Two-headed mutants of the lamprey, a basal vertebrate
© The Author(s). 2016
Received: 11 August 2016
Accepted: 17 October 2016
Published: 16 November 2016
This is the first report of two-headed (bicephaly) lamprey twins. Although lampreys sit at a crucial phylogenetic position, there are only a few reports on their teratology and developmental abnormalities.
Two-headed mutants were obtained by artificial fertilization in the laboratory as spontaneous occurrences. All mutants were derived from single fertilizations using single male and female gametes, suggestive of a genetic background. The anterio-posterior position of the axonal bifurcation and symmetricity varied in each mutant. Other malformations were coincidently observed, including pericardial edema, yolk sac edema and axial bending. Asymmetrical (lateral- branched) mutants displayed more severe abnormalities in the cranial nerves than symmetrical mutants.
Two-headed mutants of the lamprey are described. These mutants have similar malformations to dorsal blastopore lip-transplanted lamprey embryos, suggesting that they could be generated by a disorder in head-organizing activity.
KeywordsLamprey Teratology Two-headed twin Axial duplicity Bicephaly
Malformed mutants, or so-called “monsters”, have attracted the attention of morphologists since the inception of the discipline. For example, Étienne Geoffroy Saint-Hilaire and his colleagues, including his son Isidore, described many developmental anomalies and sought to explain the mechanisms underlying their production [1, 2]. As described in their works, conjoined twins, referred to as “axial duplicity” or “double monsters” in Bateson , also represent striking examples of such anomalies.
More recently, in the field of evolutionary developmental biology, researchers have focused more on malformations, as these can represent developmental constraint, variability, and evolvability [4–6]. In fact, axial bifurcation in the caudal fin of the twin-tail goldfish is caused by ventralization during early embryonic development, which is in turn due to a mutation in a chordin gene that may have occurred during domestication . Developmental malformations in lampreys, which belong to a basal group of vertebrates (cyclostomes), may similarly provide new insights into the early evolution of vertebrates. However, only a few reports on lamprey teratology and developmental abnormalities have been published to date (e.g., [8–10]).
Here I report two-headed conjoined (bicephaly) mutants in the Arctic lamprey, Lethenteron camtschaticum, which were unexpectedly obtained following artificial fertilization in the laboratory. These mutants have similar malformations to dorsal blastopore lip-transplanted embryos , suggesting that they may be generated by a disorder in head-organizing activity.
Adult lampreys (Lethenteron camtschaticum) were collected from the Shiribeshi-Toshibetsu River, Hokkaido, Japan in 2012. In the next spawning season (May to June 2013), the animals were anesthetized in ethyl,3-aminobenzoate methanesulfonate (MS-222). Mature eggs and sperm were squeezed from adults and fertilized in vitro. Embryos were cultured at 16 °C, fixed in 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) overnight, dehydrated in a graded methanol series, and stored in 100% methanol at −20 °C.
Whole-mount immunofluorescence with anti-acetylated tubulin (Sigma, T6793) antibodies was performed according to the protocol described by Kuratani et al.  with some minor modifications. Briefly, fixed embryos stored in methanol were washed in TBST containing 5% dimethylsulfoxide (TSTd). The embryos were then blocked with 5% non-fat dry milk in TSTd (TSTM). They were incubated with the primary antibody (diluted 1:1000 in TSTM) for 2–4 days at room temperature. After washing with TSTd, samples were incubated with a secondary antibody (Invitrogen, Alexa fluor 555, A21424) diluted 1:200 in TSTM. Then embryos are washed by TSTd several times, treated RNase and counter-stained by YOYO-1. After a final wash in TSTd, embryos were dehydrated and clarified in a 1:2 mixture of benzyl alcohol and benzyl benzoate (BABB) and then examined using a confocal laser microscope (LSM 510, Zeiss).
Paraffin sections were cut at a thickness of 8 μm and stained with hematoxylin and eosin, according to a standard protocol.
Results and discussion
The mutants described in this study are similar to the experimentally induced conjoined lamprey twins, produced through dorsal blastopore lip transplantation, reported by Yamada . The monumental work of Spemann and Mangold  demonstrated that transplantation of the dorsal blastopore lip in amphibians could induce conjoined twin embryos and this region acts as the head organizer. Although the developmental mechanisms for the head-organizer in lampreys are not known in detail, it is possible that the two-headed mutants described in this study were generated by some disruption of the head-organizing activity. In Xenopus, Siamois (Sia) and Twin (Twn) induce expression of head-organizer-specific genes, such as Goosecoid (Gsc), and double knockdown of Sia and Twn results in two-headed malformations . However, Sia and Twn orthologs are absent in non-amphibian vertebrates, suggesting that there is species-specific diversity in the developmental mechanisms of head-organizer activity . To investigate the degree to which these mechanisms are conserved or diversified in vertebrates, and to reveal the evolutionary origin of the vertebrate head, further comparative studies on the vertebrate head-organizer are needed.
I report two-headed mutants in a lamprey, obtained from artificial fertilization using single male and female gametes, suggestive of a genetic background. These mutants have similar malformations to dorsal blastopore lip-transplanted lamprey embryos, suggesting that they may be generated by a defect in head-organizing activity.
- V1 :
Ophthalmicus profundus nerve
- V2,3 :
I thank Prof. Hiroshi Wada (University of Tsukuba) for his critical reading of the manuscript and constructive comments.
This study was supported by Japan Society for the Promotion of Science (JSPS); Grant number: 13J00621.
Availability of data and materials
The data supporting the conclusions of this article are included within the article and its Additional file 1.
The author declares that he/she has no competing interests.
Consent for publication
This study was performed in accordance with the Regulations on Animal Experimentation at University of Tsukuba. Approval is not needed for experimentation on fishes under Japanese law (Act on Welfare and Management of Animals).
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Appel TA. The Cuvier-Geoffroy debate: French biology in the decades before Darwin. New York/Oxford: Oxford Univ Press; 1987.Google Scholar
- Geoffroy Saint-Hilaire I. Traité de tératologie. Paris: JB Baillière; 1836.Google Scholar
- Bateson W. Materials of the study of variation: treated with special regard to discontinuity in the origin of species. London: Macmillan; 1894.Google Scholar
- Alberch P. The logic of monsters: evidence for internal constraints in development and evolution. Geobios. 1989;12:21–57.View ArticleGoogle Scholar
- Guinard G. Introduction to evolutionary teratology, with an application to the forelimbs of Tyrannosauridae and Carnotaurinae (Dinosauria: Theropoda). Evol Biol. 2015;42:20–41.View ArticleGoogle Scholar
- Diogo R, Smith CM, Ziermann M. Evolutionary developmental pathology and anthropology: A new field linking development, comparative anatomy, human evolution, morphological variations and defects, and medicine. Dev Dyn. 2015;244:1357–74.View ArticlePubMedGoogle Scholar
- Abe G, Lee SH, Chang M, Liu SC, Tsai HY, Ota KG. The origin of the bifurcated axial skeletal system in the twin-tail goldfish. Nat Commun. 2014;5:3360.View ArticlePubMedPubMed CentralGoogle Scholar
- Korschelt E. Regeneration und Transplantation. Berlin: Gebrüder Bornträger; 1927/1931.
- Piavis GW. Embryological stages in the sea lamprey and effects of temperature on development. Fishery Bull. 1961;61:111–43.Google Scholar
- Hanson LH. Observations on twinning in the sea lamprey (Petromyzon marinus) in the laboratory. J Great Lakes Res. 1985;11:549–51.View ArticleGoogle Scholar
- Yamada T. Induktion der sekundären Embryonalanlage im Neunaugenkeim. Okajimas Folia Anat Jpn. 1938;17:369–88.View ArticleGoogle Scholar
- Kuratani S, Ueki T, Aizawa S, Hirano S. Peripheral development of cranial nerves in a cyclostome, Lampetra japonica: morphological distribution of nerve branches and the vertebrate body plan. J Comp Neurol. 1997;384:483–500.View ArticlePubMedGoogle Scholar
- Hardisty MW. Gonadogenesis, sex differentiation and gametogenesis. In: Hardisty MW, Potter IC, editors. The biology of lampreys, vol. 1. London: Academic; 1971. p. 295–359.Google Scholar
- Laale HW, Lerner W. Teratology and early fish development. Amer Zool. 1981;21:517–33.View ArticleGoogle Scholar
- Rothschild BM, Schultze HP, Pellegrini R. Herpetological osteopathology: Annotated bibliography of amphibians and reptiles. New York: Springer; 2012.View ArticleGoogle Scholar
- Kaufman MH. The embryology of conjoined twins. Childs Nerv Syst. 2004;20:508–25.View ArticlePubMedGoogle Scholar
- Higashiyama H, Hirasawa T, Oisi Y, Sugahara F, Hyodo S, Kanai Y, Kuratani S. On the vagal cardiac nerves, with special reference to the early evolution of the head–trunk interface. J Morphol. 2016;277:1146–58.View ArticlePubMedGoogle Scholar
- Ditrich H. The origin of vertebrates: A hypothesis based on kidney development. Zool J Linn Soc. 2007;150:435–41.View ArticleGoogle Scholar
- Spemann H, Mangold H. Induction of embryonic primordial by implantation of organizers from a different species. Roux’s Arch Entw Mech. 1924;100:599–638. reprinted and translated in Int J Dev Biol. 2001;45:13–38.Google Scholar
- Bae S, Rei CD, Kessler DS. Siamois and Twin are redundant and essential in formation of the Spemann organizer. Dev Biol. 2011;352:367–81.View ArticlePubMedPubMed CentralGoogle Scholar