Due to easy culturing, short generation time, easily accessible embryos (Sokoloff, 1972), and the fact that the beetle is suited for a large set of experi-mental methods, Tribolium castaneum became a more and more popular model system for developmental biology and a number of other fields. The accessibility to standard techniques like immunohistochemistry and in situ hybridization (Patel et al., 1994; Sommer and Tautz, 1993) was a key requirement for the study of gene functions during development. The establishment of robust RNA interference (RNAi) techniques (Brown et al., 1999a; Bucher et al., 2002) and functional trans-formation systems (Berghammer et al., 1999b; Lorenzen et al., 2003; Pavlopoulos et al., 2004) allowed comprehensive functional analyses and even a large scale insertional mutagenesis screen (Trauner et al., 2009). One of the recent and most important mile stones in Tribolium research was the publication of the genome se-quence (Richards et al., 2008). This comprehensive set of tools allowed the
func-tional analysis of genes which were described in Drosophila or vertebrates. In-deed, this ‘candidate gene approach” served very successful for many years and a large number of genes have been analyzed in a number of processes, including segmentation (see 2.4), axis formation (see 2.4 and 2.6), or head patterning (Posnien et al., 2011; Schinko et al., 2008). However, this approach is restricted to inherent limitations and depends on the identification of candidates in other spe-cies. The different modes of development between Drosophila and Tribolium make it very likely that the beetle makes use of a number of different factors than the fly.
And indeed the identification of the transcriptional repression of Tc-caudal through Tc-mex3 as well as the identification of the unusual Gap-gene Tc-mille-pattes con-firm this assumption (Savard et al., 2006; Schoppmeier et al., 2009). But also some key regulators for dorsal-ventral (2.5 and 2.6) and anterior-posterior (2.3 and 2.4) patterning are missing in Tribolium compared to Drosophila and the substi-tutes are not known in many cases. Filling the gaps in our knowledge on these de-velopmental processes requires an extensive, unbiased screening approach.
The most reasonable way to meet this demand in Tribolium is a large scale RNAi screen. The powerful and easily appliable technique of parental RNAi allows the production of a high number of embryonic loss of function phenotypes with reasonable workload (Bucher et al., 2002). RNAi has a number of advantages over genetic screens in Tribolium. The lack of a comprehensive set of balancers for the ten chromosomes in Tribolium makes a mutagenesis screen in the beetle very laborious and costly (Berghammer et al., 1999a). Since a mutagenesis screen would require long term stockkeeping, a saturating screen in Tribolium is not suitable with the limited labs working on Tribolium worldwide (Trauner et al., 2009). A genome wide RNAi screen allows the annotation of the phenotype along with the identity of the targeted gene in a database, which allows permanent ac-cess to all neac-cessary data for easy reproduction of the experiment without exten-sive stockkeeping (Schmitt-Engel, 2010; Schmitt-Engel et al., in preparation). As RNAi is a reverse genetics approach, no time consuming identification of the mu-tated gene is necessary. Furthermore, in a classical genetic screen only 25 % of the larvae are homozygous for a zygotic mutation (St Johnston, 2002). The strong RNAi response in Tribolium can reach up to 100 % efficiancy for particular effects (Bucher et al., 2002). Finally, depending on a well annotated genome, it is a lot easier to estimate the saturation of an RNAi screen compared to a mutagenesis
screen where an unequal distribution of the mutations along the genome makes this task more complicated (Pollock and Larkin, 2004).
2.7.1 The iBeetle screening concept (design of the iBeetle screen)
Several German Tribolium research groups have joined under the guidance of Gregor Bucher (Göttingen) and Martin Klingler (Erlangen) to perform a genome wide RNAi screen in the red flour beetle to identify candidate genes involved in a set of different processes. By realizing this project, Tribolium is brought forward as a model organism for developmental biology. A detailed description of the screen, the participating research groups, and the core projects can be found in (Schmitt-Engel, 2010 and under http://ibeetle.uni-goettingen.de/). The design of the iBeetle screen has been a major topic in the PhD project of C. Schmitt-Engel. During this project, which contained a detailed pre-screen, it was successfully proven that the iBeetle screening concept is doable and leads to new phenotypes (Schmitt-Engel, 2010). In this paragraph the actual concept will be shortly mentioned, the detailed screening procedure for the pupal injection screen is explained in the materials and methods section (3.6).
The screening project is split into two major funding periods of three years each. The participating PhD students screen for about 12–14 month and work af-terwards on a particular project to analyze candidate genes found during the screening process. The iBeetle screen is supposed to fulfill three major tasks:
Identification of genes for processes which are not analyzable in Drosophila or very difficult to study in the fly. This is for instance true for larval leg mus-culature (the Drosophila larva does not have legs) or embryonic head devel-opment, since Drosophila undergoes head involution (Bucher and Wimmer, 2005).
The identification of genes which are not present or have a different function in the fly.
The establishment of Tribolium as a usable screening platform for genome wide studies and the acquisition of a comprehensive set of functional data for particular developmental processes. The latter will be available via an online database and open for the entire Tribolium community (iBeetle-Base, http://ibeetle-base.uni-goettingen.de/search/phenotypeSearch.jsf).
The iBeetle screen constists of two experimentally independent screening parts: the larval injection screen, mainly focusing on insect metamorphosis and development of the odoriferous glands (which are not present during larval stages), and the pupal injection screen, having the main focus on embryonic de-velopment including embryonic muscle formation. Transgenic lines driving tissue-specific GFP expression are used in both screening parts in order to make the screen more effective.The transformation marker is in both cases an EGFP gene under control of the 3xP3 promoter (Berghammer et al., 1999b; Horn and Wimmer, 2000). In the larval injection screen, dsRNA injection is performed with female Tri-bolium L5 and L6 larvae and the injected animals are analyzed in downstream screening steps. Injection takes place in larvae of the mD17 strain which carry a not localized insertion of a minos-3xP3-EGFP construct, leading to EGFP expres-sion in the metathoracic musculature which allows screening for muscle defects in this body part (Pavlopoulos et al., 2004). Since the larval injection screen had no impact on the project described in this thesis, I refer to (Schmitt-Engel, 2010;
Schmitt-Engel et al., in preparation) for more information. In the pupal injection screen female pupae of the Pig-19 strain (Lorenzen et al., 2003; Trauner et al., 2009) were injected, crossed to male beetles, and the offspring was analyzed for developmental defects (parental RNAi) (Bucher et al., 2002). During this screening part the main focus was on the offspring of the injected animals. The Pig-19 strain expresses GFP in the larval body musculature, allowing the identification of de-fects during muscle development or mesoderm establishment in general. Dede-fects affecting ectodermal structures were analyzed by doing cuticle preparations on the progeny of injected females.
The iBeetle screen is designed as a first pass screen, which means that every injection is done only once (although in several animals), without experimen-tal replicates. In order to monitor and maintain the screening quality, continuous positive and negative controls are included in the screening process.