Systematic Reverse Genetic Screen to Identify Novel Genes Required for Anterior Patterning of the Red Flour Beetle Tribolium castaneum

by Jonas Schwirz
Doctoral thesis
Date of Examination:2014-04-29
Date of issue:2014-08-19
Advisor:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Ernst A. Wimmer
Referee:Prof. Dr. Andreas Wodarz
Persistent Address: http://dx.doi.org/10.53846/goediss-4654

 

 

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Abstract

English

The formation of two main body axis is a crucial event during bilaterian de-velopment and has been in the focus of research for many years. The vinegar fly Drosophila melanogaster served as an excellent model organism for insect devel-opmental biology. Body axis formation in the fly is today vastly understood. Never-theless, comparative studies using the so-called ‘candidate gene approach’ in oth-er species revealed that many aspects of Drosophila development seem to be evolutionarily derived. However, although very successful during the last decades, the candidate gene approach has its limitations and comprehensive and unbiased studies in species other than Drosophila are necessary in order to fill our gaps of knowlegde about axis formation and early patterning in arthropods. The red flour beetle Tribolium castaneum offers a vast tool box for descriptive and functional analysis of the molecular processes during early development and has emerged to the second best arthropod model system after Drosophila. In this study the ap-proach of a comprehensive RNA interference screen has been used to identify new factors required for anterior patterning in Tribolium. About 25 % of the Tribolium genome were screened during the first approximately 14 months of the iBeetle screen. Almost 10 % of all screened genes were annotated to result in head defects on the L1 larval cuticle after knock down. Out of this set of candidate genes a stepwise selection led to the identification of two genes for in depth analy-sis. The E3 ubiquitin ligase Tc-Smurf is necessary for patterning the blastodermal fate map in Tribolium and loss of Tc-Smurf function leads to loss of prospective head regions. Missing Tc-Smurf function during later stages can result in death of anterior body regions up to the entirety of embryonic cells. Tc-Smurf is necessary to control Decapentaplegic (Dpp) signaling during early and later stages of Tribolium embryogenesis and is especially important for restricting Dpp signaling in the anterior embryo. The second candidate gene TC004374 probably plays an important role for the formation of the anterior amnion during differentiated blastoderm stages and for maintenance of amniotic tissue at the margin of the embryo during germ band stages. Loss of TC004374 function also leads to ectopic domains of phosphorylated Mothers against Dpp (pMAD) protein in the embryonic headlobes. This indicates that both detected genes are involved in inhibition of Dpp signaling in the anterior embryonic region during Tribolium embryogenesis.
Keywords: Development; head development; Tribolium; iBeetle; RNAi screen, insect development
 
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