Extrinsic apoptosis participates to tail regression during the metamorphosis of the chordate Ciona.

Apoptosis is a regulated cell death ubiquitous in animals defined by morphological features depending on caspases. Two regulation pathways are described, currently named the intrinsic and the extrinsic apoptosis. While intrinsic apoptosis is well studied and considered ancestral among metazoans, extrinsic apoptosis is poorly studied outside mammals. Here, we address extrinsic apoptosis in the urochordates Ciona, belonging to the sister group of vertebrates. During metamorphosis, Ciona larvae undergo a tail regression depending on tissue contraction, migration and apoptosis. Apoptosis begin at the tail tip and propagates towards the trunk as a polarized wave. We identified Ci-caspase 8/10 by phylogenetic analysis as homolog to vertebrate caspases 8 and 10 that are the specific initiator of extrinsic apoptosis. We detected Ci-caspase 8/10 expression in Ciona larvae, especially at the tail tip. We showed that chemical inhibition of Ci-caspase 8/10 leads to a delay of tail regression, and Ci-caspase 8/10 loss of function induced an incomplete tail regression. The specificity between apoptotic pathways and initiator caspase suggests that extrinsic apoptosis regulates cell death during the tail regression. Our study presents rare in vivo work on extrinsic apoptosis outside mammals, and contribute to the discussion on its evolutionary history in animals.

Intrinsic apoptosis is evolutionarily divergent among metazoans.

Apoptosis is regulated cell death that depends on caspases. A specific initiator caspase is involved upstream of each apoptotic signaling pathway. Characterized in nematode, fly, and mammals, intrinsic apoptosis is considered to be ancestral, conserved among animals, and depends on shared initiators: caspase-9, Apaf-1 and Bcl-2. However, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspase activation remain highly heterogeneous and hide profound molecular divergence among apoptotic pathways in animals. Uncovering the phylogenetic history of apoptotic actors, especially caspases, is crucial to shed light on the evolutionary history of intrinsic apoptosis. Here, we demonstrate with phylogenetic analyses that caspase-9, the fundamental key of intrinsic apoptosis, is deuterostome-specific, while caspase-2 is ancestral to bilaterians. Our analysis of Bcl-2 and Apaf-1 confirms heterogeneity in functional organization of apoptotic pathways in animals. Our results support emergence of distinct intrinsic apoptotic pathways during metazoan evolution.

Senescence-induced cellular reprogramming drives cnidarian whole-body regeneration.

Cell fate stability is essential to maintaining "law and order" in complex animals. However, high stability comes at the cost of reduced plasticity and, by extension, poor regenerative ability. This evolutionary trade-off has resulted in most modern animals being rather simple and regenerative or complex and non-regenerative. The mechanisms mediating cellular plasticity and allowing for regeneration remain unknown. We show that signals emitted by senescent cells can destabilize the differentiated state of neighboring somatic cells, reprogramming them into stem cells that are capable of driving whole-body regeneration in the cnidarian Hydractinia symbiolongicarpus. Pharmacological or genetic inhibition of senescence prevents reprogramming and regeneration. Conversely, induction of transient ectopic senescence in a regenerative context results in supernumerary stem cells and faster regeneration. We propose that senescence signaling is an ancient mechanism mediating cellular plasticity. Understanding the senescence environment that promotes cellular reprogramming could provide an avenue to enhance regeneration.

Pluripotent, germ cell competent adult stem cells underlie cnidarian regenerative ability and clonal growth.

In most animals, pluripotency is irreversibly lost post gastrulation. By this stage, all embryonic cells have already committed either to one of the somatic lineages (ectoderm, endoderm, or mesoderm) or to the germline. The lack of pluripotent cells in adult life may be linked to organismal aging. Cnidarians (corals and jellyfish) are an early branch of animals that do not succumb to age, but the developmental potential of their adult stem cells remains unclear. Here, we show that adult stem cells in the cnidarian Hydractinia symbiolongicarpus (known as i-cells) are pluripotent. We transplanted single i-cells from transgenic fluorescent donors to wild-type recipients and followed them in vivo in the translucent animals. Single engrafted i-cells self-renewed and contributed to all somatic lineages and gamete production, co-existing with and eventually displacing the allogeneic recipient's cells. Hence, a fully functional, sexually competent individual can derive from a single adult i-cell. Pluripotent i-cells enable regenerative, plant-like clonal growth in these animals.

d-Serine controls epidermal vesicle release via NMDA receptor, allowing tissue migration during the metamorphosis of the chordate Ciona.

d-Serine, a free amino acid synthesized by serine racemase, is a coagonist of N-methyl-d-aspartate-type glutamate receptor (NMDAR). d-Serine in the mammalian central nervous system modulates glutamatergic transmission. Functions of d-serine in mammalian peripheral tissues such as skin have also been described. However, d-serine's functions in nonmammals are unclear. Here, we characterized d-serine-dependent vesicle release from the epidermis during metamorphosis of the tunicate Ciona. d-Serine leads to the formation of a pocket that facilitates the arrival of migrating tissue during tail regression. NMDAR is the receptor of d-serine in the formation of the epidermal pocket. The epidermal pocket is formed by the release of epidermal vesicles' content mediated by d-serine/NMDAR. This mechanism is similar to observations of keratinocyte vesicle exocytosis in mammalian skin. Our findings provide a better understanding of the maintenance of epidermal homeostasis in animals and contribute to further evolutionary perspectives of d-amino acid function among metazoans.

Comparative transcriptomic analysis reveals gene regulation mediated by caspase activity in a chordate organism.

BACKGROUND: Apoptosis is a caspase regulated cell death present in all metazoans defined by a conserved set of morphological features. A well-described function of apoptosis is the removal of excessive cells during development and homeostasis. Recent studies have shown an unexpected signalling property of apoptotic cells, affecting cell fate and/or behaviour of neighbouring cells. In contrast to the apoptotic function of cell elimination, this new role of apoptosis is not well understood but seems caspase-dependent. To deepen our understanding of apoptotic functions, it is necessary to work on a biological model with a predictable apoptosis pattern affecting cell fate and/or behaviour. The tunicate Ciona intestinalis has a bi-phasic life cycle with swimming larvae which undergo metamorphosis after settlement. Previously, we have shown that the tail regression step during metamorphosis, characterized by a predictable polarized apoptotic wave, ensures elimination of most tail cells and controls primordial germ cells survival and migration. RESULTS: We performed differential transcriptomic analysis between control metamorphosing larvae and larvae treated with the pan-caspase inhibitor Z-VAD-fmk in order to explore the transcriptional control of apoptotic cells on neighbouring cells that survive and migrate. When caspase activity was impaired, genes known to be involved in metamorphosis were downregulated along with other implicated in cell migration and survival molecular pathways. CONCLUSION: We propose these results as a confirmation that apoptotic cells can control surrounding cells fate and as a reference database to explore novel apoptotic functions in animals, including those related to migration and differentiation.

Apoptosis and cell proliferation during metamorphosis of the planula larva of Clytia hemisphaerica (Hydrozoa, Cnidaria).

BACKGROUND: Metamorphosis in marine species is characterized by profound changes at the ecophysiological, morphological, and cellular levels. The cnidarian Clytia hemisphaerica exhibits a triphasic life cycle that includes a planula larva, a colonial polyp, and a sexually reproductive medusa. Most studies so far have focused on the embryogenesis of this species, whereas its metamorphosis has been only partially studied. RESULTS: We investigated the main morphological changes of the planula larva of Clytia during the metamorphosis, and the associated cell proliferation and apoptosis. Based on our observations of planulae at successive times following artificial metamorphosis induction using GLWamide, we subdivided the Clytia's metamorphosis into a series of eight morphological stages occurring during a pre-settlement phase (from metamorphosis induction to planula ready for settlement) and the post-settlement phase (from planula settlement to primary polyp). Drastic morphological changes prior to definitive adhesion to the substrate were accompanied by specific patterns of stem-cell proliferation as well as apoptosis in both ectoderm and endoderm. Further waves of apoptosis occurring once the larva has settled were associated with morphogenesis of the primary polyp. CONCLUSION: Clytia larval metamorphosis is characterized by distinct patterns of apoptosis and cell proliferation during the pre-settlement phase and the settled planula-to-polyp transformation.