected in the papilla-forming region in the embryos injected with the us7488 construct (Fig. 2b). Next, we carried out deletion analysis on the upstream 7488 bp region. Reporter constructs of us2028 and us1211 drove lacZ expression in the papilla-forming region as the us7488 construct (Fig. 2c). In contrast, expression of lacZ was detected ectopically in the sensory vesicle but not in the papilla-forming region in the embryos injected with the construct of us849 (Fig. 2d), suggesting that the upstream 849 bp region contains a promoter of Hr-Pitx but does not contain enhancer regions that drive Hr-Pitx expression in the papilla-forming region. Then, it was suggested that a transcription regulatory region that is required for Hr-Pitx expression in the papilla-forming region is located in the region ranging from 850 to 1211 bp upstream from the translation start site of the Hr-Pitx gene.

image

Figure 2. A regulatory region of Hr-Pitx expression in the papilla-forming region is located in the upstream 1211–850 bp region. (a) Upstream region and the 1st exon of the Hr-Pitx gene are shown at the top. Numbers represent the base pair numbers from the translation start site (ATG). The scheme of deletions is shown on the left side. Upstream regions containing the ATG (black lines) were fused to the lacZ reporter gene (blue boxes). Frequency of embryos positive for β-galactosidase activity in the papilla-forming region is shown on the right side. (b–c) Embryos introduced with the constructs of us7488 (b), us1211 (c) and us849 (d). Frontal views of the trunk are shown with dorsal to the top. Scale bar indicates 100 μm.

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Putative Otx and Fox binding sites are required for activation of the papilla enhancer

To narrow down the regulatory region that is required for Hr-Pitx expression in the papilla-forming region, we carried out further deletion analysis of the us1211 construct (Fig. 3a). Although the us1134 construct drove lacZ expression in the papilla-forming region as the us1211 construct (Fig. 3b), a more deleted construct, us1034, with missing 100 bp-long of the 5′ most region of the inserted genomic DNA fragment in the us1134 construct, failed to drive lacZ expression in the papilla-forming region (Fig. 3b′). Next, we generated Δ927–856 and Δ1028–856 without 927–856 and 1028–856 bp regions, respectively, out of the us1211 construct (Fig. 3a). The Δ927–856 construct also drove lacZ expression similarly to the us1211 construct (Fig. 3c). On the other hand, expression of lacZ was hardly detected in the papilla-forming region in the embryos injected with the Δ1028–856 construct (Fig. 3c′). These results indicate that the upstream region 1134–1035 and 1028–928 bp are required for driving lacZ expression in the papilla. Then, we searched transcription factor binding sequences located in the upstream 1134–928 bp region, and found two putative Otx binding sites and three Fox binding sites (Fig. 3a). To test the importance of these putative transcription factor binding sites, we generated reporter constructs, mOtx and mFox mutated with the putative Otx and Fox binding sites, respectively, in the upstream 1134–928 bp region. In the embryos injected with mOtx or mFox construct, lacZ expression was scarcely detected in the papilla-forming region (Fig. 3d, d′). These results suggest that putative binding sites of Otx and Fox factors are required for driving Hr-Pitx expression in the papilla-forming region.

image

Figure 3. Putative Otx and Fox binding sites are required for activation of Hr-Pitx papilla enhancer. (a) The upstream region and the 1st exon of Hr-Pitx are shown at the top. Numbers represent the base pair number from the translation start site (ATG). The scheme of deletions is shown on the left side. Upstream regions containing the ATG (black lines) were fused to the lacZ reporter gene (blue boxes). Yellow and pink ovals represent putative binding sites for Otx and Fox, respectively. Point mutations were introduced into Otx or Fox binding sites in the us1211 construct. Frequency of embryos positive for β-galactosidase activity in the papilla-forming region is shown on the right side. (b, b′–d, d′) Embryos introduced with the constructs of us1134 (b), us1034 (b′), Δ927–856 (c), Δ1028–856 (c′), mOtx (d) and mFox (d′). Frontal views of the trunk are shown with dorsal to the top. Scale bar indicates 100 μm.

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Hr-Otx is required for the expression of Hr-Pitx in the papilla-forming region

In the development of the papilla in Hroretzi, Hr-Otx is expressed in the cells of the papilla and sensory vesicle forming lineages from the 32-cell stage through the swimming larva stage, though expression in the papilla-forming region becomes undetectable by the middle tailbud stage (Wada et al. 1999). We examined expression of Hr-Otx in the papilla-forming lineage cells that are recognized by the onset of Hr-Pitx expression at the neurula stage using a fluorescence two-color WISH method to detect Hr-Otx and Hr-Pitx transcripts. Transcripts of Hr-Otx was detected in eight cells expressing Hr-Pitx in the anterior trunk at the neurula stage, and Hr-Otx was also expressed in the sensory vesicle and anterior trunk epidermis as previously reported (Fig. 4a–d, Wada et al. 1999). Then, to examine whether Hr-Otx function is required for the Hr-Pitx expression in the papilla-forming region, we carried out knocking down of Hr-Otx using antisense morpholino oligonucleotide (MO) against Hr-Otx (Wada et al. 1999). Embryos injected with Hr-Otx MO were allowed to develop to the tailbud stage and were examined for the expression of Hr-Pitx. In the embryos injected with Hr-Otx MO, expression of Hr-Pitx was detected in the left epidermis similar to the control uninjected embryos, but there were no expressions of Hr-Pitx in the papilla-forming region (Fig. 5a–d). These results suggest that the function of Hr-Otx is required for the Hr-Pitx expression in the papilla-forming region in the development of Hroretzi.

image

Figure 4. Hr-Otx is co-expressed with Hr-Pitx in the papilla-forming region at the neurula stage. (a–d) Transcripts of Hr-Pitx and Hr-Otx are co-localized in eight cells in the papilla-forming region at the late neurula stage. Shown are papilla-forming regions of the mid neurula stage embryo examined for expression of Hr-Pitx (a; magenta) and Hr-Otx (b; green) by whole-mount in situ hybridization (WISH) and stained with 4´6´-diamidino-2-phenylindole dihydrochloride (DAPI) (c; blue). (d) Merged image of a, b and c. Arrowheads indicate cells that express Hr-Pitx. Scale bar indicates 100 μm.

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image

Figure 5. Function of Hr-Otx is required for the expression of Hr-Pitx in the papilla-forming region. (a–d) Expression of Hr-Pitx in the embryo injected with Hr-Otx MO (a, b) and in the control uninjected embryo (c, d). Frontal views (a, c) and dorsal views (b, d) are shown with dorsal to the top and anterior to the left, respectively. Numbers in (a) and (c) indicate the proportion of embryos with normal Hr-Pitx expression in the papilla-forming region. (e, f) Embryos introduced with the us1211 construct together with (e) or without (f) Hr-Otx MO. Frontal views of the trunk are shown with dorsal to the top. (g) Frequency of embryos positive for β-galactosidase activity in the papilla-forming region in embryos introduced with the us1211 construct together with (+) or without (−) Hr-Otx MO. (h, i) Embryos introduced with the us1211 construct and Hr-Otx MO together with (h) or without (i) Hr-Otx mRNA into the a4.2 blastomere of the 8-cell stage. Frontal views of the trunk are shown with dorsal to the top. (j) Frequency of embryos positive for β-galactosidase activity in the papilla-forming region in embryos introduced with the us1211 construct together with (+) or without (−) Hr-Otx MO and/or mRNA into the a4.2 blastomere of the 8-cell stage. Scale bars indicate 100 μm.

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Next, we tested whether Hr-Otx is required for the activity of the Hr-Pitx papilla enhancer. We injected the reporter construct of 1211 together with Hr-Otx MO and tested the lacZ expression at the tailbud stage. As described above, the us1211 construct drove lacZ expression in the papilla-forming region at the tailbud stage (Fig. 5f, g). By contrast, expression of lacZ was not detected in the papilla-forming lineages in embryos injected with the us1211 construct and Hr-Otx MO (Fig. 5e, g). Then, we carried out co-injection of Hr-Otx mRNA together with Hr-Otx MO and the us1211 construct. It has been reported that injection of Hr-Otx mRNA into fertilized eggs results in incomplete gastrulation and neurulation and causes ectopic formation of anterior neuroectoderm (Wada & Saiga 1995). To avoid the effect of morphological defects caused by overexpression of Hr-Otx, we injected Hr-Otx mRNA, Hr-Otx MO and the us1211 construct into the a4.2 blastomere of 8-cell stage, which gives rise to the right side of the papilla, trunk epidermis and sensory vesicle. In the embryos introduced with Hr-Otx mRNA and MO, lacZ expression from the us1211 construct in the papilla-forming region was recovered as embryos injected only with the us1211 construct (Fig. 5h–j). These observations indicate that functions of Hr-Otx are required for the activity of the Hr-Pitx papilla enhancer.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

Expression of Hr-Pitx in papilla-forming region is regulated by Hr-Otx

In the present study, we examined the expression pattern and the transcription regulatory mechanism of the Pitx gene in the ascidian Hroretzi. We established that Hr-Pitx is expressed in the papilla-forming region, in addition to the left epidermis, ocellus photoreceptors and the stomodeum in the development of H. roretzi. We identified an enhancer region that drives expression in the papilla-forming region and found out that both putative Otx and Fox transcriptional factor binding sites were required for the activity of the enhancer. Next, we showed that Hr-Otx was expressed in the cells expressing Hr-Pitx first in the papilla-forming region at the neurula stage and knocking down of Hr-Otx caused downregulation of Hr-Pitx expression in the papilla-forming region. We further showed that the activity of the Hr-Pitx papilla enhancer was restored by injection of Hr-Otx mRNA together with Hr-Otx MO. These results suggest that expression of Hr-Pitx in the papilla-forming region is downstream of Hr-Otx function.

At the mid neurula stage, Hr-Pitx was expressed only in the eight cells of the papilla-forming region. By contrast, Hr-Otx is expressed in the prospective sensory vesicle region and in the anterior trunk epidermis, in addition to the papilla-forming region by the neurula stage (Wada et al. 1999). Thus, it is suggested that there is an additional factor that activates region specific expression of Hr-Pitx at the neurula stage. The most probable candidate would be Fox transcription factor, because putative Fox binding sites are required for activating the Hr-Pitx papilla enhancer. In Hroretzi, three groups of Fox genes, FoxA/HNF-3, FoxB and FoxD, have been identified. Hr-FoxA/HNF3-1 is expressed in the endoderm and notochord lineage cells from the 16-cell stage through the mid tailbud stage (Shimauchi et al. 1997) and it is required for notochord formation (Kumano et al. 2006). Hr-FoxA is also expressed in the lineage of the ventral layer of the central nervous system from the late gastrula stage to the mid tailbud stage (Shimauchi et al. 1997). Hr-FoxB has been shown to be expressed in the nerve cord precursors (A7.4 and A7.8 pairs) and muscle lineage cells (B7.4 pair) at the 64-cell stage and their descendant cells at the 110-cell stage, and has been shown to repress the expression of the Brachyury gene (Hashimoto et al. 2011). Two types of FoxD, designated as Hr-FoxDa and Hr-FoxDb, have been found in H. roretzi (Kumano et al. 2006). At the 16-cell stage, Hr-FoxDa is expressed in A5.1, A5.2 and B5.1 pairs, which give rise to the notochord, endoderm and other tissues. Although its expression is restricted to the endoderm precursors at the 32- and 64-cell stage, function of Hr-FoxDa is necessary for the notochord formation. Expression of Hr-FoxDb was detectable from the neurula stage in several domains but not in the papilla-forming region (Kumano et al. 2006). Thus, at present, it remains unclear which Fox factor is involved in activating expression of Hr-Pitx in the papilla-forming region.

Conserved gene expression in the adhesive organ development in chordate

The cement gland is a transient adhesive organ of the Xenopus larva and it is derived from the ventral-most portion of the extreme anterior domain that is mesoderm-free, with ectoderm and endoderm directly juxtaposed, and defined by expression of Pitx genes (Dickinson & Sive 2007). During the development of Xenopus, Otx2 is expressed in the cement gland primordium. It has been shown that overexpression of Otx2 induces ectopic cement gland formation in the ventrolateral epidermis (Blitz & Cho 1995; Pannese et al. 1995; Gammill & Sive 1997). Conversely, the cement gland formation is prevented by blocking Otx2 function using dominant negative construct (Gammill & Sive 2001). In the cement gland, Pitx1 and Pitx2c are expressed during embryogenesis (Campione et al. 1999; Hollemann & Pieler 1999; Schweickert et al. 2001b), and it has been reported that ectopic expression of Pitx1 induces ectopic cement gland formation (Chang et al. 2001). Furthermore, knocking down of Pitx1 and Pitx2c inhibits ectopic cement gland formation induced by Otx2 (Schweickert et al. 2001a). Additionally, it has been suggested that Pitx genes act downstream of Otx2 during the development of the cement gland (Dickinson & Sive 2007). Bone morphogenetic protein (BMP) signaling is also involved in the inducing and positioning the cement gland. BMP4 is expressed in the cement gland primordium together with Otx2 (Gammill & Sive 2000). Although high concentrations of BMP4 induce epidermis in ectodermal explants, lower concentrations promote specification of the cement gland (Wilson et al. 2002). It has been suggested that BMP signaling and Otx2 work together to activate cement gland formation and that adequate levels of BMP signaling define the permissive region for cement gland formation (Gammill & Sive 2000).

Cement gland-like structures have been described in teleost fishes (Pottin et al. 2010). Larvae of the Mexican tetra, Amexicanus, have a transient adhesive organ called the casquette on the anterior head. The casquette has similar properties to the cement gland of Xenopus: it is derived from the ectoderm; is innervated by the trigeminal ganglion; and has an inhibitory function on larval swimming behavior. Moreover, BMP4, Pitx1 and Pitx2 are expressed in the casquette during embryogenesis, however, the expression pattern of Otx in the development of Amexicanus has not been examined (Pottin et al. 2010). In the bichir, Polypterus senegalus, which belongs to the basal actinopterygians, single Otx1, Otx2 and Otx5 genes have been identified, and among them, PsOtx1 and PsOtx5 are expressed in the developing attachment gland (Suda et al. 2009).

The ascidian papilla shares some structural properties with the adhesive organ in vertebrate embryos in the development. Both the papillae and the cement gland of Xenopus consist of morphologically similar tall cuboidal epithelial cells that originate from the region anterior to the field of neural fate at the gastrula stage (Keller 1975; Nishida 1987). The papillae develop in the anterior-most part of the trunk where the ectoderm and endoderm lie adjacent to each other (this is apparent in C. intestinalis, according to sagittal sections of tailbud embryos in the Four-dimensional Ascidian Body Atlas (FABA) database, http://chordate.bpni.bio.keio.ac.jp/faba/1.4/top.html; Hotta et al. 2007) like in the extreme anterior domain of Xenopus larva (Dickinson & Sive 2007). It has been reported that papillae contain glutamatergic neurons in C. intestinalis larvae (Horie et al. 2008). Although Xenopus cement gland has no neurons, it is innervated by trigeminal neurons that release an excitatory amino acid (Boothby & Roberts 1992). In the development of Hroretzi, Hr-Otx is expressed in the cells of the papillae as well as in the sensory vesicle lineages (a6.5 pair) from the 32-cell stage. Although expression of Hr-Otx in the sensory vesicle continues to the swimming larva stage, the expression of Hr-Otx in the papillae becomes undetectable by the tailbud stage (Wada et al. 1999). Knockdown of Hr-Otx using antisense morpholino oligonucleotide causes the loss of the papillae and the anterior part of central nervous system (Wada et al. 1999). In Hroretzi, two BMP genes, Hr-BMPa and Hr-BMPb, which have been identified as homologues of BMPs5–8 and BMP2/4, respectively, are expressed in the papilla-forming region of the tailbud embryo (Miya et al. 1996, 1997). Overexpression of Hr-BMPb inhibits papillae formation in the larva and induces ectopic expression of Hr-EpiC, a marker for the epidermis, in the anterior-most region of the neurula, which would give rise to papillae (Miya et al. 1997). On the other hand, overexpression of Hr-Chordin causes abnormal formation of the adhesive organ that consists only of one large papilla (Darras & Nishida 2001b). These observations suggest that BMP signaling of an adequate level is required for correct formation of the adhesive organ in the ascidian development, which further supports the notion that the papilla is the ascidian homologue of the adhesive organ of vertebrate larvae.

In this study, we have shown that Hr-Pitx is expressed in the papilla-forming region and that this expression is under control of Hr-Otx gene function. Taking the previous observations made in Xenopus (Schweickert et al. 2001a; Dickinson & Sive 2007) and our results into account, it is suggested that the involvement of Pitx and Otx in the adhesive organ development, with the former being located downstream of the latter, is conserved between H. roretzi and X. laevis. Previously, Pottin and colleagues proposed that the adhesive organ developmental module, in which functions of Bmp, Otx and Pitx genes are involved, is an ancestral characteristic of the larval development of chordates (Pottin et al. 2010; Retaux & Pottin 2011). The results of the present study are consistent with their proposal and provide further insights into the adhesive organ GRN, in which the involvement of Otx as the upstream gene in the expression of Pitx may constitute a core part, in the larval chordate development. However, in another ascidian species, C. intestinalis, Pitx gene is not expressed in the papilla-forming lineages during its development (Boorman & Shimeld 2002; Christiaen et al. 2005), suggesting that the involvement of Pitx and Otx in the papilla formation is not conserved in this species. This is not necessarily surprising because there are several examples showing that conserved cell lineage and morphologies are controlled by different regulatory networks between H. roretzi and C. intestinalis (Lemaire 2009). For instance, the mechanism of secondary notochord induction appears to differ between these two species. In H. roretzi, BMP2/4 has been shown to act as an inducer of the secondary notochord cells (Darras & Nishida 2001a). In C. intestinalis, however, inhibition of BMP signaling has no effect on the secondary notochord induction (Hudson & Yasuo 2006; Lemaire 2009). In future studies, the comparison of underlying mechanisms for papilla development between H. roretzi and C. intestinalis will provide insights into the evolutional stability and plasticity of the GRN for adhesive organ formation.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information

This research was partially supported by Grants-in-Aid from JSPS (22570207, 18370088) to HS. We thank the staff members of the International Coastal Research Center of the Ocean Research Institute, University of Tokyo, and Research Center for Marine Biology, Asamushi, Graduate School of Life Science, Tohoku University for providing us with ascidians and their hospitality.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
dgd1366-sup-0001-FigS1.tifimage/tif7612KFig. S1. Detailed analysis of the expression of Hr-Pitx at the swimming larva stage.

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