Jellyfish have existed on the earth for around 600 million years and have evolved in response to environmental changes. Hydrozoan jellyfish, members of phylum Cnidaria, exist in multiple life stages, including planula larvae, vegetatively-propagating polyps, and sexually-reproducing medusae. Although free-swimming medusae display complex morphology and exhibit increase in body size and regenerative ability, their underlying cellular mechanisms are poorly understood. Here, we investigate the roles of cell proliferation in body-size growth, appendage morphogenesis, and regeneration using Cladonema pacificum as a hydrozoan jellyfish model. By examining the distribution of S phase cells and mitotic cells, we revealed spatially distinct proliferating cell populations in medusae, uniform cell proliferation in the umbrella, and clustered cell proliferation in tentacles. Blocking cell proliferation by hydroxyurea caused inhibition of body size growth and defects in tentacle branching, nematocyte differentiation, and regeneration. Local cell proliferation in tentacle bulbs is observed in medusae of two other hydrozoan species, Cytaeis uchidae and Rathkea octopunctata, indicating that it may be a conserved feature among hydrozoan jellyfish. Altogether, our results suggest that hydrozoan medusae possess actively proliferating cells and provide experimental evidence regarding the role of cell proliferation in body-size control, tentacle morphogenesis, and regeneration.

Medusozoans, the Cnidarian subphylum, have multiple life stages including sessile polyps and free-swimming medusae or jellyfish, which are typically bell-shaped gelatinous zooplanktons that exhibit diverse morphologies. Despite having a relatively complex body structure with well-developed muscles and nervous systems, the adult medusa stage maintains a high regenerative ability that enables organ regeneration as well as whole body reconstitution from the part of the body. This remarkable regeneration potential of jellyfish has long been acknowledged in different species; however, recent studies have begun dissecting the exact processes underpinning regeneration events. In this article, we introduce the current understanding of regeneration mechanisms in medusae, particularly focusing on cellular behaviors during regeneration such as wound healing, blastema formation by stem/progenitor cells or cell fate plasticity, and the organism-level patterning that restores radial symmetry. We also discuss putative molecular mechanisms involved in regeneration processes and introduce a variety of novel model jellyfish species in the effort to understand common principles and diverse mechanisms underlying the regeneration of complex organs and the entire body.

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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.

As the sister group to bilaterians, cnidarians stand in a unique phylogenetic position that provides insights 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 established techniques of genetic manipulation. Recently, gene knockdown by delivery of short hairpin RNAs into eggs via electroporation has been introduced to two polyp-type cnidarians, Nematostella vectensis and Hydractinia symbiolongicarpus, enabling systematic loss-of-function experiments. By contrast, current methods of genetic manipulation for medusa-type cnidarians, or jellyfish, are quite limited, and reliable techniques are required to interrogate function of specific genes. Here, we present a method to knockdown target genes by small interfering RNA (siRNA) delivery 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 knockdown 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 siRNAs electroporation, which circumvents the technical difficulty of microinjecting small eggs. Unexpectedly, Wnt3 knockdown in Cladonema does not induce morphological defects in planula, suggesting a distinct mechanism that operates during embryogenesis. Our method for electroporation of siRNAs targeting specific genes is applicable to other animals, including and beyond jellyfish species, and will facilitate the investigation and understanding of myriad aspects of animal development.