Proton Microbeam Targeted Irradiation of the Gonad Primordium Region Induces Developmental Alterations Associated with Heat Shock Responses and Cuticle Defense in Caenorhabditis elegans

SIMPLE SUMMARY: We used the charged-particle microbeam of the AIFIRA facility to investigate the effects of targeted irradiation of the progenitor gonad stem region on the organogenesis of worm gonad and vulva. A dedicated experimental approach was developed to enable the manipulation and targeted irradiation of the progenitor gonad stem region. This was achieved using three MeV protons during a definite developmental stage of the worms. The outcomes revealed distinct developmental modifications and specific gene inductions involved in cellular stress and cuticle injury responses. These findings were proven through an integration of methodologies, encompassing micro-irradiation under the reversible immobilization of worms, confocal imaging, cell sorting assays, and long-read sequencing analysis. ABSTRACT: We describe a methodology to manipulate Caenorhabditis elegans (C. elegans) and irradiate the stem progenitor gonad region using three MeV protons at a specific developmental stage (L1). The consequences of the targeted irradiation were first investigated by considering the organogenesis of the vulva and gonad, two well-defined and characterized developmental systems in C. elegans. In addition, we adapted high-throughput analysis protocols, using cell-sorting assays (COPAS) and whole transcriptome analysis, to the limited number of worms (>300) imposed by the selective irradiation approach. Here, the presented status report validated protocols to (i) deliver a controlled dose in specific regions of the worms; (ii) immobilize synchronized worm populations (>300); (iii) specifically target dedicated cells; (iv) study the radiation-induced developmental alterations and gene induction involved in cellular stress (heat shock protein) and cuticle injury responses that were found.