Planarian Immobilization, Partial Irradiation, and Tissue Transplantation

Summary

An effective method for grafting tissue of defined and consistent size between planaria is described. Also included is a description of how the immobilization technique used for transplantation can be adapted, in conjunction with lead shields, for the partial irradiation of live animals.

Abstract

The planarian, a freshwater flatworm, has proven to be a powerful system for dissecting metazoan regeneration and stem cell biology^1,2. Planarian regeneration of any missing or damaged tissues is made possible by adult stem cells termed neoblasts³. Although these stem cells have been definitively shown to be pluripotent and singularly capable of reconstituting an entire animal⁴, the heterogeneity within the stem cell population and the dynamics of their cellular behaviors remain largely unresolved. Due to the large number and wide distribution of stem cells throughout the planarian body plan, advanced methods for manipulating subpopulations of stem cells for molecular and functional study in vivo are needed.

Tissue transplantation and partial irradiation are two methods by which a subpopulation of planarian stem cells can be isolated for further study. Each technique has distinct advantages. Tissue transplantation allows for the introduction of stem cells, into a naïve host, that are either inherently genetically distinct or have been previously treated pharmacologically. Alternatively, partial irradiation allows for the isolation of stem cells within a host, juxtaposed to tissue devoid of stem cells, without the introduction of a wound or any breech in tissue integrity. Using these two methods, one can investigate the cell autonomous and non-autonomous factors that control stem cell functions, such as proliferation, differentiation, and migration.

Both tissue transplantation^5,6 and partial irradiation⁷ have been used historically in defining many of the questions about planarian regeneration that remain under study today. However, these techniques have remained underused due to the laborious and inconsistent nature of previous methods. The protocols presented here represent a large step forward in decreasing the time and effort necessary to reproducibly generate large numbers of grafted or partially irradiated animals with efficacies approaching 100 percent. We cover the culture of large animals, immobilization, preparation for partial irradiation, tissue transplantation, and the optimization of animal recovery. Furthermore, the work described here demonstrates the first application of the partial irradiation method for use with the most widely studied planarian, Schmidtea mediterranea. Additionally, efficient tissue grafting in planaria opens the door for the functional testing of subpopulations of naïve or treated stem cells in repopulation assays, which has long been the gold-standard method of assaying adult stem cell potential in mammals⁸. Broad adoption of these techniques will no doubt lead to a better understanding of the cellular behaviors of adult stem cells during tissue homeostasis and regeneration.

Protocol

Note: this protocol suggests the use of potentially hazardous materials (lead and chloretone). Acquire, read, and follow MSDS for all potentially hazardous materials.

1. Animal Culture, Selection, and Preparation

1. For animal culture and handling use planarian water (1X Montjuïc salts⁹) and plastic transfer pipettes.
2. Sexual biotype Schmidtea mediterranea can be used when raised in the laboratory under normal culture conditions¹⁰. To produce asexual specimens of requisite size, S. mediterranea raised at room temperature under normal conditions⁹ should be ....

Discussion

Importance of immobilization

Immobilization is by far the most critical step for the successful completion of any of these processes. If planaria are improperly immobilized prior to partial irradiation, they may move beneath the lead shield, producing inconsistent and confounding results. Additionally, if animals are insufficiently immobilized following transplantation, the host worm will likely move away from the graft tissue, resulting in a failure of the graft to heal properly to the host. Pro.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
General Purpose Transfer Pipette	Samco	691
Capillary tubes (ID 0.75 mm)	FHC	30-30-0
Capillary tubes (OD 0.7 mm)	FHC	30-50-08
Parafilm M	VWR	52858-076
Kimwipes 34155	VWR	500029-891
Black filter paper	Schleicher Schuell	10310809
Whatman #2 filter paper 1002-055	Fisher Scientific	09-810B
Whatman #3 filter paper 1003-185	Fisher Scientific	09-820E
Cigarette rolling paper	Zig-Zag, original	NA
Petri dishes	VWR	82050-544
Forceps DUMONT, INOX #5	FST	11251-20
Chloretone	Sigma Aldrich	112054
Casein	Sigma Aldrich	C3400
Lead Shields	Alpha Systems Corp., Bluffdale, UT	Custom design
XRAD-320 Biological Irradiator	Precision X-Ray, North Branford, CT	NA