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Immunohistochemistry and Fluorescent Whole Mount RNA In Situ Hybridization in Larval and Adult Brains of Tribolium

  • Vera S. Hunnekuhl
  • Janna Siemanowski
  • Max S. Farnworth
  • Bicheng He
  • Gregor BucherEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2047)

Abstract

Arthropod brains are fascinating structures that exhibit great complexity but also contain conserved elements that can be recognized between species. There is a long tradition of research in insect neuroanatomy, cell biology, and in studying the genetics of insect brain development. Recently, the beetle Tribolium castaneum has gained attention as a model for insect head and brain development, and many anterior patterning genes have so far been characterized in beetle embryos. The outcome of embryonic anterior development is the larval and, subsequently, the adult brain. A basic requirement to understand genetic cell type diversity within these structures is the ability to localize mRNA and protein of neural genes. Here we detail our protocols for RNA in situ hybridization in combination with immunohistochemistry, optimized for dissected brains of larval and adult beetles.

Keywords

In situ hybridization Gene expression Antibody staining Fluorescence labeling Insect brain Tribolium castaneum Red flour beetle 

References

  1. 1.
    Lichtneckert R, Reichert H (2005) Insights into the urbilaterian brain: conserved genetic patterning mechanisms in insect and vertebrate brain development. Heredity (Edinb) 94:465–477CrossRefGoogle Scholar
  2. 2.
    Younossi-Hartenstein A, Green P, Liaw GJ et al (1997) Control of early neurogenesis of the Drosophila brain by the head gap genes tll, otd, ems, and btd. Dev Biol 182:270–283Google Scholar
  3. 3.
    Hirth F (2003) An urbilaterian origin of the tripartite brain: developmental genetic insights from Drosophila. Development 130:2365–2373CrossRefGoogle Scholar
  4. 4.
    Boyan G, Williams L (2011) Embryonic development of the insect central complex: insights from lineages in the grasshopper and Drosophila. Arthropod Struct Dev 40:334–348CrossRefGoogle Scholar
  5. 5.
    Boyan GS, Reichert H (2011) Mechanisms for complexity in the brain: generating the insect central complex. Trends Neurosci 34:247–257CrossRefGoogle Scholar
  6. 6.
    Ludwig P, Williams JLD, Lodde E et al (1999) Neurogenesis in the median domain of the embryonic brain of the grasshopper Schistocerca gregaria. J Comp Neurol 414:379–390CrossRefGoogle Scholar
  7. 7.
    Posnien N, Koniszewski NDB, Hein HJ, Bucher G (2011) Candidate gene screen in the red flour beetle Tribolium reveals six3 as ancient regulator of anterior median head and central complex development. PLoS Genet 7:e1002416CrossRefGoogle Scholar
  8. 8.
    Koniszewski NDB, Kollmann M, Bigham M et al (2016) The insect central complex as model for heterochronic brain development—background, concepts, and tools. Dev Genes Evol 226:209–219CrossRefGoogle Scholar
  9. 9.
    Richards S, Gibbs RA, Weinstock GM et al (2008) The genome of the model beetle and pest Tribolium castaneum. Nature 452:949–955Google Scholar
  10. 10.
    Gilles AF, Schinko JB, Averof M (2015) Efficient CRISPR-mediated gene targeting and transgene replacement in the beetle Tribolium castaneum. Development 142:2832–2839CrossRefGoogle Scholar
  11. 11.
    Schinko JB, Hillebrand K, Bucher G (2012) Heat shock-Mediated misexpression of genes in the beetle Tribolium castaneum. Dev Genes Evol 222:287–298CrossRefGoogle Scholar
  12. 12.
    Schinko JB, Weber M, Viktorinova I et al (2010) Functionality of the GAL4/UAS system in Tribolium requires the use of endogenous core promoters. BMC Dev Biol 10CrossRefGoogle Scholar
  13. 13.
    Schmitt-Engel C, Schultheis D, Schwirz J et al (2015) The iBeetle large-scale RNAi screen reveals gene functions for insect development and physiology. Nat Commun 6Google Scholar
  14. 14.
    Farnworth MS, Eckermann KN, Ahmed HM et al (2019) The red flour beetle as model for comparative neural development: genome editing to mark neural cells in Tribolium brain development. Methods Brain DevelopGoogle Scholar
  15. 15.
    Posnien N, Schinko JB, Kittelmann S, Bucher G (2010) Genetics, development and composition of the insect head—a beetle’s view. Arthropod Struct Dev 39:399–410CrossRefGoogle Scholar
  16. 16.
    Loesel R, Nässel DR, Strausfeld NJ (2002) Common design in a unique midline neuropil in the brains of arthropods. Arthropod Struct Dev 31:77–91CrossRefGoogle Scholar
  17. 17.
    Strausfeld NJ (2012) Arthropod brains: evolution, functional elegance, and historical significance. The Belknap Press of Harvard University Press, Cambridge, MAGoogle Scholar
  18. 18.
    Büscher M, Oberhofer G, Garcia-Perez NC, Bucher G (2019) A protocol for double in situ hybridization and immunohistochemistry for the study of embryonic brain development in Tribolium castaneum. Methods Brain DevelopGoogle Scholar
  19. 19.
    Hunnekuhl VS, Akam M (2014) An anterior medial cell population with an apical-organ-like transcriptional profile that pioneers the central nervous system in the centipede Strigamia maritima. Dev Biol 396:136–149CrossRefGoogle Scholar
  20. 20.
    Yoshida-Noro C, Myohara M, Kobari F, Tochinai S (2000) Nervous system dynamics during fragmentation and regeneration in Enchytraeus japonensis (Oligochaeta, Annelida). Dev Genes Evol 210:311–319CrossRefGoogle Scholar
  21. 21.
    Bodies E, Dakou E, Vanbekbergen N et al (2014) Whole-mount in situ hybridization (WISH) optimized for gene expression analysis in mouse embryos and embryoid bodies. In: Nielsen B (ed) In situ hybridization protocols, Methods in molecular biology (methods and protocols), vol 1211. Humana Press, New York, NYGoogle Scholar
  22. 22.
    Schinko J, Posnien N, Kittelmann S et al (2009) Single and double whole-mount in situ hybridization in red flour beetle (Tribolium) embryos. Cold Spring Harb Protoc 4:1–5Google Scholar
  23. 23.
    Dearden PK, Akam M (2001) Early embryo patterning in the grasshopper, Schistocerca gregaria: wingless, decapentaplegic and caudal expression. Development 128:3435–3444Google Scholar
  24. 24.
    Schindelin J, Arganda-Carreras I, Frise E et al (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Vera S. Hunnekuhl
    • 1
  • Janna Siemanowski
    • 1
  • Max S. Farnworth
    • 1
  • Bicheng He
    • 1
  • Gregor Bucher
    • 1
    Email author
  1. 1.Department of Evolutionary Developmental GeneticsGeorg-August-University GöttingenGöttingenGermany

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