Single and Double Whole-Mount In Situ Hybridization in Red Flour Beetle (Tribolium) Embryos

Abstract

INTRODUCTIONThe red flour beetle, Tribolium castaneum, has emerged as an important model system for studying the evolution of development. Studies with Tribolium complement the vast amount of research done with Drosophila. Developmental features that are conserved between Drosophila and Tribolium, such as body segmentation, are achieved by quite different means, and thus comparison of developmental mechanisms between these two insects can address many interesting questions concerning the evolution of morphology and other characters. Most in situ protocols used for Tribolium have been adapted from Drosophila studies. Whole-mount in situ hybridization is a standard technique to visualize the activity of genes in embryos. The single and double staining protocol presented here uses two nonfluorescent stains to reveal gene activity. The development of both stains can be monitored visually, allowing the strength of the signal to be adjusted as needed. Cells that express both of the genes under investigation are readily detected using a microscope. The use of EGTA during fixation increases the proportion of embryos that devitellinize upon methanol treatment.

Early asymmetries in maternal transcript distribution associated with a cortical microtubule network and a polar body in the beetle Tribolium castaneum

“…Pre-blastoderm embryos were fixed in 4% formaldehyde in PBS for 15-30 min, according to established protocols for Tribolium (Schinko et al, 2009;Shippy et al, 2009). Ovarioles were dissected and fixed according to Lynch et al (2010).…”

“…Alkaline phosphatase in situ hybridizations were carried out using DIG-labelled RNA probes and NBT/ BCIP, according to established protocols (Schinko et al, 2009;Shippy et al, 2009). The proteinase K step was found to be unnecessary on preblastoderm eggs and thus omitted.…”

Asymmetrically expressed axin required for anterior development in Tribolium

“…Off-target effects were excluded by injection of two nonoverlapping dsRNA fragments (base pairs 448-1091 and 1,168-1,617 of Tc-axin cds, respectively), which elicited the same phenotype as the injection of the long dsRNA fragments; 500-to 1,000-bp fragments were used as templates for digoxigenin-labeled riboprobes. In situ hybridization was performed as previously described (29).…”

RNAi in the Red Flour Beetle (Tribolium)

“…An introduction to the red flour beetle and its use as a model organism in evolutionary developmental biology is presented in The Red Flour Beetle, Tribolium castaneum (Coleoptera): A Model for Studies of Development and Pest Biology ). Protocols are also available for Red Flour Beetle (Tribolium) Germline Transformation and Insertional Mutagenesis (Berghammer et al 2009), Single and Double Whole-Mount In Situ Hybridization in Red Flour Beetle (Tribolium) Embryos (Schinko et al 2009) and Concurrent In Situ Hybridization and Antibody Staining in Red Flour Beetle (Tribolium) Embryos 8. Determine the concentration using a spectrophotometer (usually 300-500 ng/µL).…”

“…Older embryos can be injected to identify the late functions of a gene, but such injections may produce a higher incidence of nonspecific effects due to damage to the embryo. [Schinko et al 2009]). Staining of another gene in the same embryos and in the same color reaction is required as a positive control (Schinko et al 2008).…”