Universität zu Köln

Hellekes, Viktoria ORCID: 0000-0003-1639-9709 (2025). Nematode genetics beyond Caenorhabditis elegans: Adapting modern genetic tools to phylogenetically distant nematodes. PhD thesis, Universität zu Köln.

PDF Viktoria_Hellekes_Dissertation_01072025_for_publishing.pdf - Submitted Version Download (61MB)

Abstract

Understanding the genetic basis of development and evolution in diverse species requires tools for functional analysis that can be applied beyond traditional models. Model organisms were selected primarily for their experimental accessibility rather than for their representation of various biological traits. Insights gained from them often provide an incomplete picture of the biology and evolutionary history of broader groups of species. The phylum Nematoda comprises a broad diversity of species occupying a wide range of ecological niches. Nematodes share a conserved body plan, yet show striking differences in early development at the genetic and cellular levels, with differences in cleavage patterns, gene expression, and signaling pathways. Functional studies in other nematode species than Caenorhabditis elegans are therefore essential to understand both conserved and novel mechanisms. Panagrolaimus sp. PS1159 is a free-living, triploid nematode that reproduces parthenogenetically. It offers a promising experimental model due to its phylogenetic distance to C. elegans, its fascinating traits such as cryptobiosis, and was chosen to comparatively study development, evolution, and alternative reproductive and survival strategies. This thesis presents the first successful use of CRISPR/Cas9 in a Panagrolaimus nematode, applying both the non-homologous end joining (NHEJ) and homology-directed repair (HDR). Targeting the unc-22 orthologue induced a visible twitching phenotype that can be used as a co-CRISPR marker for future research. Protocol optimization led to editing efficiencies of up to 30%. An mEGFP knock-in into a histone H3.3 orthologue allowed live imaging of nuclei and demonstrated the feasibility of creating fluorescent reporter lines. To facilitate genotyping of modified strains after gene editing, a workflow using Oxford Nanopore Technologies (ONT) Sequencing was established, demonstrated its potential to detect complex edits and large insertions, and offered an accelerated alternative to traditional cloning and Sanger sequencing, which are highly time consuming. With an established CRISPR protocol, this thesis focused on the functional analysis of one of the most classical gene families in evolutionary developmental biology: the Hox genes, key regulators of anterior–posterior body patterning that are highly conserved amongbilaterians. In C. elegans, six Hox genes are present, the anterior ceh-13, the central lin-39 and mab-5, and the posterior egl-5, php-3 and nob-1. The canonical collinearity rule is broken in C. elegans and only three genes are essential during embryogenesis. To analyze their conservation, spatial expression patterns of the full set of Hox genes were analyzed in PS1159 and the more distantly related Plectus sambesii using in situ hybridization chain reaction (HCR), revealing strong similarities to C. elegans. Knockouts of lin-39 and mab-5 in PS1159 resulted in similar phenotypes compared to C. elegans and indicate functional conservation. In conclusion, this thesis established Panagrolaimus sp. PS1159 as a genetically tractable system and lays a methodological basis for future comparative developmental studies in nematodes beyond C. elegans.

Item Type:	Thesis (PhD thesis)
Translated title:	Title Language UNSPECIFIED English
Creators:	Creators Email ORCID ORCID Put Code Hellekes, Viktoria UNSPECIFIED orcid.org/0000-0003-1639-9709 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-789464
Date:	2025
Language:	English
Faculty:	Faculty of Mathematics and Natural Sciences
Divisions:	Faculty of Mathematics and Natural Sciences > Department of Biology > Institut für Entwicklungsbiologie
Subjects:	Life sciences
Uncontrolled Keywords:	Keywords Language CRISPR UNSPECIFIED Hox genes UNSPECIFIED Oxford Nanopore Sequencing UNSPECIFIED Nematode UNSPECIFIED
Date of oral exam:	25 September 2025
Referee:	Name Academic Title Schiffer, Philipp Dr. Gehring, Niels Prof. Dr.
Refereed:	Yes
URI:	http://kups.ub.uni-koeln.de/id/eprint/78946