Whole planarian chromosome squash

Summary Whole planarian chromosome squash allows researchers to qualitatively analyze chromosome integrity. Treatment with colchicine is used to halt dividing cells within metaphase and does not require amputation or tissue puncturing. In combination with acetic-orcein, a stain-fixative for chromosomes, this strategy is suitable for animals with friable tissues caused by drug treatment, radiation, and RNA interference phenotypes. The whole planarian squash method presented here is a minimally invasive procedure that facilitates simultaneous analysis of chromosomal integrity in control and experimental animals. For complete details on the use and execution of this protocol, please refer to Peiris et al. (2016).


SUMMARY
Whole planarian chromosome squash allows researchers to qualitatively analyze chromosome integrity. Treatment with colchicine is used to halt dividing cells within metaphase and does not require amputation or tissue puncturing. In combination with acetic-orcein, a stain-fixative for chromosomes, this strategy is suitable for animals with friable tissues caused by drug treatment, radiation, and RNA interference phenotypes. The whole planarian squash method presented here is a minimally invasive procedure that facilitates simultaneous analysis of chromosomal integrity in control and experimental animals. For complete details on the use and execution of this protocol, please refer to Peiris et al. (2016).

BEFORE YOU BEGIN
On day 1 of the protocol fresh colchicine solution should be made. Prior to the start of day 2 of the protocol, all karyotyping reagents must be made fresh.
1. Select control and/or experimental planarians of similar size that are no greater than 1 cm in length and place them into a 6 cm petri dish. 2. Remove planarian 13 Montjuic saltwater and replace with 4-5 mL of Colchicine solution. 3. Allow planarians to incubate for 12-16 h in the dark.
CRITICAL: To avoid planarian toxicity induced by colchicine, the incubation period should not exceed 16 h.

Whole planarian chromosome squash
Timing: 30-60 min Figure 2A provides a visual representation of the whole animal procedure.
Planarian tissue will be fixed and heated, assisting with the softening of tissue and separation of cells allowing for chromosomes to be stained by orcein solution. REAGENT  4. Place one animal per 1.5 mL tube and remove colchicine solution. 5. Add 0.5-1 mL of 3:1 ethanol: acetic acid fixative to each tube and incubate for 15 min at room temperature (25 C) on an orbital platform rotator.
Note: In all steps, ensure planarians are fully submerged within the solution and not floating on top.
6. Replace solution with 0.5-1 mL 1 N HCl and pre-incubate for 2 min at room temperature (25 C) . 7. Immediately incubate at 60 C in a Thermomixer-R for 6 min. 8. Quickly remove the HCl solution.
CRITICAL: At this point tissues are quite soft. Thus, remove all residual HCl without puncturing the planarian by angling the 1.5 mL tube so that the planarian will stick to the wall of the tube.
CRITICAL: The planarian will not be seen, as the 1% acetic-orcein solution is dark purple. In most cases, the planarian will be residing at the bottom of the tube. Thus, remove as much solution as possible (125-175 mL). The residual liquid will be diluted out with 60% acetic acid solution.
11. Add 500 mL of 60% acetic acid solution and incubate for 5 min. 12. With a transfer pipette gently place animals on a 24 3 60 mm cover slips.
Note: 25 3 75 mm slides are too thick to view chromosomes at 1003 thus, use 24 3 60 mm cover slips. 13. After planarians are transferred, remove residual 60% acetic acid solution. 14. Add 10-20 mL of 1:1:1 lactic acid: acetic acid: MilliQ H 2 O on top of each worm and incubate for 5 min. 15. Remove excess liquid and add 2 mL of orcein solution to each animal. 16. Take a 22 3 22 mm cover slip, quickly place it on top of the treated animal and with slight pressure and one continual movement, use your thumb to squash the animal throughout the slide. Troubleshooting 1.
Pause point: Protocol is complete. Slides can be stored at room temperature (25 C) until chromosome viewing. Slides do not require sealing and can be stored at room temperature (25 C) for long-term void of humidity.

EXPECTED OUTCOMES
Whole planarian chromosome squash is designed to obtain the maximum number of chromosomes per animal without manipulating tissue (e.g., amputations or tissue puncturing) prior to the colchicine soaking step. Phenotypes of weakened tissue integrity and lesions are commonly found within experimental groups (e.g., Ubc9(RNAi) and Rad51(RNAi) animals) (Peiris et al., 2016;Thiruvalluvan et al., 2018). Thus, reduced tissue manipulation allows for the analysis of chromosomes between control and experimental groups.

LIMITATIONS
This protocol is limited to brightfield imaging as the animal is squashed onto a slide with a coverslip. Other protocols have been optimized to allow for immunohistochemical staining and telomeric FISH protocols within planarian (Guo et al., 2018). Future work is required to optimize whole planarian chromosome squashing for immunohistochemical staining and FISH protocols with minimal disruption to squashed tissue.

TROUBLESHOOTING
There are two possible issues that may arise during this protocol. The issues will not be evident until viewing under a microscope and will result in the inability to view chromosomes. The potential issues are (1) excessive squashing of sample and (2) suboptimal orcein staining.
Problem 1: excessive squashing If too much pressure is used during the squashing process, chromosomes will be damaged, destroyed or squashed to the point that they cannot be viewed under the microscope (step 16).

Potential solution
The amount of force required to conduct planarian squash must be optimized per scientist. If thumb pressure is excessive, try using an unsharpened pencil and drop the pencil eraser down onto the planarian for squashing.
Problem 2: suboptimal orcein staining Depending on the species of planarian, 1% acetic-orcein may not be efficient for chromosome staining. Therefore, a limited number or no chromosomes will be visible (steps 9 and 14).

Potential solution
The percent of orcein must be optimized per species. Studies have shown that increasing orcein concentration to 2% could enhance visualization of chromosomes (Cour, 2009).

Resource availability
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Né stor J. Oviedo (noviedo2@ucmerced.edu). Data and code availability This study did not generate/analyze datasets/code