siRNA-mediated gene knockdown via electroporation in hydrozoan jellyfish embryos

Abstract

As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insight into evolutionary aspects of animal development, physiology, and behavior. While cnidarians are classified into two types, sessile polyps and free-swimming medusae, most studies at the cellular and molecular levels have been conducted on representative polyp-type cnidarians and have focused on establishing techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced in two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for most medusa-type cnidarians, or jellyfish, are quite limited, except for Clytia hemisphaerica, and reliable techniques are required to interrogate function of specific genes in different jellyfish species. Here, we present a method to knock down target genes by delivering small interfering RNA (siRNA) into fertilized eggs via electroporation, using the hydrozoan jellyfish, Clytia hemisphaerica and Cladonema paciificum. We show that siRNAs targeting endogenous GFP1 and Wnt3 in Clytia efficiently knock down gene expression and result in known planula phenotypes: loss of green fluorescence and defects in axial patterning, respectively. We also successfully knock down endogenous Wnt3 in Cladonema by siRNA electroporation, which circumvents the technical difficulty of microinjecting small eggs. Wnt3 knockdown in Cladonema causes gene expression changes in axial markers, suggesting a conserved Wnt/β-catenin-mediated pathway that controls axial polarity during embryogenesis. Our gene-targeting siRNA electroporation method is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.

Neuronal and non-neuronal functions of the synaptic cell adhesion molecule, neurexin, inNematostella vectensis

“…In recent years, the repertoire of neural-related gene homologs in early diverging metazoan genomes and molecular mechanisms of neural development have been analyzed (Putnam et al 2007; Marlow et al 2009; Chapman et al 2010; Nakanishi et al 2012). Additionally, our understanding of the function of the cnidarian nervous system is rapidly advancing, primarily due to application of molecular biological tools (Ikmi et al 2014; Servetnick et al 2017; Cleves et al 2018; Sanders et al 2018; Karabulut et al 2019b; Masuda-Ozawa et al 2022a). However, despite the high evolutionary conservation of synapse-related molecules in their genomes, there is little direct experimental data on their function in cnidarians; thus, little is clear about pre-bilaterian synaptic functions.…”

“…To verify Nrxn function in cnidarians, we first tested the effects of Nrxn knockdown on the developmental process of δ-Nrxn-positive neurons. We performed small interfering RNA (siRNA)-mediated gene knockdown by delivering siRNAs targeting each δ-Nrxn mRNA into N. vectensis embryos (Masuda-Ozawa et al 2022b). Loss of any of the three δ-Nrxns (NvNrxnδ1,-2, and -4) was non-lethal, and caused no visible defects in polyp development (Supplementary Figure 7).…”

Distinct stem-like cell populations facilitate functional regeneration of theCladonemamedusa tentacle

“…cDNA was synthesized from 200 ng of total RNA by Prime script RT master Mix (Takara, RR036A). RT-qPCR was performed using TB Green Premix Ex TaqII (Tli RNaseH Plus) (Takara, RR820L) and a Quantstudio6 Flex Real-Time PCR system (Thermo Fisher) using F-actin capping protein subunit beta as an internal control (Masuda-Ozawa et al, 2022). The qPCR primer sets are as follows: CpCapZbeta : 5’-AAAGAAAGCTGGAGACGGTTCA-3’ (Forward), 5’-GTAGTGGGCATTTCTTCCGC-3’ (Reverse), Nanos1 : 5’-TTCGTCAAGTGGCAGTCGTG-3’ (Forward), 5-CAAGCCCTGGTACAAACGGA-3’ (Reverse).…”

“…The hydrozoan jellyfish Cladonema pacificum is an emerging jellyfish model that has been utilized to study development and physiology (Fujiki, 2019; Masuda-Ozawa et al, 2022; Takeda et al, 2018). Because Cladonema allows for easy lab maintenance with its high spawning rate, it enables monitoring all life cycle stages and exploring organismal responses to different stimuli.…”

Cnidarian pharyngeal nervous system illustrates prebilaterian neurosecretory regulation of feeding

“…To KD genes, siRNAs were electroporated into N. vectensis eggs based on methods we recently developed 50,66 and as described previously 67 with the following modifications: the eggs were suspended in N. vectensis medium with 8% Ficoll before electroporation. After electroporation, the eggs were incubated for 2 hours with sperm-containing N. vectensis medium for fertilization.…”