Characterization of Ambra1 in asexual cycle of a non-vertebrate chordate, the colonial tunicate Botryllus schlosseri, and phylogenetic analysis of the protein group in Bilateria☆
Graphical abstract
Introduction
Autophagy is a key cellular process, used to maintain homeostasis by clearing unnecessary or injured cell components through lysosomal activity. As autophagy is involved in normal physiology as well as in embryo development, cell differentiation, and survival during starvation, the process is tightly regulated (Di Bartolomeo et al., 2010, Mizushima and Komatsu, 2011) and its failure is linked to several pathophysiological processes, including cancer, myopathies and neurodegenerative disorders (Ravikumar et al., 2010). Due to its essential role in cell survival and viability, the autophagic process is conserved throughout the eukaryotes (King, 2012). In particular, the molecular mechanism of this process has been preserved during evolution, as demonstrated by expression in different organisms of a basic set of key AuTophaGy-related genes (ATGs). These genes were initially identified in yeast (Klionsky et al., 2003), and then some of their orthologs were also found in eukaryotic organisms including insects, nematodes, mammals and plants (Levine and Klionsky, 2004). However, some differences in the autophagic machinery exist between yeast and metazoans (King, 2012, Levine and Klionsky, 2004). In mammals, the Beclin1/Vps34 complex is regulated by additional key proteins such as UVRAG, Ambra1 and Bif-1, which are not present in yeast and Dictyostelium (Calvo-Garrido et al., 2010). Among the latter genes, the activating molecule in Beclin 1-regulated autophagy (Ambra1) was identified only few years ago in mice (Fimia et al., 2007). This protein is a central positive regulator of the autophagic process thanks to its ability to bind Beclin 1 upon autophagic stimuli, thus promoting the interaction between Beclin 1 and its target kinase Vps34 (Di Bartolomeo et al., 2010, He and Levine, 2010). Since its initial characterization in mammals, Ambra1 was found to be involved not only in autophagy but also in other key processes such as apoptosis, cell proliferation and nervous system development (Fimia et al., 2007). The surprisingly varied roles of this large protein is likely derived from its ability to bind a great number of other regulatory partners (such as Beclin1, Bcl-2, DLC1, PP2A, E3 ubiquitin ligases Cullin-5 and Cullin-4), depending on the specific process under examination (Antonioli et al., 2014, Cianfanelli et al., 2015a, Cianfanelli et al., 2015b, Fimia et al., 2013). Ambra1 genes have been cloned and characterized in human, mouse and zebrafish (Benato et al., 2013, Fimia et al., 2007, Skobo et al., 2014). Due to genome duplication during teleost evolution, two paralogous genes were found in zebrafish; both are required for correct embryonic development (Benato et al., 2013, Skobo et al., 2014).
In the tunicate Ciona robusta, formerly Ciona intestinalis type A (see Brunetti et al., 2015, Pennati et al., 2015), a genome search identified the Ambra1 gene, together with other genes involved in the autophagic process (Godefroy et al., 2009). These genes were compared to their homologues in S. cerevisiae, C. elegans, D. melanogaster and H. sapiens (Godefroy et al., 2009). This analysis showed that all the non-yeast genes involved in autophagy are present in the genome of C. robusta, therefore possessing an autophagic machinery comparable to that of vertebrate. Tunicates are marine invertebrates, considered as the sister group of vertebrates (Delsuc et al., 2006). Due to their phylogenetic position, these animals are potentially strategic to the study of the evolution of autophagic proteins in vertebrates. Ascidians, the main group in the subphylum Tunicata, are characterized by a bi-phasic cycle: a swimming tadpole larva derived from a fertilized egg, which metamorphoses into a sessile individual. The larva possesses some of the specific features of the chordate body plan: a dorsal tubular nerve cord, the notochord and a muscular tail. However, the larva loses this organization at metamorphosis, becoming a sessile filter-feeding zooid, with a fissured pharynx allowing respiration and feeding. Ascidians can be solitary or colonial and the colonial species reproduce either sexually or asexually. In the colonial species the oozooid derived from larval metamorphosis begins a lifelong, recurring asexual reproduction (called also blastogenesis or budding), from which eventually genetically identical individuals, the blastozooids (zooids derived from blastogenesis), differentiate.
Among colonial ascidians, B. schlosseri is a model species: it is cosmopolitan, its genome is available, it is easily reared in laboratory, its biological cycle is well characterized and a recognized staging method is available for asexual reproduction (Fig. 1) (Manni et al., 2014, Voskoboynik et al., 2013). In B. schlosseri, each adult blastozooid has one or more buds that bear young budlets. In laboratory conditions, budding follows a weekly-synchronized cycle, marked by the regression and absorption of the filtering adults. In this process, called take-over, the adult zooids are replaced by their buds, which develop into mature zooids; budlets become buds and produce a new generation of budlets. After several blastogenetic cycles, the colony organizes in star-shaped systems of a dozen of clonal blastozooids, arranged around a common cloacal, excurrent siphon. The whole colony is embedded in the tunic, where all blastozooids are linked by a complex vascular system (Gasparini et al., 2015). During the regression phase, adult organs undergo shrinkage and collapse, circulating phagocytes massively infiltrate senescent tissues and rapidly ingest apoptotic cells (Burighel and Schiavinato, 1984). Within 24–36 h, zooids are completely resorbed (Ballarin et al., 2008, Cima et al., 2010). Since a colony is virtually immortal, many cyclical apoptotic events occur during its life span: for this reason, B. schlosseri is considered a useful model to study programmed cell death. In the present study, we describe the molecular cloning and characterization of the complete Ambra1 transcript in the non-vertebrate species B. schlosseri and compare its deduced amino acid sequence with those from C. robusta and mammals, also reporting a comprehensive analysis of the phylogenetic relationship of orthologous genes in bilaterians. Finally, we demonstrated, by qPCR analysis, that the Ambra1 gene in B. schlosseri is expressed at different levels during the ontogenetic phases.
Section snippets
Animals sampling
Colonies of B. schlosseri (Styelidae, Stolidobranchia) were collected in the Lagoon of Venice and then cultured in the laboratory in natural sea water (apart from starvation experiment, see below) according to Sabbadin’s technique (Sabbadin, 1955, Sabbadin, 1960) and fed with Liquifry Marine (Interpet, 0308).
For qPCR experiments related to quantification of gene expression during blastogenesis, four colony stages were utilized (Fig. 1D); staging was determined in vivo following the recently
Isolation and characterization of the B. schlosseri Ambra1 cDNA and its deduced protein
Tblastn searches for B. schlosseri Ambra1 cDNA sequence were initially performed in a EST collection database of B. schlosseri colonies no longer available, that was kindly provided by AW De Tomaso (University of California, Santa Barbara, USA) and that has been subsequently used to produce the assemblies retrievable at http://octopus.obs-vlfr.fr/blast/botrylle/blast_botryllus.php. These preliminary searches were performed using, as query, the Ambra1 amino acid sequence of different species: C.
Ambra1 gene identification and characterization in B. schlosseri
In this study, we report the identification, through genome bioinformatic searches and targeted cloning, of the complete Ambra1 transcript in a non-vertebrate species, the tunicate B. schlosseri. Before this study, expression of Ambra1, together with expression of other genes involved in the mammalian autophagic process but not present in yeast, was demonstrated only in the tunicate C. robusta (Godefroy et al., 2009), formerly Ciona intestinalis type A (see Brunetti et al., 2015, Pennati et
Conclusions
In this study we cloned and characterized Ambra1 cDNA in the tunicate B. schlosseri. Ambra1 expression during the asexual life cycle of this colonial tunicate results higher in early mid-cycle stage with respect to the take-over. The differential expression of Ambra1 during the blastogenetic cycle suggests a possible role in the colonial asexual development, role that needs to be analyzed by means of functional studies. Nevertheless molecular phylogeny demonstrates that Ambra1: (i) is
Acknowledgments
This work was financially supported by University of Padova – Italy (RFO-ex 60%) to LDV, by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca – Italy) PRIN 2009 Project to LM (Grant number 2009XF7TYT) and by University of Padova Senior postdoc 2012 Project to FG (Grant number GRIC120LSZ). The authors would like to thank Sara Formaggio for helping with the acquisition of data and Sebastian M. Shimeld for useful comments and suggestions. We also thank: Anthony W. De Tomaso for
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2020, Developmental and Comparative ImmunologyCitation Excerpt :The sequences of BsC3 transcript and protein were previously described (Franchi and Ballarin, 2014) and registered in GenBank under the ID KR996751.1 and ALM04210.1, respectively. Primers for PCR amplifications, designed on the sequence of BsC3 and B. schlosseri elongation factor (BsEF; Gasparini et al., 2016), are listed in Table 1. Total RNA was isolated from B. schlosseri whole colonies with the SV Total RNA Isolation System (Promega) and its quality was determined by the A260/280 ratio.
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2019, Developmental BiologyCitation Excerpt :The anatomical and developmental ontology of B. schlosseri asexual development is now available, allowing the use of a controlled and shared vocabulary among different laboratories (Manni et al., 2014). The hypothesis of the close evolutionary relationship between tunicates and vertebrates (Delsuc et al., 2018; Kocot et al., 2018; Giribet, 2018) is also supported by analysis of hundreds of nuclear genes from 15 species, including the colonial tunicate B. schlosseri (Voskoboynik et al., 2013a) and outcomes of single gene/structure/pathway studies on B. schlosseri asexual reproduction (Degasperi et al., 2009; Gasparini et al., 2013, 2016). The genomes of B. schlosseri and B. leachii have been published (Voskoboynik et al., 2013a; Blanchoud et al., 2018), and transcriptomes covering different reproductive/regenerative traits are available (Voskoboynik et al., 2013a, 2013b; Campagna et al., 2016; Corey et al., 2016; Kowarsky et al., 2017; Rosental et al., 2018; Ricci et al., 2016b).
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2017, General and Comparative EndocrinologyCitation Excerpt :Noteworthy, muscle defects were rescued after co-injection of human AMBRA1 mRNA, pointing out the conservation of Ambra1 functions through evolutionary times (Skobo et al., 2014). Moreover, the cloning and characterization of Ambra1 in the tunicate Botryllus schlosseri has recently demonstrated that Ambra1 is an ancient gene, having evolved distinctly at least before the radiation of Bilateria (Gasparini et al., 2016). Autophagy defects lead to various neurodegenerative and lysosomal-related diseases, and to oncogenesis and cancer progression (Mizushima and Komatsu, 2011).
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This paper was edited by the Associate Editor Bernd Schierwater.
- 1
Present address: CUTECH Srl, Via San Marco, 9/M – I-35129 Padova, Italy.
- 2
These authors contributed equally to this work.
- 3
These authors contributed equally to this work.