Fluorescent <em>In Situ</em> Hybridization and 5-Ethynyl-2'-Deoxyuridine Labeling for Stem-Like Cells in the Hydrozoan Jellyfish <em>Cladonema pacificum</em>

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

“…Note that, while Cladonema possess two Nanos genes, Nanos1 and Nanos2, Nanos2 + cells co-express a nematoblast marker Mcol1 , suggesting that Nanos2 is a nematoblast marker rather than a stem cell marker in Cladonema medusa (S3E and S3F Fig), similar to Hydractinia [47]. In the intact tentacle, Nanos1 + , Piwi + or Vasa1 + cells, are primarily distributed in the bulb and branching site (Figs 3Ci and S3A-S3D, S4Ai, S4Bi) [38,39]. By contrast, at 24 hpa Nanos1 + cells accumulated around the injury site where blastema formed (Fig 3Cii), and Piwi + and Vasa1 + cells were also localized in the blastema region (S4Aii and S4Bii Fig).…”

“…To molecularly characterize these blastema cells, we examined the expression of the stem cell markers Nanos1 and Piwi by fluorescent in situ hybridization (FISH). In the intact tentacle, Nanos1 + cells are primarily distributed in the bulb of the intact tentacle (Fujita et al, 2022; Hou et al, 2021). By contrast, at 24 hpa Nanos1 + cells accumulated around the injury site where blastema formed (Figure 3C ii), and Piwi + cells were also localized in the blastema region (Figure S3C).…”

“…By contrast, at 24 hpa Nanos1 + cells accumulated around the injury site where blastema formed (Figure 3C ii), and Piwi + cells were also localized in the blastema region (Figure S3C). While Nanos1 + cells accumulated to the new branching bud after 48 hpa (Figure 3C iii, Fujita et al, 2022; Hou et al, 2021), Nanos1 expression was no longer detected at the regenerating tip and was restricted to the middle and basal side of the tentacle (Figure 3C iii). We further quantified the distribution of Nanos1 + cells and found a gradual transition of their distribution from the distal to the basal side of the regenerating tentacle (Figure 3D).…”

“…The following FISH protocol was created from the published protocol (Fujita et al, 2022). The sequence of Nanos1 and Piwi was previously described (Hou et al, 2021), the presumptive sequence of Minicollagen1 ( Mcol1 ) was acquired from Dr. Kumano, and Nematocyte Outer Wall Antigen ( Nowa ) was acquired from the annotation of our RNA-seq results.…”

siRNA-mediated gene knockdown via electroporation in hydrozoan jellyfish embryos

“…The resulting plasmids were used for RNA probe synthesis with digoxigenin (DIG) labeling mix (Roche), and T7 or T3 RNA polymerase (Roche) was used, according to the insert direction. The detail for the probe synthesis was referred to the published protocol 46 .…”