Planarian stem cells sense the identity of the missing pharynx to launch its targeted regeneration.

In order to regenerate tissues successfully, stem cells must detect injuries and restore missing cell types through largely unknown mechanisms. Planarian flatworms have an extensive stem cell population responsible for regenerating any organ after amputation. Here, we compare planarian stem cell responses to different injuries by either amputation of a single organ, the pharynx, or removal of tissues from other organs by decapitation. We find that planarian stem cells adopt distinct behaviors depending on what tissue is missing to target progenitor and tissue production towards missing tissues. Loss of non-pharyngeal tissues only increases non-pharyngeal progenitors, while pharynx removal selectively triggers division and expansion of pharynx progenitors. By pharmacologically inhibiting either mitosis or activation of the MAP kinase ERK, we identify a narrow window of time during which stem cell division and ERK signaling produces pharynx progenitors necessary for regeneration. These results indicate that planarian stem cells can tailor their output to match the regenerative needs of the animal.

Many animals can repair and regrow body parts through a process called regeneration. Tiny flatworms called planaria have some of the greatest regenerative abilities and can regrow their whole bodies from just a small part. They can do this because around a fifth of their body is made of stem cells, which are cells that continuously produce new cells and turn into other cell types through a process called differentiation. Measuring the gene activity in stem cells from planaria shows that these cells are not all the same. Different groups of stem cells have specific genes turned on which are needed to regrow certain body parts. It is unclear whether all stem cells respond to injuries in the same way, or whether the stem cells that respond are specific to the type of injury. For example, stem cells needed to repair the gut may respond more specifically to gut injuries than to other damage. Bohr et al. studied how stem cells in planaria respond to different injuries, by comparing an injury to a specific organ to a more serious injury involving several organs. The specific injury was the loss of the pharynx, the feeding organ of the flatworm, while the more serious injury was the loss of the entire head. Within hours of removing the pharynx, stem cells that were poised to develop into pharyngeal cells became much more active than other stem cell types. When the head was removed, however, a wide range of stem cells became active to make the different cell types required to build a head. This suggests that stem cells monitor all body parts and respond rapidly and specifically to injuries. These findings add to the understanding of regeneration in animal species, which is of great interest for medicine given humans' limited ability to heal. Many of the genetic systems that control regeneration in planaria also exist in humans, but are only active before birth. In the long-term, understanding the key genes in these processes and how they are controlled could allow regeneration to be used to treat human injuries.

Injury Delays Stem Cell Apoptosis after Radiation in Planarians.

Stem cells are continuously exposed to multiple stresses, including radiation and tissue injury. As central drivers of tissue repair and regeneration, it is necessary to understand how their behavior is influenced by these stressors. Planarians have an abundant population of stem cells that are rapidly eliminated after radiation exposure via apoptosis. Low doses of radiation eliminate the majority of these stem cells, allowing a few to remain [1]. Here, we combine radiation with injury to define how stem cells respond to tissue damage. We find that a variety of injuries induced within a defined window of time surrounding radiation cause stem cells to outlast those in uninjured animals. Injury stimulates localized cell death adjacent to wounds [2], in the same regions where stem cells persist. This persistence occurs in the absence of proliferation. Instead, stem cells are retained near the wound due to delayed apoptosis, which we quantify by combining fluorescence-activated cell sorting (FACS) with annexin V staining. Pharmacological inhibition of the mitogen-activated protein (MAP) kinase extracellular signal-regulated kinase (ERK) prevents stem cell persistence after injury, implicating wound-induced ERK activity in this response. By combining radiation with injury, our work reveals a novel connection between dying cells and stem cells that remain. Furthermore, the ability to induce stem cell persistence after radiation provides a paradigm to study mechanisms that may contribute to unanticipated consequences of injury, such as tumorigenesis.

Shugoshin Is Essential for Meiotic Prophase Checkpoints in C. elegans.

The conserved factor Shugoshin is dispensable in C. elegans for the two-step loss of sister chromatid cohesion that directs the proper segregation of meiotic chromosomes. We show that the C. elegans ortholog of Shugoshin, SGO-1, is required for checkpoint activity in meiotic prophase. This role in checkpoint function is similar to that of conserved proteins that structure meiotic chromosome axes. Indeed, null sgo-1 mutants exhibit additional phenotypes similar to that of a partial loss-of-function allele of the axis component, HTP-3: premature synaptonemal complex disassembly, the activation of alternate DNA repair pathways, and an inability to recruit a conserved effector of the DNA damage pathway, HUS-1. SGO-1 localizes to pre-meiotic nuclei when HTP-3 is present but not yet loaded onto chromosome axes and genetically interacts with a central component of the cohesin complex, SMC-3, suggesting that it contributes to meiotic chromosome metabolism early in meiosis by regulating cohesin. We propose that SGO-1 acts during pre-meiotic replication to ensure fully functional meiotic chromosome architecture, rendering these chromosomes competent for checkpoint activity and normal progression of meiotic recombination. Given that most research on Shugoshin has focused on its regulation of sister chromatid cohesion during chromosome segregation, this novel role may be conserved but previously uncharacterized in other organisms. Further, our findings expand the repertoire of Shugoshin's functions beyond coordinating regulatory activities at the centromere.

Chemical Amputation and Regeneration of the Pharynx in the Planarian Schmidtea mediterranea.

Planarians are flatworms that are extremely efficient at regeneration. They owe this ability to a large number of stem cells that can rapidly respond to any type of injury. Common injury models in these animals remove large amounts of tissue, which damages multiple organs. To overcome this broad tissue damage, we describe here a method to selectively remove a single organ, the pharynx, in the planarian Schmidtea mediterranea. We achieve this by soaking animals in a solution containing the cytochrome oxidase inhibitor sodium azide. Brief exposure to sodium azide causes extrusion of the pharynx from the animal, which we call "chemical amputation." Chemical amputation removes the entire pharynx, and generates a small wound where the pharynx attaches to the intestine. After extensive rinsing, all amputated animals regenerate a fully functional pharynx in approximately one week. Stem cells in the rest of the body drive regeneration of the new pharynx. Here, we provide a detailed protocol for chemical amputation, and describe both histological and behavioral methods to assess successful amputation and regeneration.

Synaptonemal Complex Components Are Required for Meiotic Checkpoint Function in Caenorhabditis elegans.

Synapsis involves the assembly of a proteinaceous structure, the synaptonemal complex (SC), between paired homologous chromosomes, and is essential for proper meiotic chromosome segregation. In Caenorhabditis elegans, the synapsis checkpoint selectively removes nuclei with unsynapsed chromosomes by inducing apoptosis. This checkpoint depends on pairing centers (PCs), cis-acting sites that promote pairing and synapsis. We have hypothesized that the stability of homolog pairing at PCs is monitored by this checkpoint. Here, we report that SC components SYP-3, HTP-3, HIM-3, and HTP-1 are required for a functional synapsis checkpoint. Mutation of these components does not abolish PC function, demonstrating they are bona fide checkpoint components. Further, we identify mutant backgrounds in which the instability of homolog pairing at PCs does not correlate with the synapsis checkpoint response. Altogether, these data suggest that, in addition to homolog pairing, SC assembly may be monitored by the synapsis checkpoint.

Spindle assembly checkpoint proteins regulate and monitor meiotic synapsis in C. elegans.

Homologue synapsis is required for meiotic chromosome segregation, but how synapsis is initiated between chromosomes is poorly understood. In Caenorhabditis elegans, synapsis and a checkpoint that monitors synapsis depend on pairing centers (PCs), cis-acting loci that interact with nuclear envelope proteins, such as SUN-1, to access cytoplasmic microtubules. Here, we report that spindle assembly checkpoint (SAC) components MAD-1, MAD-2, and BUB-3 are required to negatively regulate synapsis and promote the synapsis checkpoint response. Both of these roles are independent of a conserved component of the anaphase-promoting complex, indicating a unique role for these proteins in meiotic prophase. MAD-1 and MAD-2 localize to the periphery of meiotic nuclei and interact with SUN-1, suggesting a role at PCs. Consistent with this idea, MAD-1 and BUB-3 require full PC function to inhibit synapsis. We propose that SAC proteins monitor the stability of pairing, or tension, between homologues to regulate synapsis and elicit a checkpoint response.