Regeneration has long fascinated humanity, and its documentation has progressed from simple descriptive observations to the intense molecular and cellular investigations of today. The overarching goal of this work is to make the key methods and tools being used in modern regeneration and stem cell biology research accessible to docents and students in the classroom. We have designed a series of experimental activities with accompanying protocols using four inexpensive, commercially available planarian species indigenous to North America: Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracilis. These planarians are fast and robust regenerators, and can easily be maintained in the classroom. The activities presented here can be used to guide students through hypothesis-driven experiments, and range from simple manipulations aimed at high school students (e.g., planarian husbandry, feeding, and cutting experiments) to gene expression and protein function analyses suitable for college students. Regeneration time courses, the more complex whole worm in situ hybridizations, and RNA interference for gene knock-down experiments are described for each of the four species. Cumulatively, the suggested methods and experiments will facilitate the exploration of animal regeneration biology and promote curiosity-driven, hands-on application of the scientific method.
- Girardia sp.
- Dugesia dorotocephala
- Phagocata gracilis
- Phagocata morgani
- planarian maintenance
- RNA interference
- stem cells
- tissue-specific marker
- whole worm in situ hybridization
Today, life is being interrogated at unprecedented levels of resolution spanning molecular interactions to cellular, multicellular, and organismal behavior. A recurring theme in these efforts is the essential role that curious minds observing nature play in the process of discovery and understanding. Because observation and examination of nature is central to the development of human thought, hands-on approaches are widely regarded as effective classroom methods not only to explain many biological processes, but also to encourage student curiosity. Here, we introduce an age- and ability-scalable, experimental activity program using freshwater planarians that promotes a hands-on approach for students to explore fundamental questions underlying animal regeneration, tissue homeostasis, and stem cell biology.
Regeneration and Stem Cells
Regeneration is the fascinating process that replaces damaged or lost structures in adult organisms. Many animals across the tree of life manifest this remarkable ability, chiefly among them, starfish arm regeneration, fish tail fin regeneration, and salamander limb regeneration (Figure 1). In extreme cases, such as in hydra and planarians, each tissue fragment can regenerate a complete, new organism (Sánchez Alvarado & Tsonis, 2006).
Regeneration has long captured the human imagination. Aristotle reported on the reconstitution of an amputated lizard tail circa 350 BC (Aristotle et al., 1965), and in the eighteenth century, naturalists began to study regeneration systematically by performing scientific experiments. Abraham Trembley, a Swiss naturalist, documented the regenerative capacity of hydra (Lenhoff et al., 1986), and Lazzaro Spallanzani, an Italian priest, biologist, and physiologist, reported the regenerative properties of earthworms, snails, newts, and frogs (Dinsmore, 1991; Tsonis & Fox, 2009). During the early twentieth century, Thomas H. Morgan, an American geneticist and embryologist, wrote on this topic in his book, Regeneration (1901). Researchers suspected and later confirmed that many forms of regeneration often rely upon the activities of persistent and undifferentiated post-birth cells referred to as adult stem cells (Coutu et al., 2011; Randolph, 1891). These cells (see Glossary) both replenish themselves and differentiate anew into tissue-specific cells that replace those lost to physiological wear and tear and/or injury (Smith et al., 1991).
The presence and proliferation of highly potent stem cells in both vertebrates and invertebrates are frequent mechanisms underpinning animal regeneration (Gurley & Sánchez Alvarado, 2008). Stem cell potency is defined by the ability of these cells to generate different cell types. Four general classes have been described: (1) totipotent stem cells can give rise to any of the varied cell types of extra-embryonic and embryonic tissues of an organism; (2) pluripotent stem cells produce most, but not all cell types; (3) multipotent stem cells produce only a few different cell types; and (4) unipotent stem cells generate only a single cell type (Stocum, 2001). Like differentiated cells, the morphological and functional features of undifferentiated stem cells can be defined by gene expression activities. The activation and repression of genes is a finely regulated molecular choreography involving transcription factors, i.e., molecules that bind DNA. For instance, we now know that the pluripotency of mammalian embryonic stem cells depends in great part on the activities of four transcription factors, known as OCT4, SOX2, cMYC and KLF4. These proteins regulate the expression of such genes as Wnt, Hedgehog, BMP/TGF-β, and Notch, which are essential for the self-renewal, maintenance, and differentiation of stem cells into specific tissue types (Liu et al., 2008). Together, OCT4, SOX2, cMYC, and KLF4 are also known as the Yamanaka factors, because of Dr. Yamanaka's discovery that the presence of these four factors could convert adult differentiated cells into pluripotent stem cells (Takahashi & Yamanaka, 2006). Although very important, these transcription factors are not the only molecules involved in the maintenance of stem cell pluripotency. Scientists are continually studying the role in stem cell biology of previously identified and newly discovered molecules.
Planarians as a Model for Regeneration Studies
Planarians are flatworms belonging to the phylum Platyhelminthes. Thousands of species have been described; some live in freshwater, like the species introduced here, others in marine environments, and some on land (Schockaert et al., 2008). Planarians are bilaterally symmetric and contain many complex tissue and organ systems, including photoreceptors (eyes), epidermis, muscle, a bilobed brain, two ventral nerve cords, protonephridia (kidneys), and a digestive system (Figure 2) (Newmark & Sánchez Alvarado, 2002). The blind gut is a highly branched gastrovascular cavity composed of one anterior and two posterior branches. The gut connects to the pharynx, an extendable muscular tube used for feeding and defecation (Figure 2) (Elliott & Sánchez Alvarado, 2012). Planarians are also highly motile and move via the coordinated movement of cilia localized on their ventral epidermis (Elliott & Sánchez Alvarado, 2012).
Planarians have been adopted as a model system for studying regeneration and stem cell biology because they can regenerate an entire organism from virtually any portion of their bodies in a relatively short period of time. This terrific capability depends on an abundant population of adult pluripotent stem cells called neoblasts, which are found throughout the planarian body plan, with the exception of the area in front of the photoreceptors and the pharynx (Figure 3A). The vast majority of neoblasts are found in the mesenchyme, occupying the space between the gut branches (Reddien & Sánchez Alvarado, 2004). After amputation, neoblasts increase their proliferation rate, and their progeny form an unpigmented mass of new tissue called the regeneration blastema, in which cellular differentiation is orchestrated to ultimately restore the missing body parts (Figure 3B) (Reddien & Sánchez Alvarado, 2004). Almost concurrently, the old tissues undergo a remodeling process, which facilitates the functional integration of the newer differentiating blastema to the preexisting anatomy. In the end, this process reestablishes a properly proportioned and functioning new organism (Reddien & Sánchez Alvarado, 2004).
Another interesting characteristic of planarians is their capacity to scale their bodies depending on nutrient availability. Planarians can survive prolonged starvation periods (multiple months), and during this time reduce their size while remaining a functioning, proportioned, and regeneration-capable worm. Besides being caused not by changes in cell size, but rather cell number, this “degrowth” is fully reversible: once food is available again, the animals regrow to the original size (Newmark & Sánchez Alvarado, 2002).
Currently, the most studied planarian species is Schmidtea mediterranea, thanks to many laboratories that optimized molecular and morphological tools (Newmark & Sánchez Alvarado, 2002). However, S. mediterranea is not endemic to the United States, and specific regulations are in place for the handling of this species to avoid its accidental propagation in North America. This fact, therefore, limits the routine use of S. mediterranea in the classroom.
The aim of this paper is to overcome this limitation by presenting optimized experimental protocols for four North American planarian species, Girardia sp., Dugesia dorotocephala, Phagocata morgani, and Phagocata gracilis. These species can be readily and inexpensively purchased from commercial vendors (Carolina Biological Supply Company or Ward's Science, see References for urls) and easily maintained in the classroom (Kenk et al., 1972). Here we show results for three different experiments of varying expertise from high school to college levels: (1) amputation strategies and regeneration time courses; (2) analyses of gene expression by whole mount in situ hybridization (WISH); and (3) evaluation of gene function using RNA interference (RNAi), that specifically inhibits expression of a gene of interest. Husbandry information, protocols, lists of required materials, bioinformatic resources, and other tools designed to facilitate experimentation are available at http://cuttingclass.stowers.org.
Characteristic Attributes of Four Commercially Available Planarian Species
Four planarian species (Girardia sp., D. dorotocephala, P. morgani, and P. gracilis) are available from American vendors (Carolina Biological Supply Company or Ward's Science, see References), and each can be readily distinguished based upon a number of easily observable traits (Table 1) (Figure 4) (Kenk et al., 1972).
Planarians are remarkably well-suited organisms for educational purposes. Not only are their regenerative capacities saliently manifested, but they are also easy to manipulate, inexpensive, and easy to maintain and expand if needed. The animals can be purchased, then amplified by amputation as needed. Here we provide a list with several activities related to planarian regeneration, gene expression, and loss-of-function. The protocols for these experiments are available on the website http://cuttingclass.stowers.org, and they do not require sophisticated equipment and/or extensive training. Moreover, activities could also involve the use of planarians for behavioral experiments, histological sample processing, morphological or antibody staining, DNA and RNA purification, protein extraction, and isolation and culture of bacterial strains.
The goals of these activities are to teach: (1) how to maintain a model system, with a rigorous and consistent protocol that guarantees experimental reproducibility; (2) how to set up an experiment with indispensable controls; (3) how to decide which are the quantitative/qualitative data that should be collected; (4) how to make observations and discuss the obtained data; and (5) how to develop an explanation/model that justifies the collected data.
Specifically, in the sections below (specifically “WISH: A Method to Visualize Gene Expression” and “RNAi: Disruption of Gene Function”), the students will acquire knowledge of and abilities in molecular biology. The section on WISH shows that cell characteristics and functions are defined by specific gene expressions, and that gene expression patterns differ across tissues. The section on RNAi shows that the proteins produced from mRNA do the majority of the work inside the cells, and that the degradation of specific mRNA could seriously change/affect cell functionality.
Maintaining, Feeding, and Amplifying Planarians
Students can participate in the maintenance, feeding, and amplification of their planarians, which requires amputation of large worms and subsequent regeneration of the fragments. Related activities are mentioned in Table 2. “Planarian Maintenance” protocol is available on the website http://cuttingclass.stowers.org.
Amputation Strategies and Regeneration Time Courses
Referred to as “almost immortal under the edge of a knife” (Dalyell, 1814), planarians can be cut into fragments, most of which can regenerate into a complete new organism (Newmark & Sánchez Alvarado, 2002). Students can systematically cut the worms and follow the fate of the fragments. Here, time courses of three different amputation strategies are provided (Figures 5–8). This set of experiments demonstrates the robustness and reproducibility of regeneration in planarians. Notably, each species rebuilds missing body part(s) while preserving the orientation of body axes. Related activities are mentioned in Table 2. “Observing Planarian Regeneration” protocol is available on the website http://cuttingclass.stowers.org.
WISH: A Method to Visualize Gene Expression
WISH is a technique used to detect expression of any gene of interest in both intact and regenerating worms. The WISH protocol exploits specific, synthetically made riboprobe, whose sequence is complementary to the mRNA of the gene of interest. The riboprobe binds the mRNA, and then specific antibodies detect the mRNA-riboprobe complex. Finally, antibodies are visualized through a chemical reaction that produces blue staining in the cells that contain the mRNA-riboprobe-antibody complex (King & Newmark, 2013). Here, the expression of eleven genes is shown, enabling students to visualize the stem cells (neoblasts), body axis domains, and major organ systems (Table 3) (Figures 9–12). Related activities are mentioned in Table 2. “Whole mount in situ Hybridization (WISH)” protocol is available at http://cuttingclass.stowers.org.
RNAi: Disruption of Gene Function
To understand the function of a given gene, biologists disrupt its activity and observe the deficiencies (phenotypes) that result. RNAi is the standard technique applied to planarians to dissect gene function (Sánchez Alvarado & Newmark, 1999; Newmark et al., 2003; Rouhana et al., 2013). The RNAi exploits a specific double-strand RNA (dsRNA), complementary to the mRNA of interest that must be either produced in bacteria or synthesized in vitro. The dsRNA is delivered into the worm by either feeding or injection, and diffuses into the cytoplasm of the cells. The dsRNA activates a defense mechanism that induces the degradation of both dsRNA and its complementary mRNA (Agrawal et al., 2003). Thus, the function of any given gene during regeneration can be studied through systematic RNAi knock-down (Hannon, 2002).
Here, two RNAi experiments are described: the ß-catenin(RNAi) illustrates a polarity phenotype, and odf2(RNAi) gives rise to a behavioral phenotype (Figure 13) (Gurley et al., 2008; Petersen & Reddien, 2008; Reddien et al., 2005). Related activities are mentioned in Table 2. “RNA Interference (RNAi)” protocol is available at http://cuttingclass.stowers.org.
Regeneration is documented for Girardia sp. (Figure 5), D. dorotocephala (Figure 6), P. morgani (Figure 7), and P. gracilis (Figure 8) following three different amputation paradigms: (1) two transverse amputations perpendicular to the anteroposterior (A/P) axis; (2) one lateral amputation parallel to the A/P axis; and (3) one oblique cut made at an approximate 45° angle to the A/P axis.
Wound healing: Following amputations, the first important step is the rapid contraction of the wound that reduces exposure of internal tissues to the environment (Figures 5–8). Specifically, after the second amputation paradigm, the contraction of the body causes the formation of a curved, U-like body shape, and the fragments swim in a circle. Similarly, after the third amputation paradigm, the musculature contraction causes the formation of a curl where the tissue is thinner, and also in this case, the posterior fragment cannot swim straight.
Blastema formation and regeneration of new anatomy: The next step during regeneration involves the formation of a blastema, which is a special type of tissue found in many regeneration contexts. It consists of a single-layered epidermis surrounding an internal mass of proliferating and differentiating cells able to regenerate all missing tissues (Newmark & Sánchez Alvarado, 2000). In all species, the blastema is unpigmented (white) and it gradually increases in size as new anatomy is rebuilt (Figures 5–8). The most obvious anatomical feature that emerges from the blastema during regeneration are the eyes, which increase in size and pigmentation over the time.
Tissue remodeling: The process of integrating newly regenerated structures with preexisting anatomy through tissue remodeling is an important step. Tissue remodeling is most evident in the case of the pharynx after the first amputation paradigm. The new pharynx appears in the blastema, and eventually becomes positioned in the new animal's midsection as the scale and proportion of the body is reestablished. Although the preexisting pharynx is in the central fragment, it shrinks in size over the course of tissue remodeling to properly scale for the size of the new animal body. The pharynx can be easily seen and monitored from the dorsal side of the worms.
Completion of regeneration: Regeneration is considered complete when the blastema becomes fully pigmented, the body proportions are reestablished, and the animal is capable of eating. Girardia sp., D. dorotocephala, and P. morgani (Figures 5–7) complete regeneration in 14 days at room temperature. In contrast, P. gracilis (Figure 8) requires two more weeks to fully regenerate. Notably, during the entire regeneration process, the polarity of the axes is preserved, as is the ability of the fragments to swim.
WISH: Localization of Gene Expression
The WISH protocol, through the use of riboprobes, detects and stains the cells that are expressing a gene of interest. In this paper, markers for planarian body axes and organs were selected and used to illustrate their anatomy and distribution (Table 3).
Stem cells: piwi-1 is a gene expressed specifically in neoblasts and is most commonly used to visualize the planarian stem cells (Reddien et al., 2005). Neoblasts are spread throughout the body of all four planarian species. Girardia sp. (Figure 9), D. dorotocephala (Figure 10), and P. morgani (Figure 11) neoblasts are distributed everywhere except in the pharynx and the most anterior part of the worms. In contrast, P. gracilis (Figure 12) neoblasts appear to be more broadly distributed with particular enrichment in the brain.
Boundaries and axes: The expression of the genes ifb and slit define the mediolateral (M/L) axis. ifb defines the edge of the worms, while slit delineates the midline of the animals (Figures 9–12) (Molina et al., 2011; Cebrià et al., 2007). Additionally, sfrp-1 and frizzled-like represent good markers of the anterior and posterior ends of the planarian, respectively (Figures 9–12) (Gurley et al., 2008).
Nervous system: The central nervous system (CNS) is composed of two cephalic ganglia that form the brain, and two ventral nerve cords that reach the tip of the tail (Figure 2). The CNS can be visualized by the expression of the gene prohormone convertase 2 (pc2) (Figures 9–12) (Collins et al., 2010). opsin-like, coding for a light-sensitive protein, is specifically expressed in the eye's photoreceptor cells (Figures 9–12) (Sánchez Alvarado & Newmark, 1999).
Digestive system: The porcupine-like (Figure 9, 10, 12) and tnxb (Figure 11) expression patterns reveal the three main branches of the planarian gut (one projecting anteriorly and two posteriorly) and its complex ramification (Gurley et al., 2008; Vu et al., 2015). Expression of laminin-like defines the pharynx, a muscular feeding tube connected to the gut (Figures 9–12) (Cebrià & Newmark, 2007). Girardia sp., D. dorotocephala, and P. morgani typically possess one pharynx (Figures 9–11). However, P. gracilis possesses multiple pharynges (Figure 12).
Excretory system: Regulation of water/salt balance, or osmoregulation, in planarians is maintained by the excretory system, which can be visualized with the protonephridia marker innexin that stains the flame cells (Oviedo & Levin, 2007). The protonephridia are the single functional units of the excretory system homologous to the nephridia of vertebrate kidneys. The planarian protonephridia are tubular structures spread throughout the entire body of the worms (Figures 9–12).
Body wall musculature: The muscular system expresses collagen and a higher concentration of muscle cells (area with more intense signal) can be observed in the head (Witchley et al., 2013). Muscle cells are also spread in the posterior part of the planarians and in the pharynx (Figures 9–12). Girardia sp. shows an intense staining in the pre-pharyngeal region (Figure 9), whereas P. morgani has more concentrated muscle cells in the anterior part of the body (Figure 11).
RNAi: Disruption of Gene Function
RNAi specifically degrades mRNA for genes of interest, revealing the function of the targeted gene product through the phenotypes that emerge in the absence of its protein. Here, ß-catenin (in each species) and odf2 (only in P. morgani) genes were targeted for RNAi. After three feedings, the animals were cut perpendicularly to the A/P axis into three fragments and observed during their regeneration.
Upon silencing the ß-catenin gene, each species displays a disruption in regeneration polarity. Each fragment regenerates an ectopic head from posterior wounds instead of a tail, showing a “double-head” or “Janus” phenotype (Figure 13A). Furthermore, uninjured animals of each species subjected to ß-catenin(RNAi) will likely become anteriorized, converting their tails into a second head and sprouting ectopic heads along the body edge, as has been observed in S. mediterranea. This phenotype indicates that ß-catenin is required for the specification and maintenance of posterior tissues in planarians (Gurley et al., 2008; Petersen & Reddien, 2008; Adell et al., 2009).
Upon silencing of the odf2 gene, all fragments display a behavioral phenotype. The worms appear morphologically normal, except that instead of swimming forward, they move toward their right side (Figure 13B). This “sidewinder” phenotype is caused by the misorientation of the cilia on the ventral side of the animals whose beating allows their normal locomotion. This phenotype shows that odf2 is involved in the orientation of the ventral cilia, and when it is interfered with via RNAi, the direction of animal locomotion is misoriented (Reddien et al., 2005).
Collectively, the information and experimental manipulations presented here provide students with a set of carefully thought out and controlled, curiosity-driven, hands-on experiences in science. The activities are composed of questions, experimental approaches to answer them, data collection, and data analyses, allowing students to apply the scientific method to solve complex biological problems using planarians, invertebrates that are routinely used in biomedical research laboratories. Students will also gain an in-depth knowledge of key topics in the rapidly moving fields of regeneration and stem cell biology, allowing them to better understand and critically evaluate experiments in regenerative medicine and stem cell technologies. Ultimately, we hope this teaching paper will spark student excitement about science in general, and the field of regeneration and stem cell biology in particular.
We thank all members of the Sánchez Alvarado laboratory for their support, especially Erin Davies, Kai Lei, and Stephanie Nowotarski for helpful comments on the manuscript. We also thank Mark Miller for illustration assistance, all the members of the Aquatics Facility and the Molecular Biology Facility at the Stowers Institute for Medical Research for their support. We are equally grateful to the Science Education Department at the Howard Hughes Medical Institute for their support in disseminating and advertising this project and for HHMI EXROP.
Transcriptomes are deposited with the TSA (NCBI). Accession numbers: GEHB00000000 (Girardia sp.), GEIG00000000 (Dugesia dorotocephala), GEKK00000000 (Phagocata morgani) and GEGP0000 0000 (Phagocata gracilis). Funding for this project was provided by Howard Hughes Medical Institute and Stowers Institute for Medical Research.
Appendix 1. Glossary.
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