Amphioxus mouth after dorso-ventral inversion
© Kaji et al. 2016
Received: 14 August 2015
Accepted: 3 February 2016
Published: 6 February 2016
Deuterostomes (animals with ‘secondary mouths’) are generally accepted to develop the mouth independently of the blastopore. However, it remains largely unknown whether mouths are homologous among all deuterostome groups. Unlike other bilaterians, in amphioxus the mouth initially opens on the left lateral side. This peculiar morphology has not been fully explained in the evolutionary developmental context. We studied the developmental process of the amphioxus mouth to understand whether amphioxus acquired a new mouth, and if so, how it is related to or differs from mouths in other deuterostomes.
The left first somite in amphioxus produces a coelomic vesicle between the epidermis and pharynx that plays a crucial role in the mouth opening. The vesicle develops in association with the amphioxus-specific Hatschek nephridium, and first opens into the pharynx and then into the exterior as a mouth. This asymmetrical development of the anterior-most somites depends on the Nodal-Pitx signaling unit, and the perturbation of laterality-determining Nodal signaling led to the disappearance of the vesicle, producing a symmetric pair of anterior-most somites that resulted in larvae lacking orobranchial structures. The vesicle expressed bmp2/4, as seen in ambulacrarian coelomic pore-canals, and the mouth did not open when Bmp2/4 signaling was blocked.
We conclude that the amphioxus mouth, which uniquely involves a mesodermal coelomic vesicle, shares its evolutionary origins with the ambulacrarian coelomic pore-canal. Our observations suggest that there are at least three types of mouths in deuterostomes, and that the new acquisition of chordate mouths was likely related to the dorso-ventral inversion that occurred in the last common ancestor of chordates.
The mouth opening is evolutionarily related to the blastopore, the first opening to connect the gut and exterior during development. In cnidarians, gastrulation occurs at the animal pole and the blastopore directly gives rise to the mouth/anus . In contrast, in most bilaterians, the blastopore forms at the vegetal pole, and primarily determines the anteroposterior body axis . As the blastopore is at the posterior in their developmental system, bilaterians exhibit various patterns of mouth formation in the anterior body.
Some protostomes utilize the blastopore as the mouth after shifting the blastopore towards the anterior during development [3, 4], which is referred to as protostomy. In contrast, deuterostome ambulacrarians (echinoderms + hemichordates) and chordates open their mouths independently of the blastopore, a process called deuterostomy. The new setting of the oral site in ambulacrarian larvae is suggested to be caused by the separation of mouth-forming gene regulatory networks (GRN) from the ancestral blastoporal GRN that specifies mouth formation, axis determination, and germ layer specification . This uncoupling with a gene set comparable to ambulacrarians is also found in protostome animals [5, 6] and thus seems to represent an ancestral state.
In this study, we identified a coelomic vesicle that developed from the posterior wall of the left anterior-most somite. This vesicle accompanies the Hatschek nephridium and contributes to the formation of the larval mouth. The oral development of amphioxus was controlled under the evolutionarily conserved Nodal-Pitx signaling unit [16–18] on the left side and has no relation to the mouth-forming GRN found in ambulacrarians and protostomes. The developmental process of the amphioxus mouth strongly suggests its similarity to that of ambulacrarian coelomic pore-canals and their common evolutionary origin. Thus the amphioxus mouth is distinct from olfactorean (urochordates and vertebrates)  mouths that develop from a specific region called the anterior pan-placode at the anterior extremity of the body  or its equivalent . We suggest that deuterostomes display at least three types of different mouths, of which at least two types have been acquired by chordates, all developing independently of the blastopore.
Materials and methods
Amphioxus specimens studied in the present research were from a Japanese population of Branchiostoma japonicum (formerly B. belcheri) [21, 22]. All embryos and larvae were collected during breeding season from first laboratory generations bred by parental animals collected from a wild habitat in the Ariake Sea, Japan, that were then maintained in a laboratory culture system . The amphioxus colony is maintained and all embryos and larvae subjected to the present study were manipulated according to guidelines established by Hiroshima University for the care and use of experimental animals.
Preparation of RNA probes for whole-mount in situ hybridization
List of forward and reverse primer sets for PCR amplification
Name of gene
Zhang and Mao (2010) 
Putnam et al. (2008) 
Langeland et al. (2006) 
Zhang et al. (2007) 
Hiruta et al. (2005) 
Holland et al. (1999) 
Candiani et al. (2006) 
Whole-mount in situ hybridization (WISH)
Embryos and larvae for WISH were fixed at 4 °C overnight with freshly prepared 4 % paraformaldehyde in 0.1 M 3-(N-morpholino) propanesulfonic acid (MOPS) buffer, pH 7.5, containing 0.5 M NaCl, then washed with 50 % ethanol, and stored in 75 % ethanol at -20 °C until use. WISH was performed with a setting temperature of 60 °C for prehybridization and hybridization according to previously described protocol . Expression patterns were observed after changing solution from phosphate buffered saline (PBS, pH 7.4) to 80 % glycerol in PBS. To obtain rendering images of confocal laser scanning microscopy (LSM), double fluorescence WISH was performed with anti-sense riboprobes for mef2 and pou4 or pax3/7. Anti-DIG-POD (Roche Applied Science, Germany) and anti-fluorescein-POD (PerkinElmer, Massachusetts) antibodies were used to detect the anti-sense riboprobes and then the TSA Plus Cyanine 3 & Fluorescein system (PerkinElmer, Massachusetts) was utilized to amplify the fluorescent signal . For diffusion of labeled fluorescein into the whole body, labeled specimens were stored in 80 % glycerol in PBS for more than one month in dark at 4 °C. Subsequently, rendering images were obtained by using an LSM (ECLIPS C1, Nikon, Japan).
Semi-thin plastic sections and transmission electron microscopy (TEM)
Larvae for TEM were fixed with 1.25 % glutaraldehyde and 1 % paraformaldehyde in Millipore filtered seawater (MPFSW) at 4 °C overnight. Fixed specimens were washed twice with MPFSW and stored in MPFSW with a few drops of the same fixative as for WISH at 4 °C. After washing with MPFSW, specimens were subjected to a conductive staining with 1 % tannic acid in MPFSW for 30 min at 4 °C, washed with MPFSW, and then postfixed with 1 % osmium tetroxide for 1 hour at 4 °C. Postfixed specimens were dehydrated conventionally, then passed through pure propylene oxide, and finally embedded into Epon 812 following a previous study . Knife-shaped larvae fixed at various times (24-30 hours post fertilization (hpf) at 25 °C) were transversely serially cut at 1-μm thickness and stained with toluidine blue to optically observe the developmental changes of the anterior part of the body. Based on observation of the semi-thin sections, appropriate specimens in resin were trimmed into blocks for TEM observation with a JEM1400 (JEOL, Japan) at Hiroshima University.
Specimens for immunostaining were obtained from those fixed for WISH. Antibodies used for detecting nerves, ventral muscles, and basal laminae were anti-acetylated tubulin antibody (T6793, Sigma-Aldrich, Missouri), anti-αSM1 antibody (NCL-SMA, Leica, UK), and anti-laminin antibody (L9393, Sigma-Aldrich, Missouri), respectively. The antibodies were diluted to 1/200 with Can Get Signal (Toyobo, Japan). As secondary antibodies, anti-mouse or -rabbit IgG antibodies labeled with Alexa Fluor 488 or 594 (Life Technologies, California) were used at 1/500 dilution. Specimens immunoreacted with the anti-laminin antibody were also stained DNA with Hoechst (Life Technologies, California) at 5 μg/ml while washing the secondary antibody. Fluorescent images were obtained as optical sections or rendering images that covered more than half an amphioxus body width by using an LSM (ECLIPS C1, Nikon, Japan).
To block Nodal signaling, embryos were treated with SB505124 (S4696, Sigma-Aldrich, Missouri)  at 5 μM in MPFSW from the prehatching early neurula stage to the hatching neurula stage (2 hours from 10 hpf at 25 °C). The treatment was stopped by washing twice with MPFSW. Dorsomorphin (045-31221, Wako, Japan) was used to perturb BMP signaling before the mouth opened . The treatments at 5-, 10-, and 25-μM concentrations were started at the knife-shaped larval stage (24 hpf at 25 °C) and stopped at 30 hpf. Treated specimens and those reared in MPFSW with the same volume of dimethyl sulfoxide added were fixed under the same protocol as for WISH specimens at 16, 24, 36, and 66 hpf for Nodal-blocked specimens and 72 hpf for BMP-blocked specimens.
Image data processing
Digital images in JPEG or TIFF format were obtained by an optical microscope (TE2000, Nikon, Japan), LSM (ECLIPS C1, Nikon, Japan), or TEM (JEM1400, JEOL, Japan). They were visually optimized and edited with Adobe Photoshop and Illustrator CS6 (Adobe, California).
Results and discussion
To further understand the developmental process of the vesicle formation, we performed fluorescent WISH with pax3/7, an upstream muscle specification gene , and pou4 probes for a developmental series of early larvae. Fluorescent rendering images of LSM clarified that the vesicle developed from the posterior ventral corner of the left first somite (Fig. 2e–g). The posterior wall first became thickened and bulged ventrally at the late neurula stage (18–20 hpf at 25 °C). The thickened wall expressed pax3/7, and subsequently the ventral bulge was extruded from the wall, incorporating the somitocoelic lumen as a vesicle, and began to express pou4 (Fig. 2e–g). In the pax3/7-expressing region, a canal newly formed to connect the somitocoel and cavity of the vesicle. These observations confirmed that this vesicle (hereafter referred to as oral mesovesicle, OMV) is mesodermal as previously suggested [30, 31], and that the OMV is the definitive source of the larval mouth in amphioxus.
Within the above-noted canal, TEM observations identified cell(s) bearing a flagellum with a whorl of microvilli, a character of amphioxus excretory cells called cyrtopodocytes  (Fig. 3f, g). The canal initially opened into the pharynx through the cavity of the OMV. Our observations highlight the close developmental relationship between the Hatschek nephridium (Hn) and the mouth in amphioxus, although classical studies inferred that the mouth opened independently from the bulge of the left first somite even as they described the bulge [9, 33].
The primordial oral site in vertebrate embryos commonly appears as a median pan-placode at the anterior extremity of the body, and the pan-placode contains the olfactory, pituitary, and stomodaeum subdomains from the dorsal to ventral direction . The stomodeal ectoderm is directly in contact with the underlying endoderm without any intervening mesodermal cells, and dissolves the basal lamina between itself and the pharyngeal endoderm to form an oropharyngeal membrane that opens to the exterior as a mouth . Since the dissolution of basal laminae is essential for fusion and intercalation between the two epithelia, we examined this by TEM observations and immunolabeling with an anti-laminin antibody.
When the OMV became an isolated vesicle, it was tightly in contact with both the epidermis and endoderm without any basal laminae as in the vertebrate stomodaeum, as confirmed by the TEM observations and immunostaining against laminin (Fig. 3a–c, f, g). The cell mass of the Hatschek nephridium mounted the OMV and developed basal lamina on the surface except in the area in contact with the OMV (Fig. 3c, f). The perforation between the OMV and pharynx occurred without intercalation of the two epithelia, but the OMV appeared to be incorporated into the pharyngeal endoderm (Fig. 3d, e). At the final stage, intercalation occurred between the thinned epidermis and the remnant of the OMV that was regionally restricted to the future perforation site. The opening of the Hatschek nephridium was located dorsal to the mouth opening site (Fig. 3e).
Dorso-ventral inversion is not only an issue of body axes, but also of mouth formation. Gene expression analyses suggest that a dorso-ventral inversion occurred in deuterostomes, possibly in a chordate ancestor . When chordates acquired dorsal structures such as the notochord and epithelial neural tube, they utilized a signaling center at the dorsal margin of the blastopore with GRN including nodal, goosecoid, chordin, and brachyury. Interestingly, the chordate dorsalizing GRN is comparable to that determining the oral side of sea urchin embryos . If this similarity is related to the dorso-ventral inversion, chordates needed a new means of mouth formation. Gastrulation in chordates permits new mouth formation by having the developing primitive gut underlie the epiblast (outer layer), especially in the anterior region, contrary to ambulacrarian gastrulation, in which the primitive gut does not fill the blastocoel or rapidly differentiate a protocoel at the anterior end in some enteropneust embryos [48, 49]. To open a new mouth, olfactoreans (urochordates + vertebrates) developed an anterior placode or stomodaeum under a median Pitx function [20, 38], whereas amphioxus utilizes an ancestral coelomic canal coupled with the early Pitx function on the anterior left side, probably owing to the inability to form placodes . In mouth formation, the Nodal-Pitx signaling unit has likely been co-opted variously as this unit is also utilized in the molluscan stomodaeum . The amphioxus mouth does not represent an ancestral condition of the vertebrate mouth, but instead may display a close relationship to the deep origin of gill openings in deuterostomes.
We have studied the development of the amphioxus mouth that initially opens on the left lateral side of the pharynx. The unusual location of the amphioxus mouth is caused by unique development that involves the coelomic mesoderm. We conclude that the opening of the amphioxus mouth is mediated by a coelomic vesicle (OMV) that develops from the posterior ventral corner of the left first somite in association with the Hatschek nephridium. This OMV development is controlled by the Nodal-Pitx unit that gives rise to the left-right asymmetrical development. The developmental pattern of the amphioxus mouth leads us to hypothesize on the common evolutionary origin of the amphioxus mouth and ambulacrarian pore-canals. This unique mouth is parsimoniously regarded an apomorphic character most likely acquired at the appearance of the amphioxus lineage and has no relation to the olfactorean mouths. As the olfactorean group also develops a mouth under a GRN different from that in ambulacrarians, which is likely ancestral in chordates, we also hypothesize that new chordate mouths were acquired in relation to the dorso-ventral inversion that occurred in the last common ancestor of chordates.
We thank Y. Henmi of Kumamoto University for the generous acceptance of the amphioxus culture; A. Maenaka, K. Shiohira, and H. Shimasaki of Kumamoto University for collecting wild amphioxus and daily care of the amphioxus colony; and K. Koike of Hiroshima University for transmission electron microscopic images. We also thank K. B. Artinger of University of Colorado Denver and Y. Yaoita of Hiroshima University for their critical reading of the manuscript.
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