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

Co-staining of Nanos1 expression with EdU labeling makes it possible to distinguish actively proliferating stem-like cells. We can identify stem-like cells distribution at the single cell level in the jellyfish Cladonema. We can visualize stem-like cells and proliferating cells in the same jellyfish sample, which allows for the detection of proliferating and non-proliferating stem-like cells, leading to understanding of stem cell heterogeneity.

Developmental biology and regeneration studies using animal models that includes stem cells. The hydrozoan jellyfish Cladonema pacificum is an emerging model organism that can be kept in a laboratory environment without any specialized system. Cladonema medusae have branched tentacles.

The branching occurs at a new site along the adaxial side of the tentacle. Over time, the tentacles continue to elongate and branch, with older branches being pushed out toward the tip. The tentacles can also regenerate within a few days of amputation.

Small size, easy handling, and the presence of adult stem cells or stem-like cells make Cladonema a good system to investigate the role of stem cells in different processes. To begin, place Cladonema medusae into 1.5 milliliter tubes using a 3.5 milliliter transfer pipette and add ASW up to a total volume of 500 microliters. Add 7.5 microliters of 10 millimolar EdU stock solution and incubate the samples for one hour at 22 degrees Celsius.

Wait for an hour and remove as much ASW containing EdU as possible. After anesthetizing the medusae, incubate for five minutes. Fix the medusae overnight at four degrees Celsius with 4%paraformaldehyde in ASW.

For proteinase treatment and post-fixation, remove the paraformaldehyde and wash the samples with PBS containing 0.1%Tween 20 thrice for 10 minutes each. Remove the PBST and add the hybridization buffer. Incubate the samples in the hybridization buffer for 15 minutes at room temperature.

Remove the hybridization buffer and add the fresh hybridization buffer. Pre-hybridize at 55 degrees Celsius for at least two hours in a hybridization incubator. Remove the hybridization buffer and incubate with the hybridization buffer containing the probes.

Hybridize at 55 degrees Celsius for 18 to 24 hours in a hybridization incubator. Next, proceed with probe removal. Start with removing the hybridization buffer containing probes and then add wash buffer one.

Wash the samples with wash buffer twice for 15 minutes at 55 degrees Celsius. Remove wash buffer one and add wash buffer two. Wash the samples with wash buffer two twice for 15 minutes at 55 degrees Celsius.

Remove wash buffer two and then add 2X SSC. Wash the samples with 2X SSC twice for 15 minutes at 55 degrees Celsius. Remove 2X SSC and then add preheated PBST.

Wash the samples for 15 minutes with PBST at room temperature. For anti-DIG antibody incubation, start by removing PBST and then add 1%blocking buffer. Incubate the samples for at least one hour at room temperature while shaking slowly on a rocker.

After blocking, remove the 1%blocking buffer. Add anti-DIG-POD solution and incubate the samples overnight at four degrees Celsius. To detect the DIG labeled probe, remove the anti-DIG-POD solution and add tris sodium chloride Tween buffer.

Wash the samples with tris sodium chloride Tween buffer three times for 10 minutes at room temperature. Dilute fluorescent dye conjugated tyramide stock solution in the amplification diluent buffer to make the active Cy5 tyramide solution. Remove as much tris sodium chloride Tween buffer as possible and then add the active Cy5 tyramide solution.

Incubate the samples for 10 minutes in the dark. Wash the samples with PBST thrice for 10 minutes in the dark. To prepare the EdU detection cocktail, mix the components.

Remove PBST and then add the EdU detection cocktail. Incubate the sample in the dark for 30 minutes. Wash with PBST three times for 10 minutes in the dark.

For DNA staining, dilute Hoechst in PBST to prepare the Hoechst solution. Remove the PBST and then add the Hoechst solution. Incubate the samples for 30 minutes in the dark.

Wash the samples with PBST three to four times for 10 minutes in the dark. Next for mounting, transfer the medusae to the bank on the slide glass using a transfer pipette with the tip cut off. Gently place a coverslip on the medusae with forceps and seal the side of the coverslip with clear nail polish.

The co-labeling of Nanos1 expression and EdU positive cells revealed the spatial pattern of stem-like cells and proliferative cells in the tentacle. EdU positive cells were more widely distributed throughout the tentacle bulb, while Nanos1 positive cells accumulated more locally at the tentacle bulb and the new branching site, suggest that distinct distributions of stem-like cells and proliferating cells are detected depending on the developmental timing and different stages. The co-labeling of EdU and Nanos1 was observed in 19.79%of the cells, suggesting that these cells are an actively proliferating stem cell population.

Intriguingly, 14.46%of the cells were found to be EdU positive Nanos1 negative in the middle of the bulb and at the new branching site, suggesting the presence of non-stem-like proliferative cells. In contrast, 26.32%of cells were observed to be EdU negative Nanos1 positive at the base of the bulb and at the new branching site, indicating the presence of a stem cell population that is either slow cycling or quiescent, neither of which is detected by EdU pulse labeling. EdU pulse labeling with short incubation time allows for detection of only proliferative cells among stem-like cells and non-stem-like cells.

When changing the incubation time for EdU and combining long-time chasing experiments, slow cycling stem cells or other cell types including progeny and differentiated cells can be marked.