Cristina González-Estévez

Gtdap-1 promotes autophagy and is required for planarian remodeling during regeneration and starvation

Remodeling is an integral component of tissue homeostasis and regeneration. In planarians, these processes occur constantly in a simple tractable model organism as part of the animals normal life history. Here, we have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1, together with DAP-kinase, has been identified as a positive mediator of programmed cell death induced by γ-IFN in HeLa cells. Although the function of DAP-kinase is well characterized, the role of DAP-1 has not been studied in detail. Our findings suggest that Gtdap-1 is involved in autophagy in planarians, and that autophagy plays an essential role in the remodeling of the organism that occurs during regeneration and starvation, providing the necessary energy and building blocks to the neoblasts for cell proliferation and differentiation. The gene functions at the interface between survival and cell death during stress-inducing processes like regeneration and starvation in sexual and asexual races of planarians. Our findings provide insights into the complex interconnections among cell proliferation, homeostasis, and cell death in planarians and perspectives for the understanding of neoblast stem cell dynamics.

Diverse miRNA spatial expression patterns suggest important roles in homeostasis and regeneration in planarians.

miRNAs are an important class of non-protein coding small RNAs whose specific functions in animals are rapidly being elucidated. It is clear that miRNAs can play crucial roles in stem cell maintenance, cell fate determination and differentiation. We use planarians, which possess a large population of pluripotent somatic stem cells, as a powerful model system to study many aspects of stem cell biology and regeneration. In particular we wish to investigate the regulatory role miRNAs may have in planarian stem cell self renewal, proliferation and differentiation. Here, we characterized the differential spatial patterns of expression of miRNAs in whole and regenerating planarians by in situ hybridization to nascent miRNA transcripts. These miRNA expression patterns are the first which have been determined for a Lophotrocozoan animal. We have characterized the expression patterns of 42 miRNAs in adult planarians, constituting a complete range of tissue specific expression patterns. We also followed miRNA expression during planarian regeneration. The majority of planarian miRNAs were expressed either in areas where stem cells (neoblasts) are located and/or in the nervous system. Some miRNAs were definitively expressed in stem cells and dividing cells as confirmed by in situ hybridisation after irradiation. We also found miRNAs to be expressed in germ stem cells of the sexual strain. Together, these data suggest an important role for miRNAs in stem cell regulation and in neural cell differentiation in planarians.

Decreased neoblast progeny and increased cell death during starvation-induced planarian degrowth

The development of a complex multicellular organism requires a careful coordination of growth, cell division, cell differentiation and cell death. All these processes must be under intricate and coordinated control, as they have to be integrated across all tissues. Freshwater planarians are especially plastic, in that they constantly replace somatic tissues from a pool of adult somatic stem cells and continuously undergo growth and degrowth as adult animals in response to nutrient availability. During these processes they appear to maintain perfect scale of tissues and organs. These life history traits make them an ideal model system to study growth and degrowth. We have studied the unique planarian process of degrowth. When food is not available, planarians are able to degrow to a minimum size, without any signs of adverse physiological outcomes. For example they maintain full regenerative capacity. Our current knowledge of how this is regulated at the molecular and cellular level is very limited. Planarian degrowth has been reported to result from a decrease in cell number rather than a decrease in cell size. Thus one obvious explanation for degrowth would be a decrease in stem cell proliferation. However evidence in the literature suggests this is not the case. We show that planarians maintain normal basal mitotic rates during degrowth but that the number of stem cell progeny decreases during starvation and degrowth. These observations are reversed upon feeding, indicating that they are dependent on nutritional status. An increase in cell death is also observed during degrowth, which is not rapidly reversed upon feeding. We conclude that degrowth is a result of cell death decreasing cell numbers and that the dynamics of neoblast self-renewal and differentiation adapt to nutrient conditions to allow maintenance of the neoblast population during the period of starvation.

Autophagy in invertebrates: insights into development, regeneration and body remodeling.

Autophagy is a process in which eukaryotic cells sequester and degrade cytoplasm and organelles via the lysosomal pathway. This process allows turnover of intracellular organelles, participates in the maintenance of cellular homeostasis and prevents accumulation of defective cellular structures. Increased autophagy is normally induced by environmental cues such as starvation and hormones, while excessive levels of autophagy can lead to autophagic programmed cell death (PCD), with features that differ from those of the apoptotic PCD process. Since autophagic PCD plays a key role in development, morphogenesis and regeneration in several animal taxa, identification of evolutionarily conserved components of the autophagic machinery is a basic starting point in order to unravel the role of autophagy under both physiological and pathological conditions. Here we summarize recent findings on the role of autophagy in two different invertebrate taxa, Platyhelminthes and Insects, focusing attention on two complex events occurring in those systems, namely planarian regeneration and insect metamorphosis. Both represent good models in which to investigate the process of autophagy and its relationship with other PCD mechanisms.

Autophagy and apoptosis in planarians

Adult planarians are capable of undergoing regeneration and body remodelling in order to adapt to physical damage or extreme environmental conditions. Moreover, most planarians can tolerate long periods of starvation and during this time, they shrink from an adult size to, and sometimes beyond, the initial size at hatching. Indeed, these properties have made them a classic model to study stem cells and regeneration. Under such stressful conditions, food reserves from the gastrodermis and parenchyma are first used up and later the testes, copulatory organs and ovaries are digested. More surprisingly, when food is again made available to shrunken individuals, they grow back to adult size and all their reproductive structures reappear. These cycles of growth and shrinkage may occur over long periods without any apparent impairment to the individual, or to its future maturation and breeding capacities. This plasticity resides in a mesoderm tissue known as the parenchyma, which is formed by several differentiated non-proliferating cell types and only one mitotically active cell type, the neoblasts, which represent approximately 20–30% of the cells in the parenchyma. Neoblasts are generally thought to be somatic stem-cells that participate in the normal continuous turnover of all cell types in planarians. Hence, planarians are organisms that continuously adapt their bodies (morphallaxis) to different environmental stresses (i.e.: injury or starvation). This adaptation involves a variety of processes including proliferation, differentiation, apoptosis and autophagy, all of which are perfectly orchestrated and tightly regulated to remodel or restore the body pattern. While neoblast biology and body re-patterning are currently the subject of intense research, apoptosis and autophagy remain much less studied. In this review we will summarize our current understanding and hypotheses regarding where and when apoptosis and autophagy occur and fulfil an essential role in planarians.

Gtdap-1 and the Role of Autophagy During Planarian Regeneration and Starvation

Planarians have been established as an ideal model organism for stem cell research and regeneration. Planarian regeneration and homeostasis require an exquisite balancing act between cell death and cell proliferation as new tissues are made (epimorphosis) and existing tissues remodeled (morphallaxis). Some of the genes and mechanisms that control cell proliferation and pattern formation are known. However, studies about cell death during remodeling are few and far between. We have studied the gene Gtdap-1, the planarian ortholog of human death-associated protein-1 or DAP-1. DAP-1 together with DAP-kinase has been identified as a positive mediator of programmed cell death induced by gamma-interferon in HeLa cells. We have found that the gene functions at the interface between autophagy and cell death in the remodeling of the organism that occurs during regeneration and starvation in sexual and asexual races of planarians. Our data suggest that autophagy of existing cells may be essential to fuel the continued proliferation and differentiation of stem cells by providing the necessary energy and building blocks to neoblasts. Addendum to: Gtdap-1 Promotes Autophagy and is Required for Planarian Remodeling During Regeneration and Starvation C. González-Estévez, D.A. Felix, A.A. Aboobaker and E. Saló Proc Natl Acad Sci USA 2007; 104:13373-8

Autophagy in freshwater planarians.

Planarians provide a new and emergent in vivo model organism to study autophagy. On the whole, maintaining the normal homeostatic balance in planarians requires continuous dynamic adjustment of many processes, including proliferation, apoptosis, differentiation, and autophagy. This makes them very different from other models where autophagy only occurs at very specific times and/or in very specific organs. This chapter aims to offer a general vision of planarians as a model organism, placing more emphasis on those characteristics related to autophagy and describing how autophagy fits into the processes of body remodeling during regeneration and starvation. We also define exactly what is known about autophagy in these organisms and we discuss the techniques available to study the relevant processes, as well as the techniques that are currently being developed. As such, this chapter will serve as a compilation of the techniques available to investigate autophagy in planarians.

Autophagy meets planarians

This review aims to demonstrate the importance of freshwater planarians as model organisms, particularly emphasizing those characteristics of the animal that make them a good model to study autophagy. The aim of this review is to provide a better understanding of autophagy in this model for the non-planarian reader, and elucidate the relevance of autophagy research in this peculiar model organism. Furthermore, I will try to synthesize the evidence showing the importance of autophagy in planarian body remodelling, and I will discuss some ideas about the role of autophagy in stem cell biology. In light of these new developments, it is likely that the planarian field will make an important contribution to the study of the molecular mechanisms involved in autophagy in the future.