Planarian Fission Induction Protocol

doi:10.21203/rs.2.10324/v1

Method Article

Planarian Fission Induction Protocol

https://doi.org/10.21203/rs.2.10324/v1

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Planarian asexual reproduction via transverse fission is a size-dependent animal behavior that offers a robust context to investigate mechanisms coordinating growth, patterning, and function. During fission, planarians mechanically break off a portion of posterior tissue which will go on to regenerate into a clonal progeny. By optimizing husbandry techniques of the planarian Schmidtea mediterranea, we developed a protocol that robustly induces fission behavior. This technique permits the investigation of the effects of different experimental perturbations on planarian fission. Using visual scoring, successful fissions can be scored daily by counting fission progeny. While this protocol only measures successful fissions, constant video recording permits the measurement of unsuccessful fission attempts as well. The protocol consists of the optimal husbandry of worms, transfer of animals to dishes, and scoring of animal fissions over a 2-week period.

Developmental biology

size

behavior

planaria

fission

When population density and nutrient availability are optimal, planarians begin to exponentially increase their numbers via an asexual reproductive behavior known as fission. During fission, planarians mechanically break off a portion of posterior tissue which will go on to regenerate into a clonal progeny. This animal behavior has been linked to size and yields an easily scoreable functional output. However, the infrequency of fission behavior under standard laboratory conditions has precluded its mechanistic dissection. By optimizing husbandry techniques of the planarian Schmidtea mediterranea, we developed a protocol that elicits a robust and rapid induction of fission behavior.

1X Montjuic salts

Dechlorinated tap water supplemented with 2mM NaCl, 0.1mM KCl, 0.1mM MgSO4, 0.12mM NaHCO3.

Planaria Liver Paste

A liver paste is prepared by obtaining fresh calf liver, trimming it of all major vessels, fat, and connective tissue, and homogenizing it in a blender. The homogenate is then aliquoted into Petri dishes in a -80°C freezer. Storage life is approximately 6 months.

150mm x 25mm Culture Dishes (Corning 430599)

Planarian Recirculation Culture System (Custom-built)

The planarian recirculation culture system is composed of three culture trays (96' L × 24' W × 12' H) stacked vertically on top of each other over a sump. Planarian water flows from the sump pump through a chiller, canister filter, and a UV sterilizer into the top tray. Water subsequently flows down drains at the opposite ends of the source through the series of three trays. Water then re-enters the sump where it is filtered through two vertically stacked 400μm and 200μm sieves. Beyond the sieves, water is gravity fed and mechanically filtered through a set of filter/floss pads. Finally, water passes through Water Garden Oasis Pond Matrix and Kaldness media to remove nitrogenous waste. Water is then able to flow back through the chiller, canister filter, and UV sterilizer back into the top tray (Fig 1).

Sanyo MIR-553 Refrigerated Incubator

1. Maintain clonal CIW4 Schmidtea mediterranea in 1X Montjuic salts in a recirculatory culture system in the dark at 20C (Fig 1).

2. Feed animals with beef liver every 3 days with beef liver paste for at least 9 days. When animals reach 5mm in length they will begin to fission and increase the periodicity of this behavior as they grow larger. Typically animals of 10mm length or longer are ideal.

3. Remove animals from re-circulation culture system and wash 5-10 times with fresh 1X Montjuic salts.

4. Place individual animals in 150mm x 25mm tissue culture dishes with 50mL 1X Montjuic salts.

5. Measure animal length and place dishes in Sanyo Incubator in the dark at 20C (Fig 2).

6. Every day check the dishes to count fissions and remove the fission progeny (Fig 2). Removal is not necessary but makes daily scoring much easier. Replace 1X Montjuic salt media weekly.

7. For data analysis, divide the number of daily cumulative fissions by initial body length and then normalized to the average of the control RNAi fissions (Fig 2).

Planaria that are not over 5mm in length or are of suboptimal health will not fission. Sick planarians exhibit a lack of appetite, dorsal tissue lesions, and head regression. Healthy planarians will consume large amounts of beef liver, grow at a steady rate, and show no visible signs of tissue degeneration. Optimize planarian health prior to fission induction and avoid culturing worms at high density following removal from the recirculation culutre system. Animals are most susceptible to tissue degeneration following removal from the recirculation culture system due to expansion of endogenous pathogenic bacteria. Additionally, culture in antibiotics, such as Gentamycin, can also reduce fission rate.

Preparation of animals by feeding in recirculation system should take at least 9 days. Washing, measuring, and plating worms should take about 30 minutes per group of 12 worms. Worms should be scored for fissions every day over 2 weeks. Scoring should take 10 minutes per group of 12 worms.

Animals of 10mm length or greater should produce fission progeny within 48 hours. Over the course of 2 weeks, animals should fission 50-90% of their body mass.

Arnold, C. P. et al. Pathogenic shifts in endogenous microbiota impede tissue regeneration via distinct activation of TAK1/MKK/p38. eLife 5, (2016).

This paper details the recirculation system and optimization of planarian health.

CPA and BWBP worked together in the development of the fission protocol under the supervision and funding acquisition of ASA.

The author declares no competing interests

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Planarian Fission Induction Protocol

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Planarian Fission Induction Protocol

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Abstract

Figures

Introduction

Reagents

Equipment

Procedure

Troubleshooting

Time Taken

Anticipated Results

References

Acknowledgements

Additional Declarations

Associated Publications

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