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.
KeywordsLancelet Homology of mouth Coelom Hydropore Nodal-signaling Gill (branchial) slits
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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- Lee PN, Kumburegama S, Marlow HQ, Martindale MQ, Wikramanayake AH. Asymmetric developmental potential along the animal-vegetal axis in the anthozoan cnidarian, Nematostella vectensis, is mediated by Dishevelled. Dev Biol. 2007;310:169–86.View ArticlePubMedGoogle Scholar
- Hejnol A, Martindale MQ. The mouth, the anus, and the blastopore --- open questions about questionable openings. In: Telford MJ, Littlewood DTJ, editors. Animal Evolution: Genomes, Fossils, and Trees. Oxford: Oxford Univ Press; 2009. p. 33–40.View ArticleGoogle Scholar
- van den Biggelaar J, Dictus WJAG. Gastrulation in the molluscan embryo. In: Stern CD, editor. Gastrulation: From Cells to Embryo. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 2004. p. 63–78.Google Scholar
- Hejnol A, Schnabel R. The eutardigrade Thulinia stephaniae has an indeterminate development and the potential to regulate early blastomere ablations. Development. 2004;132:1349–61.View ArticleGoogle Scholar
- Hejnol A, Martindale MQ. Acoel development indicates the independent evolution of the bilaterian mouth and anus. Nature. 2008;456:382–6.View ArticlePubMedGoogle Scholar
- Arendt D, Technau U, Wittbrodt J. Evolution of the bilaterian larval foregut. Nature. 2001;409:81–5.View ArticlePubMedGoogle Scholar
- Dickinson A, Sive H. Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary. Sem Cell Dev Biol. 2007;18:525–33.View ArticleGoogle Scholar
- Lowe CJ, Terasaki M, Wu M, Freeman Jr RM, Runft L, Kwan K, et al. Dorsoventral patterning in hemichordates: Insights into early chordate evolution. PLoS Biol. 2006;4:e291.PubMed CentralView ArticlePubMedGoogle Scholar
- Legros R. Sur quelque points de l’anatomie et du développement de l’Amphioxus. Anat Anz. 1910;35:561–87.Google Scholar
- van Wijhe JW. Beiträge zur Anatomie der Kopfregion des Amphioxus lanceolatus. Petrus Camper. 1901;1:109–94.Google Scholar
- Ruppert EE. Morphology of Hatschek’s nephridium in larval and juvenile stages of Branchiostoma virginiae (Cephalochordata). Isr J Zool. 1996;42 Suppl 1:161–82.Google Scholar
- MacBride EW. The formation of the layers in amphioxus and its bearing on the interpretation of the early ontogenetic processes in other vertebrates. Q J Micr Sci. 1909;54:279–345.Google Scholar
- Willey A. Amphioxus and the Ancestry of Vertebrates. Osborn HF, editors. New York: Macmillan; 1894. p. 1-316.
- Medawar PB. Asymmetry of larval Amphioxus. Nature. 1951;167:852–3.View ArticlePubMedGoogle Scholar
- Yasui K, Kaji T. The lancelet and ammocoete mouths. Zool Sci. 2008;25:1012–9.View ArticlePubMedGoogle Scholar
- Grande C, Martín-Durán JM, Kenny NJ, Truchado-García M, Hejnol A. Evolution, divergence and loss of the Nodal signaling pathway: new data and a synthesis across the Bilateria. Int J Dev Biol. 2014;58:521–32.View ArticlePubMedGoogle Scholar
- Watanabe H, Schmidt HA, Kuhn A, Höger SK, Kocagöz Y, Laumann-Lipp N, et al. Nodal signaling determines bilateral asymmetry in Hydra. Nature. 2014;515:112–5.View ArticlePubMedGoogle Scholar
- Vladimir Soukup V, Yong LW, Lu TM, Huang SW, Kozmik Z, Yu JK. The Nodal signaling pathway controls left-right asymmetric development in amphioxus. EvoDevo. 2015;6:5. doi:10.1186/2041-9139-6-5.PubMed CentralView ArticlePubMedGoogle Scholar
- Jefferies RPS. Two types of bilateral symmetry in the Metazoa: Chordate and bilaterian. In: Bock GR, Marsh J, editors. Biological Asymmetry and Handedness. Chichester: Wiley; 1991. p. 94–127.Google Scholar
- Christiaen L, Bourrat F, Joly JS. A modular cis-regulatory system controls isoform-specific Pitx expression in the ascidian stomodaeum. Dev Biol. 2005;277:557–66.View ArticlePubMedGoogle Scholar
- Zhang Q, Zhong J, Fang S, Wang Y. Branchiostoma japonicum and B. belcheri are distinct lancelets (Cephalochordata) in Xiamen waters in China. Zool Sci. 2006;23:573–9.View ArticlePubMedGoogle Scholar
- Yasui K, Igawa T, Kaji T, Henmi Y. Stable aquaculture of the Japanese lancelet Branchiostoma japonicum for 7 years. J Exp Zool (Mol Dev Evol). 2013;320B:538–47.View ArticleGoogle Scholar
- Yasui K, Zhang SC, Uemura M, Saiga H. Left-right asymmetric expression of BbPtx, a Ptx-related gene, in a lancelet species and the developmental left-sidedness in deuterostomes. Development. 2000;127:187–95.PubMedGoogle Scholar
- Wu H, Chen Y, Su Y, Luo Y, Holland LZ, Yu J. Asymmetric localization of germline markers Vasa and Nanos during early development in the amphioxus Branchiostoma floridae. Dev Biol. 2011;353:147–59.View ArticlePubMedGoogle Scholar
- Yasui K, Tabata S, Ueki T, Uemura M, Zhang S. Early development of the peripheral nervous system in a lancelet species. J Comp Neurol. 1998;393:415–25.View ArticlePubMedGoogle Scholar
- DaCosta Byfield S, Major C, Laping NJ, Roberts AB. SB-505124 is a selective inhibitor of transforming growth factor-β type 1 receptors ALK4, ALK5, and ALK7. Mol Pharmacol. 2004;65:744–52.View ArticlePubMedGoogle Scholar
- Shimizu K, Sarashina I, Kagi H, Endo K. Possible functions of Dpp in gastropod shell formation and shell coiling. Dev Gene Evol. 2011;221:59–68.View ArticleGoogle Scholar
- Candiani S, Oliveri D, Parodi M, Bertini E, Pestarino M. Expression of AmphiPOU-IV in the developing neural tube and epidermal sensory neural precursors in amphioxus supports a conserved role of class IV POU genes in the sensory cells development. Dev Gene Evol. 2006;216:623–33.View ArticleGoogle Scholar
- Hammond CL, Hinits Y, Osborn DPS, Minchin JEN, Tettamanti G, Hughes SM, et al. Signals and myogenic regulatory factors restrict pax3 and pax7 expression to dermomyotome-like tissue in zebrafish. Dev Biol. 2007;302:504–21.PubMed CentralView ArticlePubMedGoogle Scholar
- Langeland JA, Holland LZ, Chastain RA, Holland ND. An amphioxus LIM-homeobox gene, AmphiLim1/5, expressed early in the invaginating organizer region and later in differentiating cells of the kidney and central nervous system. Int J Biol Sci. 2006;2:110–6.PubMed CentralView ArticlePubMedGoogle Scholar
- Stach T, Eisler K. The ontogeny of the nephridial system of the larval amphioxus (Branchiostoma lanceolatum). Acta Zool (Stockholm). 1998;79:113–8.View ArticleGoogle Scholar
- Kümmel G. Zwei neue Formen von Cyrtocyten. Vergleich der bisher bekannten Cyrtocyten und Erörterung des Begriffes “Zelltyp”. Z Zellforsch. 1962;62:468–84.View ArticleGoogle Scholar
- Goodrich ES. The early development of the nephridia in amphioxus: introduction and part I, Hatschek’s nephridium. Q J Micr Sci. 1934;76:499–510.Google Scholar
- Dickinson AJG, Sive H. The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth. Development. 2009;136:1071–81.PubMed CentralView ArticlePubMedGoogle Scholar
- Zhang Y, Mao B. Embryonic expression and evolutionary analysis of the amphioxus Dickkopf and Kremen family genes. J Genet Genomics. 2010;37:637–45.View ArticlePubMedGoogle Scholar
- Edelman GM, Jones FS. Developmental control of N-CAM expression by Hox and Pax gene products. Phil Trans Roy Soc Lond B. 1995;349:305–12.View ArticleGoogle Scholar
- Kozmik Z, Holland ND, Kalousova A, Paces J, Schubert M, Holland LZ. Characterization of an amphioxus paired box gene, AmphiPax2/5/8: developmental expression patterns in optic support cells, nephridium, thyroid-like structures and pharyngeal gill slits, but not in the midbrain-hindbrain boundary region. Development. 1999;126:1295–304.PubMedGoogle Scholar
- Oisi Y, Ota KG, Kuraku S, Fujimoto S, Kuratani S. Craniofacial development of hagfishes and the evolution of vertebrates. Nature. 2013;493:175–80.View ArticlePubMedGoogle Scholar
- Yu J-K, Holland LZ, Holland ND. An amphioxus nodal gene (AmphiNodal) with early symmetrical expression in the organizer and mesoderm and later asymmetrical expression associated with left–right axis formation. Evo Dev. 2003;4:418–25.View ArticleGoogle Scholar
- Bertrand S, Aldea D, Oulion S, Subirana L, de Lera AR, Somorjai I, et al. Evolution of the role of RA and FGF signals in the control of somitogenesis in chordates. PLoS ONE. 2015;10:e0136587.PubMed CentralView ArticlePubMedGoogle Scholar
- Lacalli TC, Gilmour THJ, Kelly SJ. The oral nerve plexus in amphioxus larvae: function, cell types and phylogenetic significance. Proc R Soc Lond B. 1999;266:1461–70.View ArticleGoogle Scholar
- Yasui K, Kaji T, Morov AR, Yonemura S. Development of oral and branchial muscles in lancelet larvae of Branchiostoma japonicum. J Morphol. 2014;275:465–77.View ArticlePubMedGoogle Scholar
- Ruppert EE, Balser EJ. Nephridia in the larvae of hemichordates and echinoderms. Biol Bull. 1986;171:188–96.View ArticleGoogle Scholar
- Harada Y, Shoguchi E, Taguchi S, Okai N, Humphreys T, Tagawa K, et al. Conserved expression pattern of BMP-2/4 in hemichordate acorn worm and echinoderm sea cucumber embryos. Zool Sci. 2002;19:1113–21.View ArticlePubMedGoogle Scholar
- Luo Y, Su Y. Opposing Nodal and BMP signals regulate left-right asymmetry in the sea urchin larva. PLoS Biol. 2012;10:e1001402.PubMed CentralView ArticlePubMedGoogle Scholar
- Goodrich ES. The early development of the nephridia in amphioxus: Part II, the paired nephridia. Q J Micr Sci. 1934;76:655–74.Google Scholar
- Molina MD, de Crozél N, Emmanuel Haillot E, Lepage T. Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo. Cur Opi Gen Dev. 2013;23:445–53.View ArticleGoogle Scholar
- Röttinger E, Martindale MQ. Ventralization of an indirect developing hemichordate by NiCl2 suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms). Dev Biol. 2011;354:173–90.View ArticlePubMedGoogle Scholar
- Röttinger E, DuBuc TQ, Amiel AR, Martindale MQ. Nodal signaling is required for mesodermal and ventral but not for dorsal fates in the indirect developing hemichordate, Ptychodera flava. Biol Open. 2015;00:1–13. doi:10.1242/bio.011809.Google Scholar
- Meulemans D, Bronner-Fraser M. The amphioxus SoxB family: Implications for the evolution of vertebrate placodes. Int J Biol Sci. 2007;3:356–64.PubMed CentralView ArticlePubMedGoogle Scholar
- Grande C, Patel NH. Nodal signalling is involved in left–right asymmetry in snails. Nature. 2009;457:1007–11.PubMed CentralView ArticlePubMedGoogle Scholar
- Putnam NH, Butts T, Ferrier DEK, Furlong RF, Hellsten U, Kawashima T, et al. The amphioxus genome and the evolution of the chordate karyotype. Nature. 2008;453:1064–71.View ArticlePubMedGoogle Scholar
- Zhang Y, Wang LF, Shao M, Zhang HW. Characterization and developmental expression of AmphiMef2 gene in amphioxus. Sci China Ser C-Life Sci. 2007;50:637–41.View ArticleGoogle Scholar
- Hiruta J, Mazet F, Yasui K, Zhang P, Ogasawara M. Comparative expression analysis of transcription factor genes in the endostyle of invertebrate chordates. Dev Dyn. 2005;233:1031–7.View ArticlePubMedGoogle Scholar
- Holland LZ, Schubert M, Kozmik Z, Holland ND. AmphiPax3/7, an amphioxus paired box gene: insights into chordate myogenesis, neurogenesis, and the possible evolutionary precursor of definitive vertebrate neural crest. Evo Dev. 1999;1:153–65.View ArticleGoogle Scholar