Investigating the Re-initiation of Segmentation with Temporally Restricted RNAi in Tribolium castaneum

by Felix Kaufholz
Doctoral thesis
Date of Examination:2020-07-06
Date of issue:2020-10-01
Advisor:Prof. Dr. Gregor Bucher
Referee:Prof. Dr. Jörg Großhans
Referee:Prof. Dr. Gregor Eichele
Referee:Dr. Nico Posnien
Referee:Prof. Dr. Daniel John Jackson
Referee:Prof. Dr. Bleidorn Christoph
Persistent Address: http://dx.doi.org/10.53846/goediss-8229

 

 

Files in this item

Name:PhD-Thesis_Felix-Kaufholz-for_ediss.pdf
Size:21.5Mb
Format:PDF
Description:Dissertation main text
Name:Cuticles and HCR data.xlsx
Size:165.Kb
Format:VND.OPENXMLFORMATS-OFFICEDOCUMENT.SPREADSHEETML.SHEET
Description:Supplementary file #1: according to table S7.1
Name:hcr classes all embryos_high.pdf
Size:150.Mb
Format:PDF
Description:Supplementary file #2: High-quality images according to Fig. S7.12
Name:clones_phd_FK.docx
Size:344.Kb
Format:VND.OPENXMLFORMATS-OFFICEDOCUMENT.WORDPROCESSINGML.DOCUMENT
Description:Supplementary file #3: sequences of used clones

The following license files are associated with this item:

Abstract

English

Much of the success of arthropods is attributed to their body’s segmentation. Segmentation provides various opportunities for nature to evolve new structures without greatly impacting overall fitness of the animal. Most knowledge about the development of segmentation comes from the vinegar fly, Drosophila melanogaster. Drosophila has a derived long-germ embryogenesis with simultaneous segmentation. Short-germ embryogenesis or sequential segmentation as in the red flour beetle, Tribolium castaneum, however, is regarded as a more ancestral state of segmentation. Much less is known about the genetic processes underlying this sequential segmentation. Recently, a segmentation clock was identified in Tribolium. This clock utilizes oscillatory expression of the primary pair-rule genes (pPRGs) to pattern the body axis during both the static blastoderm and the elongating germband. The segmentation clock receives input from the upstream “posterior signaling center” and Tc-caudal (Tc-cad). Downstream of the segmentation clock, the secondary PRGs and the segment polarity genes interpret the pPRG input and provide further positional information along the AP axis. Studies in Tribolium revealed great insights into the molecular mechanism of sequential segmentation. However, most of these findings are based on RNAi leading to the permanent knockdown of gene function. Thus, they are not suited for studying gene interactions at later stages of the dynamic process of segmentation and the segmentation clock. I utilized a Viral Suppressors of RNAi (VSR) as a novel tool to temporally restrict RNAi in Tribolium. This novel tool, hsVSR (heat shock VSR), allowed me to investigate the segmentation processes in more depth. Specifically, I aimed to answer the question whether RNAi-induced breakdown of segmentation is irreversible or if re-initiation of segmentation is possible. With proof-of-concept experiments, I confirmed the functionality and specificity of the hsVSR system to investigate segmentation. I could then show that a rescue of segmentation after RNAi-mediated breakdown is possible by re-initiating the segmentation clock itself. However, rescue of segmentation by inhibiting RNAi of upstream factors of the segmentation clock was not possible. Once the “posterior signaling center” is lost, it cannot re-initiate. Additionally, a possible negative autoregulation of the pPRG Tc-even-skipped was uncovered. Taken together, I showed the functionality of the hsVSR system during segmentation. I identified the level at which RNAi inhibition can rescue segmentation within the segmentation process and provided molecular evidence for the nature of the rescue.
Keywords: Developmental Biology; Tribolium; Segmentation; Arthropods; RNAi; EvoDevo; Hybridization Chain Reaction
 
Statistik