Molecular complexity and gene expression controlling cell turnover during a digestive cycle of carnivorous sponge Lycopodina hypogea.

Lycopodina hypogea is a carnivorous sponge that tolerates laboratory husbandry very well. During a digestion cycle, performed without any digestive cavity, this species undergoes spectacular morphological changes leading to a total regression of long filaments that ensure the capture of prey and their reformation at the end of the cycle. This phenomenon is a unique opportunity to analyze the molecular and cellular determinants that ensure digestion in the sister group of all other metazoans. Using differential transcriptomic analysis coupled with cell biology studies of proliferation, differentiation, and programmed cell deaths (i.e., autophagy and the destructive/constructive function of apoptosis), we demonstrate that the molecular and cellular actors that ensure digestive homeostasis in a sister group of all remaining animals are similar in variety and complexity to those controlling tissue homeostasis in higher vertebrates. During a digestion cycle, most of these actors are finely tuned in a coordinated manner. Our data benefits from complementary approaches coupling in silico and cell biology studies and demonstrate that the nutritive function is provided by the coordination of molecular network that impacts the cells turnover in the entire organism.

Transdifferentiation and mesenchymal-to-epithelial transition during regeneration in Demospongiae (Porifera).

Origin and early evolution of regeneration mechanisms remain among the most pressing questions in animal regeneration biology. Porifera have exceptional regenerative capacities and, as early Metazoan lineage, are a promising model for studying evolutionary aspects of regeneration. Here, we focus on reparative regeneration of the body wall in the Mediterranean demosponge Aplysina cavernicola. The epithelialization of the wound surface is completed within 2 days, and the wound is completely healed within 2 weeks. The regeneration is accompanied with the formation of a mass of undifferentiated cells (blastema), which consists of archaeocytes, dedifferentiated choanocytes, anucleated amoebocytes, and differentiated spherulous cells. The main mechanisms of A. cavernicola regeneration are cell dedifferentiation with active migration and subsequent redifferentiation or transdifferentiation of polypotent cells through the mesenchymal-to-epithelial transformation. The main cell sources of the regeneration are archaeocytes and choanocytes. At early stages of the regeneration, the blastema almost devoid of cell proliferation, but after 24 hr postoperation (hpo) and up to 72 hpo numerous DNA-synthesizing cells appear there. In contrast to intact tissues, where vast majority of DNA-synthesizing cells are choanocytes, all 5-ethynyl-2'-deoxyuridine-labeled cells in the blastema are mesohyl cells. Intact tissues, distant from the wound, retains intact level of cell proliferation during whole regeneration process. For the first time, the apoptosis was studied during the regeneration of sponges. Two waves of apoptosis were detected during A. cavernicola regeneration: The first wave at 6-12 hpo and the second wave at 48-72 hpo.

Sponge digestive system diversity and evolution: filter feeding to carnivory.

Sponges are an ancient basal life form, so understanding their evolution is key to understanding all metazoan evolution. Sponges have very unusual feeding mechanisms, with an intricate network of progressively optimized filtration units: from the simple choanocyte lining of a central cavity, or spongocoel, to more complex chambers and canals. Furthermore, in a single evolutionary event, a group of sponges transitioned to carnivory. This major evolutionary transition involved replacing the filter-feeding apparatus with mobile phagocytic cells that migrate collectively towards the trapped prey. Here, we focus on the diversity and evolution of sponge nutrition systems and the amazing adaptation to carnivory.

Topological balance of cell distributions in plane monolayers.

Most of normal proliferative epithelia of plants and metazoans are topologically invariant and characterized by similar cell distributions according to the number of cell neighbors (DCNs). Here we study peculiarities of these distributions and explain why the DCN obtained from the location of intercellular boundaries and that based on the Voronoi tessellation with nodes located on cell nuclei may differ from each other. As we demonstrate, special microdomains where four or more intercellular boundaries converge are topologically charged. Using this fact, we deduce a new equation describing the topological balance of the DCNs. The developed theory is applied for a series of microphotographs of non-tumoral epithelial cells of the human cervix (HCerEpiC) to improve the image processing near the edges of microphotographs and reveal the topological invariance of the examined monolayers. Special contact microdomains may be present in epithelia of various natures, however, considering the well-known vertex model of epithelium, we show that such contacts are absent in the usual solid-like state of the model and appear only in the liquid-like cancer state. Also, we discuss a possible biological role of special contacts in context of proliferative epithelium dynamics and tissue morphogenesis.

A comparative ultrastructure study of the tardigrade Ramazzottius varieornatus in the hydrated state, after desiccation and during the process of rehydration.

Tardigrades can survive hostile environments such as desiccation by adopting a state of anhydrobiosis. Numerous tardigrade species have been described thus far, and recent genome and transcriptome analyses revealed that several distinct strategies were employed to cope with harsh environments depending on the evolutionary lineages. Detailed analyses at the cellular and subcellular levels are essential to complete these data. In this work, we analyzed a tardigrade species that can withstand rapid dehydration, Ramazzottius varieornatus. Surprisingly, we noted an absence of the anhydrobiotic-specific extracellular structure previously described for the Hypsibius exemplaris species. Both Ramazzottius varieornatus and Hypsibius exemplaris belong to the same evolutionary class of Eutardigrada. Nevertheless, our observations reveal discrepancies in the anhydrobiotic structures correlated with the variation in the anhydrobiotic mechanisms.

Tissue-specific alternative splicing of Tak1 is conserved in deuterostomes.

Alternative splicing allows organisms to rapidly modulate protein functions to physiological changes and therefore represents a highly versatile adaptive process. We investigated the conservation of the evolutionary history of the "Fox" family of RNA-binding splicing factors (RBFOX) as well as the conservation of regulated alternative splicing of the genes they control. We found that the RBFOX proteins are conserved in all metazoans examined. In humans, Fox proteins control muscle-specific alternative splicing of many genes but despite the conservation of splicing factors, conservation of regulation of alternative splicing has never been demonstrated between man and nonvertebrate species. Therefore, we studied 40 known Fox-regulated human exons and found that 22 had a tissue-specific splicing pattern in muscle and heart. Of these, 11 were spliced in the same tissue-specific manner in mouse tissues and 4 were tissue-specifically spliced in muscle and heart of the frog Xenopus laevis. The inclusion of two of these alternative exons was also downregulated during tadpole development. Of the 40 in the starting set, the most conserved alternative splicing event was in the transforming growth factor (TGF) beta-activated kinase Tak1 (MAP3K7) as this was also muscle specific in urochordates and in Ambulacraria, the most ancient deuterostome clade. We found exclusion of the muscle-specific exon of Tak1 was itself under control of TGF beta in cell culture and consistently that TGF beta caused an upregulation of Fox2 (RBFOX2) expression. The alternative exon, which codes for an in-frame 27 amino acids between the kinase and known regulatory domain of TAK1, contains conserved features in all organisms including potential phosphorylation sites and likely has an important conserved function in TGF beta signaling and development. This study establishes that deuterostomes share a remarkable conserved physiological process that involves a splicing factor and expression of tissue-specific isoforms of a target gene that expedites a highly conserved signaling pathway.

[Would D'Arcy Thompson have predicted a topological control of apoptosis?]. / D'Arcy Thompson aurait-il prédit un contrôle topologique de l'apoptose ?

The laws that drive morphogenesis remain a major biological question. Today's views emphasize molecular autonomous processes rather than physical and mechanical constraints proposed by d'Arcy Thompson earlier on. In Ciona intestinalis oocyte, follicular cells formed by two distinct sets of geometrically-ordered epithelial monolayers positioned over the egg control apoptosis, implying that physically-predetermined shapes play a role in the control of cell determinism. In follicular cells ideally positioned over the spherical geometry of the egg, a drastic, optimized and polarized inward apoptosis sequence directly results from this positioning, suggesting the existence of some apoptotic master cells which control the destiny of neighboring cells. This concept could shed a new light on the origin of massive apoptosis phases that take place during embryogenesis in vertebrates (e.g., cavitation, inter-digitation). It could also be applied to specific therapeutic strategies to fight cancer.

Topological properties and shape of proliferative and nonproliferative cell monolayers.

During embryonic development, structures with complex geometry can emerge from planar epithelial monolayers; studying these shape transitions is of key importance for revealing the biophysical laws involved in the morphogenesis of biological systems. Here, using the example of normal proliferative monkey kidney (COS) cell monolayers, we investigate global and local topological characteristics of this model system in dependence on its shape. The obtained distributions of cells by their valence demonstrate a difference between the spherical and planar monolayers. In addition, in both spherical and planar monolayers, the probability of observing a pair of neighboring cells with certain valences depends on the topological charge of the pair. The zero topological charge of the cell pair can increase the probability for the cells to be the nearest neighbors. We then test and confirm that analogous relationships take place in a more ordered spherical system with a larger fraction of 6-valent cells, namely, in the nonproliferative epithelium (follicular system) of ascidian species oocytes. However, unlike spherical COS cell monolayers, ascidian monolayers are prone to nonrandom agglomeration of 6-valent cells and have linear topological defects called scars and pleats. The reasons for this difference in morphology are discussed. The morphological peculiarities found are compared with predictions of the widely used vertex model of epithelium.

Cell death and renewal during prey capture and digestion in the carnivorous sponge Asbestopluma hypogea (Porifera: Poecilosclerida).

The sponge Asbestopluma hypogea is unusual among sponges due to its peculiar carnivorous feeding habit. During various stages of its nutrition cycle, the sponge is subjected to spectacular morphological modifications. Starved animals are characterized by many elongated filaments, which are crucial for the capture of prey. After capture, and during the digestion process, these filaments actively regress before being regenerated during a subsequent period of starvation. Here, we demonstrate that these morphological events rely on a highly dynamic cellular turnover, implying a coordinated sequence of programmed cell death (apoptosis and autophagy), cell proliferation and cell migration. A candidate niche for cell renewal by stem cell proliferation and differentiation was identified at the base of the sponge peduncle, characterized by higher levels of BrdU/EdU incorporation. Therefore, BrdU/EdU-positive cells of the peduncle base are candidate motile cells responsible for the regeneration of the prey-capturing main sponge body, i.e. the dynamic filaments. Altogether, our results demonstrate that dynamics of cell renewal in sponge appear to be regulated by cellular mechanisms as multiple and complex as those already identified in bilaterian metazoans.

Random nature of epithelial cancer cell monolayers.

Although the polygonal shape of epithelial cells has been drawing the attention of scientists for several centuries, only a decade and a half ago it was demonstrated that distributions of polygon types (DOPTs) are similar in proliferative epithelia of many different plant and animal species. In this study, we show that hyper-proliferation of cancer cells disrupts this universal paradigm and results in randomly organized epithelial structures. Examining non-synchronized and synchronized HeLa cervix cells, we suppose that the spread of cell sizes is the main parameter controlling the DOPT in the cancer cell monolayers. To test this hypothesis, we develop a theory of morphologically similar random polygonal packings. By analysing differences between tumoural and normal epithelial cell monolayers, we conclude that the latter have more ordered structures because of their lower proliferation rates and, consequently, more effective relaxation of mechanical stress associated with cell division and growth. To explain the structural features of normal proliferative epithelium, we take into account the spread of cell sizes in the monolayer. The proposed theory also rationalizes some highly ordered unconventional post-mitotic epithelia.

Staining and Tracking Methods for Studying Sponge Cell Dynamics.

To better understand the origin of animal cell types, body plans, and other morphological features, further biological knowledge and understanding are needed from non-bilaterian phyla, namely, Placozoa, Ctenophora, and Porifera. This chapter describes recent cell staining approaches that have been developed in three phylogenetically distinct sponge species-the homoscleromorph Oscarella lobularis, and the demosponges Amphimedon queenslandica and Lycopodina hypogea-to enable analyses of cell death, proliferation, and migration. These methods allow for a more detailed understanding of cellular behaviors and fates, and morphogenetic processes in poriferans, building on current knowledge of sponge cell biology that relies chiefly on classical (static) histological observations.

Cell shape and TGF-beta signaling define the choice of lineage during in vitro differentiation of mouse primary hepatic precursors.

Cellular differentiation relies on both physical and chemical environmental cues. The bipotential mouse embryonic liver (BMEL) cells are early progenitors of liver epithelial cells with an apparently infinite proliferative potential. These cells, which remain undifferentiated in a monolayer culture, differentiate upon release from geometrical constraints imposed by growth on a stiff plastic plate. In a complex three dimensional environment of a Matrigel extracellular matrix, BMEL cells form two types of polarized organoids of distinct morphologies: cyst-like structures suggesting cholangiocyte-type organization or complex organoids, reminiscent of liver parenchyma and associated with acquisition of hepatocyte-specific phenotypic markers. The choice of the in vitro differentiation lineage is governed by Transforming Growth Factor-beta (TGF-beta) signaling. Our results suggest that morphological cues initiate the differentiation of early hepatic precursors and confirm the inhibitory role of TGF-beta on hepatocytic lineage differentiation.

Catalytically inactive human cathepsin D triggers fibroblast invasive growth.

The aspartyl-protease cathepsin D (cath-D) is overexpressed and hypersecreted by epithelial breast cancer cells and stimulates their proliferation. As tumor epithelial-fibroblast cell interactions are important events in cancer progression, we investigated whether cath-D overexpression affects also fibroblast behavior. We demonstrate a requirement of cath-D for fibroblast invasive growth using a three-dimensional (3D) coculture assay with cancer cells secreting or not pro-cath-D. Ectopic expression of cath-D in cath-D-deficient fibroblasts stimulates 3D outgrowth that is associated with a significant increase in fibroblast proliferation, survival, motility, and invasive capacity, accompanied by activation of the ras-MAPK pathway. Interestingly, all these stimulatory effects on fibroblasts are independent of cath-D proteolytic activity. Finally, we show that pro-cath-D secreted by cancer cells is captured by fibroblasts and partially mimics effects of transfected cath-D. We conclude that cath-D is crucial for fibroblast invasive outgrowth and could act as a key paracrine communicator between cancer and stromal cells, independently of its catalytic activity.

Crystal-like order and defects in metazoan epithelia with spherical geometry.

Since Robert Hooke studied cork cell patterns in 1665, scientists have been puzzled by why cells form such ordered structures. The laws underlying this type of organization are universal, and we study them comparing the living and non-living two-dimensional systems self-organizing at the spherical surface. Such-type physical systems often possess trigonal order with specific elongated defects, scars and pleats, where the 5-valence and 7-valence vertices alternate. In spite of the fact that the same physical and topological rules are involved in the structural organization of biological systems, such topological defects were never reported in epithelia. We have discovered them in the follicular spherical epithelium of ascidians that are emerging models in developmental biology. Surprisingly, the considered defects appear in the epithelium even when the number of cells in it is significantly less than the previously known threshold value. We explain this result by differences in the cell sizes and check our hypothesis considering the self-assembly of different random size particles on the spherical surface. Scars, pleats and other complex defects found in ascidian samples can play an unexpected and decisive role in the permanent renewal and reorganization of epithelia, which forms or lines many tissues and organs in metazoans.

Ultrastructural analysis of the dehydrated tardigrade Hypsibius exemplaris unveils an anhydrobiotic-specific architecture.

Tardigrades can cope with adverse environmental conditions by turning into anhydrobiotes with a characteristic tun shape. Tun formation is an essential morphological adaptation for tardigrade entry into the anhydrobiotic state. The tun cell structure and ultrastructure have rarely been explored in tardigrades in general and never in Hypsibius exemplaris. We used transmission electron microscopy to compare cellular organization and ultrastructures between hydrated and anhydrobiotic H. exemplaris. Despite a globally similar cell organelle structure and a number of cells not significantly different between hydrated and desiccated tardigrades, reductions in the sizes of both cells and mitochondria were detected in dehydrated animals. Moreover, in anhydrobiotes, secretory active cells with a dense endoplasmic reticulum network were observed. Interestingly, these anhydrobiote-specific cells are in a close relationship with a specific extracellular structure surrounding each cell. It is possible that this rampart-like extracellular structure resulted from the accumulation of anhydrobiotic-specific material to protect the cells. Interestingly, after five hours of rehydration, the number of secretory cells decreased, and the specific extracellular structure began to disappear. Twenty-four hours after the beginning of rehydration, the cellular structure and ultrastructure were comparable to those observed in hydrated tardigrades.

NF-kappaB-dependent expression of the antiapoptotic factor c-FLIP is regulated by calpain 3, the protein involved in limb-girdle muscular dystrophy type 2A.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is a recessive genetic disorder caused by mutations in the cysteine protease calpain 3 (CAPN3) that leads to selective muscle wasting. We previously showed that CAPN3 deficiency is associated with a profound perturbation of the NF-kappaB/IkappaB alpha survival pathway. In this study, the consequences of altered NF-kappaB/IkappaB alpha pathway were investigated using biological materials from LGMD2A patients. We first show that the antiapoptotic factor cellular-FLICE inhibitory protein (c-FLIP), which is dependent on the NF-kappaB pathway in normal muscle cells, is down-regulated in LGMD2A biopsies. In muscle cells isolated from LGMD2A patients, NF-kappaB is readily activated on cytokine induction as shown by an increase in its DNA binding activity. However, we observed discrepant transcriptional responses depending on the NF-kappaB target genes. IkappaB alpha is expressed following NF-kappaB activation independent of the CAPN3 status, whereas expression of c-FLIP is obtained only when CAPN3 is present. These data lead us to postulate that CAPN3 intervenes in the regulation of the expression of NF-kappaB-dependent survival genes to prevent apoptosis in skeletal muscle. Deregulations in the NF-kappaB pathway could be part of the mechanism responsible for the muscle wasting resulting from CAPN3 deficiency.

Connecting mitochondrial dynamics and life-or-death events via Bcl-2 family proteins.

The morphology of a population of mitochondria is the result of several interacting dynamical phenomena, including fission, fusion, movement, elimination and biogenesis. Each of these phenomena is controlled by underlying molecular machinery, and when defective can cause disease. New understanding of the relationships between form and function of mitochondria in health and disease is beginning to be unraveled on several fronts. Studies in mammals and model organisms have revealed that mitochondrial morphology, dynamics and function appear to be subject to regulation by the same proteins that regulate apoptotic cell death. One protein family that influences mitochondrial dynamics in both healthy and dying cells is the Bcl-2 protein family. Connecting mitochondrial dynamics with life-death pathway forks may arise from the intersection of Bcl-2 family proteins with the proteins and lipids that determine mitochondrial shape and function. Bcl-2 family proteins also have multifaceted influences on cells and mitochondria, including calcium handling, autophagy and energetics, as well as the subcellular localization of mitochondrial organelles to neuronal synapses. The remarkable range of physical or functional interactions by Bcl-2 family proteins is challenging to assimilate into a cohesive understanding. Most of their effects may be distinct from their direct roles in apoptotic cell death and are particularly apparent in the nervous system. Dual roles in mitochondrial dynamics and cell death extend beyond BCL-2 family proteins. In this review, we discuss many processes that govern mitochondrial structure and function in health and disease, and how Bcl-2 family proteins integrate into some of these processes.

Enhancer of zeste acts as a major developmental regulator of Ciona intestinalis embryogenesis.

The paradigm of developmental regulation by Polycomb group (PcG) proteins posits that they maintain silencing outside the spatial expression domains of their target genes, particularly of Hox genes, starting from mid embryogenesis. The Enhancer of zeste [E(z)] PcG protein is the catalytic subunit of the PRC2 complex, which silences its targets via deposition of the H3K27me3 mark. Here, we studied the ascidian Ciona intestinalis counterpart of E(z). Ci-E(z) is detected by immunohistochemistry as soon as the 2- and 4-cell stages as a cytoplasmic form and becomes exclusively nuclear thereafter, whereas the H3K27me3 mark is detected starting from the gastrula stage and later. Morpholino invalidation of Ci-E(z) leads to the total disappearance of both Ci-E(z) protein and its H3K27me3 mark. Ci-E(z) morphants display a severe phenotype. Strikingly, the earliest defects occur at the 4-cell stage with the dysregulation of cell positioning and mitotic impairment. At later stages, Ci-E(z)-deficient embryos are affected by terminal differentiation defects of neural, epidermal and muscle tissues, by the failure to form a notochord and by the absence of caudal nerve. These major phenotypic defects are specifically rescued by injection of a morpholino-resistant Ci-E(z) mRNA, which restores expression of Ci-E(z) protein and re-deposition of the H3K27me3 mark. As observed by qPCR analyses, Ci-E(z) invalidation leads to the early derepression of tissue-specific developmental genes, whereas late-acting developmental genes are generally down-regulated. Altogether, our results suggest that Ci-E(z) plays a major role during embryonic development in Ciona intestinalis by silencing early-acting developmental genes in a Hox-independent manner.

The non-proliferative nature of ascidian folliculogenesis as a model of highly ordered cellular topology distinct from proliferative epithelia.

Previous studies have addressed why and how mono-stratified epithelia adopt a polygonal topology. One major additional, and yet unanswered question is how the frequency of different cell shapes is achieved and whether the same distribution applies between non-proliferative and proliferative epithelia. We compared different proliferative and non-proliferative epithelia from a range of organisms as well as Drosophila melanogaster mutants, deficient for apoptosis or hyperproliferative. We show that the distribution of cell shapes in non-proliferative epithelia (follicular cells of five species of tunicates) is distinctly, and more stringently organized than proliferative ones (cultured epithelial cells and Drosophila melanogaster imaginal discs). The discrepancy is not supported by geometrical constraints (spherical versus flat monolayers), number of cells, or apoptosis events. We have developed a theoretical model of epithelial morphogenesis, based on the physics of divided media, that takes into account biological parameters such as cell-cell contact adhesions and tensions, cell and tissue growth, and which reproduces the effects of proliferation by increasing the topological heterogeneity observed experimentally. We therefore present a model for the morphogenesis of epithelia where, in a proliferative context, an extended distribution of cell shapes (range of 4 to 10 neighbors per cell) contrasts with the narrower range of 5-7 neighbors per cell that characterizes non proliferative epithelia.

Applying ecological and evolutionary theory to cancer: a long and winding road.

Since the mid 1970s, cancer has been described as a process of Darwinian evolution, with somatic cellular selection and evolution being the fundamental processes leading to malignancy and its many manifestations (neoangiogenesis, evasion of the immune system, metastasis, and resistance to therapies). Historically, little attention has been placed on applications of evolutionary biology to understanding and controlling neoplastic progression and to prevent therapeutic failures. This is now beginning to change, and there is a growing international interest in the interface between cancer and evolutionary biology. The objective of this introduction is first to describe the basic ideas and concepts linking evolutionary biology to cancer. We then present four major fronts where the evolutionary perspective is most developed, namely laboratory and clinical models, mathematical models, databases, and techniques and assays. Finally, we discuss several of the most promising challenges and future prospects in this interdisciplinary research direction in the war against cancer.