Skip to main content

Immunostaining and In Situ Hybridization of the Developing Acoel Nervous System

  • Protocol
  • First Online:

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2047))

Abstract

The study of acoel morphologies has been recently stimulated by the knowledge that this group of animals represents an early offshoot of the Bilateria. Understanding how organ systems and tissues develop and the molecular underpinnings of the processes involved has become an area of new research. The microscopic anatomy of these organisms is best understood through the systematic use of immunochemistry and in situ hybridization procedures. These methods allow us to map, in precise detail, the expression patterns of genes and proteins, in space and time. With the additional use of genomic resources, they provide us with insights on how a group of “early” bilaterians have diversified over time. As these animals are new to the world of molecular studies, the protocols have involved a lot of new and specific adaptations to their specific anatomical-histological characteristics. Here we explain some of these protocols in detail, with the aim that should prove useful in our much-needed understanding of the origins of bilaterian animals. An anatomical sketch is provided at the beginning as a necessary guide for those not familiar with the Acoela.

This is a preview of subscription content, log in via an institution.

Buying options

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   169.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   219.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   299.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Cannon JT, Vellutini BC, Smith J, Ronquist F, Jondelius U, Hejnol A (2016) Xenacoelomorpha is the sister group to Nephrozoa. Nature 530:89–93. https://doi.org/10.1038/nature16520

    Article  CAS  PubMed  Google Scholar 

  2. Philippe H, Brinkmann H, Copley RR, Moroz LL, Nakano H, Poustka AJ, Wallberg A, Peterson KJ, Telford MJ (2011) Acoelomorph flatworms are deuterostomes related to Xenoturbella. Nature 470:255–260. https://doi.org/10.1038/nature09676

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Baguñà J, Riutort M (2004) The dawn of bilaterian animals: the case of acoelomorph flatworms. BioEssays 26:1046–1057

    Article  Google Scholar 

  4. Jondelius U, Wallberg A, Hooge M, Raikova OI (2011) How the worm got its pharynx: phylogeny, classification and bayesian assessment of character evolution in acoela. Syst Biol 60:845–871. https://doi.org/10.1093/sysbio/syr073

    Article  PubMed  Google Scholar 

  5. Achatz JG, Chiodin M, Salvenmoser W, Tyler S, Martinez P (2012) The Acoela: on their kind and kinships, especially with nemertodermatids and xenoturbellids (Bilateria incertae sedis). Org Divers Evol 13:267–286

    Article  Google Scholar 

  6. Arroyo AS, López-Escardó D, de Vargas C, Ruiz-Trillo I (2016) Hidden diversity of Acoelomorpha revealed through metabarcoding. Biol Lett 12:20160674. https://doi.org/10.1098/rsbl.2016.0674

    Article  PubMed  PubMed Central  Google Scholar 

  7. Smith J, Tyler S (1986) Frontal organs in the Acoelomorpha (Turbellaria): ultrastructure and phylogenetic significance. Hydrobiologia 132:71–78

    Article  Google Scholar 

  8. Ehlers U (1991) Comparative morphology of the statocysts in the Plathelmithes and the Xenoturbellida. Hydrobiologia 227:263–271

    Article  Google Scholar 

  9. Yamasu T (1991) Fine structure and function of ocelli and sagittocysts of acoel flatworms. Hydrobiologia 227:273–282. https://doi.org/10.1007/BF00027612

    Article  Google Scholar 

  10. Rieger RM, Tyler S, Smith JPS, Rieger GE (1991) Platyhelminthes: Turbellaria. In: Harrsion FW, Bogitsch BJ (eds) Microscopic anatomy of invertebrates. Wiley, New York

    Google Scholar 

  11. Raikova OI, Reuter M, Kotikova EA, Gustafsson MKS (1998) A commissural brain! The pattern of 5-HT immunoreactivity in acoela (Plathelminthes). Zoomorphology 118:69–77. https://doi.org/10.1007/s004350050058

    Article  Google Scholar 

  12. Reisinger E (1925) Ein landbewohnender Archiannelide. (Zugleich ein Beitrag zur Systematik der Archianneliden). Z Morphol Tiere 3:197–254

    Article  Google Scholar 

  13. Martínez P, Hartenstein V, Sprecher SG (2017) Xenacoelomorpha nervous systems. In Oxford Research Encyclopedia of Neuroscience. Ed. S. Murray Sherman. New York: Oxford University Press

    Google Scholar 

  14. Jennings JB (1957) Studies on feeding, digestion, and food storage in free-living flatworms (Platyhelminthes: Turbellaria). Biol Bull 112:63–80

    Article  Google Scholar 

  15. Achatz J, Gschwentner R, Rieger R (2005) Symsagittifera smaragdina n. spec., a new acoel (Acoela, Acoelomorpha) of the Mediterranean Sea. Zootaxa 1085:33–45

    Article  Google Scholar 

  16. Pedersen KJ (1964) The cellular organization of Convoluta convoluta, an Acoel Turbellarian: a cytological, histochemical and fine structural study. Z Zellforsch 64:655–687

    Article  CAS  Google Scholar 

  17. Hyman LH (1951) The invertebrates: Platyhelminthes and Rhynchocoela. The acoelomate bilateria vol II. McGraw-Hill Book Company, Inc, New York

    Google Scholar 

  18. Hooge M (2001) Evolution of body-wall musculature in the Platyhelminthes (Acoelomorpha, Catenulida, Rhabditophora). J Morphol 249:171–194

    Article  CAS  Google Scholar 

  19. Hooge M, Tyler S (2006) Concordance of molecular and morphological data: The example of the Acoela. Integr Comp Biol 46:118–124

    Article  CAS  Google Scholar 

  20. Semmler H, Bailly X, Wanninger A (2008) Myogenesis in the basal bilaterian Symsagittifera roscoffensis (Acoela). Front Zool 5:1–15. https://doi.org/10.1186/1742-9994-5-14

    Article  Google Scholar 

  21. Henry JQ, Martindale MQ, Boyer BC (2000) The unique developmental program of the acoel flatworm, Neochildia fusca. Dev Biol 220:285–295. https://doi.org/10.1006/dbio.2000.9628

    Article  CAS  PubMed  Google Scholar 

  22. Hejnol A (2015) Acoelomorpha and Xenoturbellida. In: Evolutionary developmental biology of invertebrates, vol 1. Springer Verlag, New York, pp 203–214

    Chapter  Google Scholar 

  23. Raikova O, Reuter M, Gustafsson MKS, Maule AG, Halton DW, Jondelius U (2004) Evolution of the nervous system in Paraphanostoma (Acoela). Zool Scr:71–88

    Article  Google Scholar 

  24. Semmler H, Chiodin M, Bailly X, Martinez P, Wanninger A (2010) Steps towards a centralized nervous system in basal bilaterians: Insights from neurogenesis of the acoel Symsagittifera roscoffensis. Develop Growth Differ 52:701–713. https://doi.org/10.1111/j.1440-169X.2010.01207.x

    Article  CAS  Google Scholar 

  25. Achatz JG, Martinez P (2012) The nervous system of Isodiametra pulchra (Acoela) with a discussion on the neuroanatomy of the Xenacoelomorpha and its evolutionary implications. Front Zool 9:27

    Article  Google Scholar 

  26. Raikova OI (2004) Neuroanatomy of basal bilaterians (Xenoturbellida, Nemertodermatida, Acoela) and its phylogenetic implications (PhD thesis). Åbo Akademi University

    Google Scholar 

  27. Perea-Atienza E, Gavilan B, Chiodin M, Abril JF, Hoff KJ, Poustka AJ, Martinez P (2015) The nervous system of Xenacoelomorpha: a genomic perspective. J Exp Biol 218:618–628. https://doi.org/10.1242/jeb.110379

    Article  PubMed  Google Scholar 

  28. Gavilán B, Perea-Atienza E, Martínez P (2016) Xenacoelomorpha: a case of independent nervous system centralization? Philos Trans R Soc B Biol Sci 371. https://doi.org/10.1098/rstb.2015.0039

    Article  Google Scholar 

  29. De Mulder K, Kuales G, Pfister D, Willems M, Egger B, Salvenmoser W, Thaler M, Gorny AK, Hrouda M, Borgonie G, Ladurner P (2009) Characterization of the stem cell system of the acoel Isodiametra pulchra. BMC Dev Biol 9:1–17. https://doi.org/10.1186/1471-213X-9-69

    Article  CAS  Google Scholar 

  30. Srivastava M, Mazza-Curll KL, Van Wolfswinkel JC, Reddien PW (2014) Whole-body acoel regeneration is controlled by Wnt and Bmp-Admp signaling. Curr Biol 24:1107–1113. https://doi.org/10.1016/j.cub.2014.03.042

    Article  CAS  PubMed  Google Scholar 

  31. Chiodin M, Børve A, Berezikov E, Ladurner P, Martinez P, Hejnol A (2013) Mesodermal gene expression in the acoel Isodiametra pulchra indicates a low number of mesodermal cell types and the endomesodermal origin of the gonads. PLoS One 8:e55499. https://doi.org/10.1371/journal.pone.0055499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Albuixech-Crespo B, López-Blanch L, Burguera D, Maeso I, Sánchez-Arrones L, Moreno-Bravo JA, Somorjai I, Pascual-Anaya J, Puelles E, Bovolenta P, Garcia-Fernàndez J, Puelles L, Irimia M, Ferran JL (2017) Molecular regionalization of the developing amphioxus neural tube challenges major partitions of the vertebrate brain. PLoS Biol 15. https://doi.org/10.1371/journal.pbio.2001573

    Article  Google Scholar 

  33. Hejnol A, Martindale MQ (2008) Acoel development indicates the independent evolution of the bilaterian mouth and anus. Nature 456:382–386. https://doi.org/10.1038/nature07309

    Article  CAS  PubMed  Google Scholar 

  34. Perea-Atienza E, Sprecher SG, Martínez P (2018) Characterization of the bHLH family of transcriptional regulators in the acoel S. roscoffensis and their putative role in neurogenesis. EvoDevo 9:1–16. https://doi.org/10.1186/s13227-018-0097-y

    Article  Google Scholar 

Download references

Acknowledgments

The research in P. Martínez laboratory was carried out with the support of the Spanish Ministry of Science, Grants BFU2006-00898, BFU2009-07383, and BFU2012-32806. E. Perea-Atienza and B. Gavilán were supported by PhD fellowships from the Universitat de Barcelona (APIF). S.G. Sprecher acknowledges the Swiss National Science Foundation 31003A_169993. Elena Perea-Atienza and Brenda Gavilán contributed equally to the development of the described methodology. The authors would also like to thank Kathryn Apse and Prof. Seth Tyler (University of Maine) for letting us publish the acoel morphology diagrams in Fig. 1 of this chapter.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Pedro Martinez .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2020 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Perea-Atienza, E., Gavilán, B., Sprecher, S.G., Martinez, P. (2020). Immunostaining and In Situ Hybridization of the Developing Acoel Nervous System. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_4

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9732-9_4

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9731-2

  • Online ISBN: 978-1-4939-9732-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics