The TALE Class Homeobox Gene Smed-prep Defines the Anterior Compartment for Head Regeneration

Planaria continue to blossom as a model system for understanding all aspects of regeneration. They provide an opportunity to understand how the replacement of missing tissues from preexisting adult tissue is orchestrated at the molecular level. When amputated along any plane, planaria are capable of regenerating all missing tissue and rescaling all structures to the new size of the animal. Recently, rapid progress has been made in understanding the developmental pathways that control planarian regeneration. In particular Wnt/beta-catenin signaling is central in promoting posterior fates and inhibiting anterior identity. Currently the mechanisms that actively promote anterior identity remain unknown. Here, Smed-prep, encoding a TALE class homeodomain, is described as the first gene necessary for correct anterior fate and patterning during planarian regeneration. Smed-prep is expressed at high levels in the anterior portion of whole animals, and Smed-prep(RNAi) leads to loss of the whole brain during anterior regeneration, but not during lateral regeneration or homeostasis in intact worms. Expression of markers of different anterior fated cells are greatly reduced or lost in Smed-prep(RNAi) animals. We find that the ectopic anterior structures induced by abrogation of Wnt signaling also require Smed-prep to form. We use double knockdown experiments with the S. mediterranea ortholog of nou-darake (that when knocked down induces ectopic brain formation) to show that Smed-prep defines an anterior fated compartment within which stem cells are permitted to assume brain fate, but is not required directly for this differentiation process. Smed-prep is the first gene clearly implicated as being necessary for promoting anterior fate and the first homeobox gene implicated in establishing positional identity during regeneration. Together our results suggest that Smed-prep is required in stem cell progeny as they form the anterior regenerative blastema and is required for specifying anterior cell fates and correct patterning

The regeneration capacity of the flatworm Macrostomum lignano—on repeated regeneration, rejuvenation, and the minimal size needed for regeneration

The lion’s share of studies on regeneration in Plathelminthes (flatworms) has been so far carried out on a derived taxon of rhabditophorans, the freshwater planarians (Tricladida), and has shown this group’s outstanding regeneration capabilities in detail. Sharing a likely totipotent stem cell system, many other flatworm taxa are capable of regeneration as well. In this paper, we present the regeneration capacity of Macrostomum lignano, a representative of the Macrostomorpha, the basal-most taxon of rhabditophoran flatworms and one of the most basal extant bilaterian protostomes. Amputated or incised transversally, obliquely, and longitudinally at various cutting levels, M. lignano is able to regenerate the anterior-most body part (the rostrum) and any part posterior of the pharynx, but cannot regenerate a head. Repeated regeneration was observed for 29 successive amputations over a period of almost 12 months. Besides adults, also first-day hatchlings and older juveniles were shown to regenerate after transversal cutting. The minimum number of cells required for regeneration in adults (with a total of 25,000 cells) is 4,000, including 160 neoblasts. In hatchlings only 1,500 cells, including 50 neoblasts, are needed for regeneration. The life span of untreated M. lignano was determined to be about 10 months

Potential of Macrostomum lignano to recover from γ-ray irradiation

Stem cells are the only proliferating cells in flatworms and can be eliminated by irradiation with no damage to differentiated cells. We investigated the effect of fractionated irradiation schemes on Macrostomum lignano, namely, on survival, gene expression, morphology and regeneration. Proliferating cells were almost undetectable during the first week post-treatment. Cell proliferation and gene expression were restored within 1 month in a dose-dependent manner following exposure to up to 150 Gy irradiation. During recovery, stem cells did not cross the midline but were restricted within lateral compartments. An accumulated dose of 210 Gy resulted in a lethal phenotype. Our findings demonstrate that M. lignano represents a suitable model system for elucidating the effect of irradiation on the stem cell system in flatworms and for improving our understanding of the recovery potential of severely damaged stem-cell systems

Combining Classical and Molecular Approaches Elaborates on the Complexity of Mechanisms Underpinning Anterior Regeneration

The current model of planarian anterior regeneration evokes the establishment of low levels of Wnt signalling at anterior wounds, promoting anterior polarity and subsequent elaboration of anterior fate through the action of the TALE class homeodomain PREP. The classical observation that decapitations positioned anteriorly will regenerate heads more rapidly than posteriorly positioned decapitations was among the first to lead to the proposal of gradients along an anteroposterior (AP) axis in a developmental context. An explicit understanding of this phenomenon is not included in the current model of anterior regeneration. This raises the question what the underlying molecular and cellular basis of this temporal gradient is, whether it can be explained by current models and whether understanding the gradient will shed light on regenerative events. Differences in anterior regeneration rate are established very early after amputation and this gradient is dependent on the activity of Hedgehog (Hh) signalling. Animals induced to produce two tails by either Smed-APC-1(RNAi) or Smed-ptc(RNAi) lose anterior fate but form previously described ectopic anterior brain structures. Later these animals form peri-pharyngeal brain structures, which in Smed-ptc(RNAi) grow out of the body establishing a new A/P axis. Combining double amputation and hydroxyurea treatment with RNAi experiments indicates that early ectopic brain structures are formed by uncommitted stem cells that have progressed through S-phase of the cell cycle at the time of amputation. Our results elaborate on the current simplistic model of both AP axis and brain regeneration. We find evidence of a gradient of hedgehog signalling that promotes posterior fate and temporarily inhibits anterior regeneration. Our data supports a model for anterior brain regeneration with distinct early and later phases of regeneration. Together these insights start to delineate the interplay between discrete existing, new, and then later homeostatic signals in AP axis regeneration

A Low Percent Ethanol Method for Immobilizing Planarians

Planarians have recently become a popular model system for the study of adult stem cells, regeneration and polarity. The system is attractive for both undergraduate and graduate research labs, since planarian colonies are low cost and easy to maintain. Also in situ hybridization, immunofluorescence and RNA-interference (RNAi) gene knockdown techniques have been developed for planarian studies. However, imaging of live worms (particularly at high magnifications) is difficult because animals are strongly photophobic; they quickly move away from light sources and out of frame. The current methods available to inhibit movement in planarians include RNAi injection and exposure to cold temperatures. The former is labor and time intensive, while the latter precludes the use of many fluorescent reporter dyes. Here, we report a simple, inexpensive and reversible method to immobilize planarians for live imaging. Our data show that a short 1 hour treatment with 3% ethanol (EtOH) is sufficient to inhibit both the fine and gross movements of Schmidtea mediterranea planarians, of the typical size used (4–6 mm), with full recovery of movement within 3–4 hours. Importantly, EtOH treatment did not interfere with regeneration, even after repeated exposure, nor lyse epithelial cells (as assayed by H&E staining). We demonstrate that a short exposure to a low concentration of EtOH is a quick and effective method of immobilizing planarians, one that is easily adaptable to planarians of all sizes and will increase the accessibility of live imaging assays to planarian researchers

SLI-1 Cbl Inhibits the Engulfment of Apoptotic Cells in C. elegans through a Ligase-Independent Function

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway, which includes the small GTPase CED-10 Rac and the cytoskeletal regulator ABI-1, acts to rearrange the cytoskeleton of the engulfing cell. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The second pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Cbl, the mammalian homolog of the C. elegans E3 ubiquitin ligase and adaptor protein SLI-1, interacts with Rac and Abi2 and modulates the actin cytoskeleton, suggesting it might act in engulfment. Our genetic studies indicate that SLI-1 inhibits apoptotic cell engulfment and DTC migration independently of the CED-10 Rac and CED-1 pathways. We found that the RING finger domain of SLI-1 is not essential to rescue the effects of SLI-1 deletion on cell migration, suggesting that its role in this process is ubiquitin ligase-independent. We propose that SLI-1 opposes the engulfment of apoptotic cells via a previously unidentified pathway.National Cancer Institute (U.S.) (Award K08CA104890

SMG-1 and mTORC1 Act Antagonistically to Regulate Response to Injury and Growth in Planarians

Planarian flatworms are able to both regenerate their whole bodies and continuously adapt their size to nutrient status. Tight control of stem cell proliferation and differentiation during these processes is the key feature of planarian biology. Here we show that the planarian homolog of the phosphoinositide 3-kinase-related kinase (PIKK) family member SMG-1 and mTOR complex 1 components are required for this tight control. Loss of smg-1 results in a hyper-responsiveness to injury and growth and the formation of regenerative blastemas that remain undifferentiated and that lead to lethal ectopic outgrowths. Invasive stem cell hyper-proliferation, hyperplasia, hypertrophy, and differentiation defects are hallmarks of this uncontrolled growth. These data imply a previously unappreciated and novel physiological function for this PIKK family member. In contrast we found that planarian members of the mTOR complex 1, tor and raptor, are required for the initial response to injury and blastema formation. Double smg-1 RNAi experiments with tor or raptor show that abnormal growth requires mTOR signalling. We also found that the macrolide rapamycin, a natural compound inhibitor of mTORC1, is able to increase the survival rate of smg-1 RNAi animals by decreasing cell proliferation. Our findings support a model where Smg-1 acts as a novel regulator of both the response to injury and growth control mechanisms. Our data suggest the possibility that this may be by suppressing mTOR signalling. Characterisation of both the planarian mTORC1 signalling components and another PIKK family member as key regulators of regeneration and growth will influence future work on regeneration, growth control, and the development of anti-cancer therapies that target mTOR signalling

c-Crk proto-oncogene contributes to transcriptional repression of p120-catenin in non-small cell lung cancer cells

As a member of adherens junction, p120-catenin (p120ctn) plays a major role in cell adhesions through stabilization of E-cadherin. p120ctn is transcriptionally down-regulated in non-small cell lung cancer (NSCLC), although the molecular mechanisms underlying p120ctn repression are incompletely defined. Here we further investigated transcriptional regulation of p120ctn in NSCLC. We prepared a promoter reporter plasmid construct that contained p120ctn promoter region from position −1082 to +320 relative to transcription start site. Through serial deletion mutation analysis of the p120ctn promoter, we pinpointed cis-acting elements involved in regulation of p120ctn. We identified transcription factor SP1 as a transcriptional repressor of p120ctn that directly binds to segment (−9 to +36) of the p120ctn promoter. SP1 can receive multiple signals from several intracellular signaling pathways. Through examination of SP1 binding partners, we identified proto-oncogene c-Crk to be involved in transcriptional down-regulation of p120ctn. RNAi mediated silencing of CRK in A549, H157 and H358 cells increased p120ctn protein levels. On the other hand, over-expression of CRK-I and CRK-II in NSCLC cells down-regulated p120ctn, an effect that was abrogated by simultaneous silencing of SP1. In summary, our data provide evidence for the role of c-Crk proto-oncogene in transcriptional repression of p120ctn that further clarifies the mechanism by which this biochemical signal promotes metastasis in NSCLC

Glutamine synthetase gene expression during the regeneration of the annelid Enchytraeus japonensis

Enchytraeus japonensis is a highly regenerative oligochaete annelid that can regenerate a complete individual from a small body fragment in 4–5 days. In our previous study, we performed complementary deoxyribonucleic acid subtraction cloning to isolate genes that are upregulated during E. japonensis regeneration and identified glutamine synthetase (gs) as one of the most abundantly expressed genes during this process. In the present study, we show that the full-length sequence of E. japonensis glutamine synthetase (EjGS), which is the first reported annelid glutamine synthetase, is highly similar to other known class II glutamine synthetases. EjGS shows a 61–71% overall amino acid sequence identity with its counterparts in various other animal species, including Drosophila and mouse. We performed detailed expression analysis by in situ hybridization and reveal that strong gs expression occurs in the blastemal regions of regenerating E. japonensis soon after amputation. gs expression was detectable at the cell layer covering the wound and was found to persist in the epidermal cells during the formation and elongation of the blastema. Furthermore, in the elongated blastema, gs expression was detectable also in the presumptive regions of the brain, ventral nerve cord, and stomodeum. In the fully formed intact head, gs expression was also evident in the prostomium, brain, the anterior end of the ventral nerve cord, the epithelium of buccal and pharyngeal cavities, the pharyngeal pad, and in the esophageal appendages. In intact E. japonensis tails, gs expression was found in the growth zone in actively growing worms but not in full-grown individuals. In the nonblastemal regions of regenerating fragments and in intact worms, gs expression was also detected in the nephridia, chloragocytes, gut epithelium, epidermis, spermatids, and oocytes. These results suggest that EjGS may play roles in regeneration, nerve function, cell proliferation, nitrogenous waste excretion, macromolecule synthesis, and gametogenesis

To Be or Not to Be a Flatworm: The Acoel Controversy

Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora - have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives