Investigation into gene regulatory networks governing the development and maintenance of the planarian reproductive system

by Thomas Marie Frédéric Nathalan Brochier
Doctoral thesis
Date of Examination:2024-01-12
Date of issue:2024-03-20
Advisor:Prof. Dr. Jochen Rink
Referee:Prof. Dr. Argyris Papantonis
Referee:Dr. Nico Posnien
Referee:Dr. Marieke Oudelaar
Referee:Dr. Ufuk Günesdogan
Referee:Prof. Dr. Carsten Lüder
Persistent Address: http://dx.doi.org/10.53846/goediss-10399

 

 

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Abstract

English

Regulation of gene expression is a fundamental mechanism allowing the existence of complex living systems. This regulation takes shape in the form of hierarchical gene regulatory networks (GRNs), culminating in the binding of transcription factors (TFs) to regulatory elements (REs). Uncovering the structures of these GRNs has shed light on the mechanistic basis of complex spatiotemporal processes like embryonic development. The reconstruction of such GRN, for any biological process, is therefore the ultimate goal for its understanding. The planarian Schmidtea mediterranea is an important model organism for the study of adult stem cell systems, cell differentiation and regeneration. However, very little is known about the structure of GRNs in this organism due to the historical lack of adequate tools to study regulatory elements. At the beginning of my thesis, I developed a robust Start-seq protocol to study RE activity in S. mediterranea by probing for transcription initiation events. Following this, I characterized the identified putative REs on the basis of their distribution, chromatin context as well as motif content. Putative REs identified using Start-seq possess characteristic epigenomic signatures as they are situated within regions of open chromatin and are enriched for active epigenetic marks such as H3K27Ac, H3K4me3 and H3k4me1. I next showed that both planarian putative enhancers and promoter are enriched for core promoter motifs that are mostly situated at the expected position. Interestingly, the DPE motif was enriched at the transcription initiation site in both types of REs instead of its described position at ± +30 nt. Following this, I showed that most of the identified REs showed sign of bidirectional transcription initiation which is considered as a characteristic feature of REs. After characterizing the REs, I leveraged the existence of two naturally occurring biotypes within S. mediterranea (sexual and asexual) to investigate gene regulatory networks (GRNs) responsible for the development and maintenance of the planarian reproductive system. These biotypes are genetically similar, but one fails to develop sexual organs. Comparing their transcription initiation landscapes allowed me to identify multiple transcription factors potentially part of in these GRNs. Notably, the majority of the selected TF candidates exhibited expression in sexual organs, with half of the candidates being specifically expressed in these tissues. Knockdown of three candidates (tead 1, thap and cebp 4 ) showed dysregulation of many reproductive genes. In situ hybridizations on sexual markers after RNAi of the three candidates confirmed the abrogation of sexual tissues in these conditions. The results indicated that tead 1 produced a severe phenotype, where most sexual organs were absent, except for the ovaries. The thap candidate exhibited a defect in shell gland formation and yolk tissue patterning, while the cebp 4 candidate displayed an impairment in sperm differentiation. Overall, this study was not able to recreate a GRN governing the development and maintenance of the reproductive tissue in S. mediterranea. However, it lays a foundation for further research on GRNs in planaria as it shows that identification of differentially active REs using the established protocol was successful in obtaining important regulators the planarian reproductive system.
Keywords: Planarians; Epigenomics
 
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