Identification of the orphan gene Prod 1 in basal and other salamander families
- Jie Geng†1,
- Phillip B Gates†2,
- Anoop Kumar2,
- Stefan Guenther3,
- Acely Garza-Garcia4,
- Carsten Kuenne3,
- Peng Zhang1,
- Mario Looso3 and
- Jeremy P Brockes2Email author
© Geng et al.; licensee BioMed Central. 2015
Received: 9 February 2015
Accepted: 24 March 2015
Published: 11 April 2015
The urodele amphibians (salamanders) are the only adult tetrapods able to regenerate the limb. It is unclear if this is an ancestral property that is retained in salamanders but lost in other tetrapods or if it evolved in salamanders. The three-finger protein Prod 1 is implicated in the mechanism of newt limb regeneration, and no orthologs have been found in other vertebrates, thus providing evidence for the second viewpoint. It has also been suggested that this protein could play a role in salamander-specific aspects of limb development. There are ten families of extant salamanders, and Prod 1 has only been identified in two of them to date. It is important to determine if it is present in other families and, particularly, the basal group of two families which diverged approximately 200 MYA.
We have used polymerase chain reaction (PCR) to identify Prod 1 in a Chinese hynobiid species Batrachuperus longdongensis. We obtained an intestinal transcriptome of the plethodontid Aneides lugubris and, from this, identified a primer which allowed PCR of two Prod 1 genes from this species. All known Prod 1 sequences from nine species in four families have been aligned, and a phylogenetic tree has been derived.
Prod 1 is found in basal salamanders of the family Hynobiidae, and in at least three other families, so it may be present in all extant salamanders. It remains a plausible candidate to have been involved in the origins of limb regeneration, as well as the apomorphic aspects of limb development.
KeywordsThree-finger protein Phylogeny Limb regeneration Plethodontid Hynobiid
Prod 1 was originally identified as a retinoid-inducible gene expressed during newt limb regeneration . It is a member of the three-finger protein superfamily that is attached to the cell surface with a glycosylphosphatidylinositol (GPI) anchor and is expressed in the adult newt limb in a shallow proximodistal gradient . It has been shown to have activities during regeneration that are relevant for both nerve dependence and positional identity of the limb blastema [3,4]. The 3D structure of the protein in solution has been solved by NMR and has a distinctive uninterrupted 12-residue α-helical stretch in the third finger . The molecular phylogeny, based on both sequence and structural criteria, indicates that Prod 1 has no known orthologues in other vertebrate taxa. In particular, exhaustive searches and phylogenetic analyses of three-finger proteins (TFPs) from Xenopus and zebrafish suggest that no Prod 1 ortholog is present . Thus, it is apparently a salamander orphan gene implicated in limb regeneration.
Salamanders (urodeles) are the only adult tetrapods able to regenerate the limb. It is unclear if limb regeneration evolved in salamanders or if it is an ancestral property for vertebrates that is retained in salamanders and lost in other tetrapods . The example of Prod 1, as well as other less studied candidates derived from proteomic or transcriptomic analysis of salamander regeneration [7,8], provides evidence for the hypothesis of local evolution, although many questions remain to be answered . It has also been suggested that Prod 1 could be implicated in salamander-specific aspects of limb development such as pre-axial dominance , which is considered to be apomorphic for urodeles .
There are ten families of extant salamanders, and a recent phylogenetic analysis, based on 30 different nuclear genes in 19 species, has concluded that the basal group of salamanders are the Cryptobranchoidea encompassing the two families Hynobiidae and Cryptobranchidae . Limb regeneration has been detected in these salamanders , and the question has been raised as to whether Prod 1 is present in this group and, hence, presumably in the other families . This protein has only been studied to date in newt and Ambystoma species (families Salamandridae and Ambystomatidae) , and we report here that it is also present in the Hynobiidae and in one other family, the Plethodontidae, the most derived and most speciose family of salamanders. During the preparation of this manuscript, transcriptomic data from Hynobius chinensis became available , and we have also included the sequence of Prod 1 from this species in our alignment and analyses. These results support the hypothesis that Prod 1 is present in all extant salamanders and is implicated in the evolution of limb regeneration.
Results and discussion
Expression of long and short forms of Prod 1 in tissues of A . lugubris
It is noteworthy that Prod 1 is found in two species of basal salamander and was therefore presumably present in the last common ancestor of crown group salamanders at the time of divergence, estimated to be at the beginning of the Jurassic. In recent analysis of fossils, evidence for the salamander-specific phenotypes of pre-axial dominance [17,11], and limb regeneration , has been detected in dissorophoid temnospondyl amphibians of the early Permian (300 to 290 MYA). This may have been close to the origin of salamanders in tetrapod evolution , and it is possible that this also coincided with the origin of Prod 1 . Prod 1 could have been present in Lower Permian dissorophoids and subsequently lost in anurans.
- A. mac:
- A. mex:
- A. tig:
glyceraldehyde phosphate dehydrogenase
million years ago
polymerase chain reaction
JPB was supported by a MRC Non-clinical Research Professorship, and the work in this lab was supported by an MRC Programme Grant. The work performed in China was supported by a National Natural Science Foundation of China grant to PZ (No. 31172075).
- Morais Da Silva S, Gates PB, Brockes JP. The newt ortholog of CD59 is implicated in proximodistal identity during amphibian limb regeneration. Dev Cell. 2002;3(4):547–55.View ArticleGoogle Scholar
- Kumar A, Gates PB, Brockes JP. Positional identity of adult stem cells in salamander limb regeneration. C R Biol. 2007;330(6–7):485–90. doi:10.1016/j.crvi.2007.01.006.View ArticlePubMedGoogle Scholar
- Echeverri K, Tanaka EM. Proximodistal patterning during limb regeneration. Dev Biol. 2005;279(2):391–401. doi:10.1016/j.ydbio.2004.12.029.View ArticlePubMedGoogle Scholar
- Kumar A, Godwin JW, Gates PB, Garza-Garcia AA, Brockes JP. Molecular basis for the nerve dependence of limb regeneration in an adult vertebrate. Science. 2007;318(5851):772–7. doi: 10.1126/science.1147710.View ArticlePubMed CentralPubMedGoogle Scholar
- Garza-Garcia A, Harris R, Esposito D, Gates PB, Driscoll PC. Solution structure and phylogenetics of Prod1, a member of the three-finger protein superfamily implicated in salamander limb regeneration. PLoS One. 2009;4(9), e7123. doi: 10.1371/journal.pone.0007123.View ArticlePubMed CentralPubMedGoogle Scholar
- Garza-Garcia AA, Driscoll PC, Brockes JP. Evidence for the local evolution of mechanisms underlying limb regeneration in salamanders. Integr Comp Biol. 2010;50(4):528–35. doi: 10.1093/icb/icq022.View ArticlePubMedGoogle Scholar
- Looso M, Michel CS, Konzer A, Bruckskotten M, Borchardt T, Kruger M, et al. Spiked-in pulsed in vivo labeling identifies a new member of the CCN family in regenerating newt hearts. J Proteome Res. 2012;11(9):4693–704. doi:10.1021/pr300521p.View ArticlePubMedGoogle Scholar
- Looso M, Preussner J, Sousounis K, Bruckskotten M, Michel CS, Lignelli E, et al. A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration. Genome Biol. 2013;14(2):R16. doi: 10.1186/gb-2013-14-2-r16.View ArticlePubMed CentralPubMedGoogle Scholar
- Mihaylova Y, Aboobaker AA. What is it about ‘eye of newt’? Genome Biol. 2013;14(2):106. doi:10.1186/gb-2013-14-2-106.View ArticlePubMed CentralPubMedGoogle Scholar
- Brockes JP, Gates PB. Mechanisms underlying vertebrate limb regeneration: lessons from the salamander. Biochem Soc Trans. 2014;42(3):625–30. doi:10.1042/BST20140002.View ArticlePubMedGoogle Scholar
- Frobisch NB, Shubin NH. Salamander limb development: integrating genes, morphology, and fossils. Developmental dynamics : an official publication of the American Association of Anatomists. 2011;240(5):1087–99. doi:10.1002/dvdy.22629.View ArticleGoogle Scholar
- Shen XX, Liang D, Feng YJ, Chen MY, Zhang P. A versatile and highly efficient toolkit including 102 nuclear markers for vertebrate phylogenomics, tested by resolving the higher level relationships of the caudata. Mol Biol Evol. 2013;30(10):2235–48. doi:10.1093/molbev/mst122.View ArticlePubMedGoogle Scholar
- Griffin PC, Solkin VA. Ecology and conservation of Onychodactylus fischeri (Caudata, Hynobiidae) in the Russian Far East. Asiatic Herpetol Res. 1995;6:53–61.Google Scholar
- Sanchez AA. Q&A: what is regeneration, and why look to planarians for answers? BMC Biol. 2012;10:88.View ArticleGoogle Scholar
- Blassberg RA, Garza-Garcia A, Janmohamed A, Gates PB, Brockes JP. Functional convergence of signalling by GPI-anchored and anchorless forms of a salamander protein implicated in limb regeneration. J Cell Sci. 2011;124(Pt 1):47–56. doi:10.1242/jcs.076331.View ArticlePubMed CentralPubMedGoogle Scholar
- Che R, Sun Y, Wang R, Xu T. Transcriptomic analysis of endangered Chinese salamander: identification of immune, sex and reproduction-related genes and genetic markers. PLoS One. 2014;9(1), e87940. doi:10.1371/journal.pone.0087940.View ArticlePubMed CentralPubMedGoogle Scholar
- Frobisch NB, Carroll RL, Schoch RR. Limb ossification in the Paleozoic branchiosaurid Apateon (Temnospondyli) and the early evolution of preaxial dominance in tetrapod limb development. Evol Dev. 2007;9(1):69–75. doi:10.1111/j.1525-142X.2006.00138.x.View ArticlePubMedGoogle Scholar
- Frobisch NB, Bickelmann C, Witzmann F. Early evolution of limb regeneration in tetrapods: evidence from a 300-million-year-old amphibian. Proceedings Biological sciences/The Royal Society. 2014;281(1794):20141550. doi:10.1098/rspb.2014.1550.View ArticlePubMed CentralPubMedGoogle Scholar
- Schoch RR. Amphibian evolution: the life of early land vertebrates. Wiley Blackwell: Topics in Paleobiology; 2014
- Brockes JP. Variation in salamanders: an essay on genomes, development and evolution. In: Kumar A, Simon A, editors. Salamanders in Regeneration Research: Methods and Protocols. USA: Springer; 2015.Google Scholar
- Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. doi:10.1093/sysbio/syq010.View ArticlePubMedGoogle Scholar
- Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9(8):772. doi:10.1038/nmeth.2109.View ArticlePubMedGoogle Scholar
- Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4.View ArticlePubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. 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.