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

https://doi.org/10.1016/j.ympev.2015.11.001Get rights and content

Highlights

  • We characterized the first ambra1 transcript in an invertebrate chordate: B. schlosseri.

  • Its expression has been quantified all along the asexual life cycle of B. schlosseri.

  • We identified Wdr5 and Katnb1 as proteins evolutionarily related to Ambra1.

  • We resolved that ambra1 evolved at least before the radiation of Bilateria.

  • Ancient duplications of WD40 repeats are related to a N-terminal domain.

Abstract

Ambra1 is a positive regulator of autophagy, a lysosome-mediated degradative process involved both in physiological and pathological conditions. Nowadays, Ambra1 has been characterized only in mammals and zebrafish. Through bioinformatics searches and targeted cloning, we report the identification of the complete Ambra1 transcript in a non-vertebrate chordate, the tunicate Botryllus schlosseri. Tunicata is the sister group of Vertebrata and the only chordate group possessing species that reproduce also by blastogenesis (asexual reproduction). B. schlosseri Ambra1 deduced amino acid sequence is shorter than vertebrate homologues but still contains the typical WD40 domain. qPCR analyses revealed that the level of B. schlosseri Ambra1 transcription is temporally regulated along the colonial blastogenetic cycle. By means of similarity searches we identified Wdr5 and Katnb1 as proteins evolutionarily associated to Ambra1. Phylogenetic analyses on Bilateria indicate that: (i) Wdr5 is the most related to Ambra1, so that they may derive from an ancestral gene, (ii) Ambra1 forms a group of ancient genes evolved before the radiation of the taxon, (iii) these orthologous Ambra1 share the two conserved WD40/YVTN repeat-like-containing domains, and (iv) they are characterized by ancient duplications of WD40 repeats within the N-terminal domain.

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

References (69)

  • C.U. Stirnimann et al.

    WD40 proteins propel cellular networks

    Trends Biochem. Sci.

    (2010)
  • B. Xue et al.

    PONDR-FIT: a meta-predictor of intrinsically disordered amino acids

    Biochim. Biophys. Acta

    (2010)
  • S.F. Altschul et al.

    Protein database searches using compositionally adjusted substitution matrices

    FEBS J.

    (2005)
  • D. Ando et al.

    Physical motif clustering within intrinsically disordered nucleoporin sequences reveals universal functional features

    PLoS ONE

    (2013)
  • L. Ballarin et al.

    Haemocytes and blastogenetic cycle in the colonial ascidian Botryllus schlosseri: a matter of life and death

    Cell Tissue Res.

    (2008)
  • A. Bateman et al.

    UniProt: a hub for protein information

    Nucleic Acids Res.

    (2015)
  • F. Benato et al.

    Ambra1 knockdown in zebrafish leads to incomplete development due to severe defects in organogenesis

    Autophagy

    (2013)
  • J. Bergsten

    A review of long-branch attraction

    Cladistics

    (2005)
  • R. Brunetti et al.

    Morphological evidence that the molecularly determined Ciona intestinalis type A and type B are different species: Ciona robusta and Ciona intestinalis

    J. Zool. Syst. Evol. Res.

    (2015)
  • P. Burighel et al.

    Neurogenic role of the neural gland in the development of the ascidian, Botryllus schlosseri (Tunicata, Urochordata)

    J. Comp. Neurol.

    (1998)
  • P. Burighel et al.

    Fine structure of the gastric epithelium of the ascidian Botryllus schlosseri. Vacuolated and zymogenic cells

    Z. Zellforsch. Mikrosk. Anat.

    (1973)
  • P. Burighel et al.

    Degenerative regression of the digestive tract in the colonial ascidian Botryllus schlosseri (Pallas)

    Cell Tissue Res.

    (1984)
  • J. Calvo-Garrido et al.

    Autophagy in Dictyostelium: Genes and pathways, cell death and infection

    Autophagy

    (2010)
  • C. Canestro et al.

    Consequences of lineage-specific gene loss on functional evolution of surviving paralogs: ALDH1A and retinoic acid signaling in vertebrate genomes

    PLoS Genet.

    (2009)
  • X. Chen et al.

    Upregulated WDR5 promotes proliferation, self-renewal and chemoresistance in bladder cancer via mediating H3K4 trimethylation

    Sci. Rep-Uk

    (2015)
  • V. Cianfanelli et al.

    AMBRA1 links autophagy to cell proliferation and tumorigenesis by promoting c-Myc dephosphorylation and degradation

    Nat. Cell Biol.

    (2015)
  • V. Cianfanelli et al.

    Connecting autophagy: AMBRA1 and its network of regulation

    Mol. Cell. Oncol.

    (2015)
  • D. Darriba et al.

    ProtTest 3: fast selection of best-fit models of protein evolution

    Bioinformatics

    (2011)
  • F. Delsuc et al.

    Tunicates and not cephalochordates are the closest living relatives of vertebrates

    Nature

    (2006)
  • S. Di Bartolomeo et al.

    The dynamic interaction of AMBRA1 with the dynein motor complex regulates mammalian autophagy

    J. Cell Biol.

    (2010)
  • C.W. Dunn et al.

    Broad phylogenomic sampling improves resolution of the animal tree of life

    Nature

    (2008)
  • G.M. Fimia et al.

    Ambra1 at the crossroad between autophagy and cell death

    Oncogene

    (2013)
  • G.M. Fimia et al.

    Ambra1 regulates autophagy and development of the nervous system

    Nature

    (2007)
  • F. Gasparini et al.

    Sexual and asexual reproduction in the colonial ascidian Botryllus schlosseri

    Genesis

    (2015)
  • Cited by (5)

    • Complement system and phagocytosis in a colonial protochordate

      2020, Developmental and Comparative Immunology
      Citation 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.

    • Sixty years of experimental studies on the blastogenesis of the colonial tunicate Botryllus schlosseri

      2019, Developmental Biology
      Citation 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).

    • The maternal control in the embryonic development of zebrafish

      2017, General and Comparative Endocrinology
      Citation 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).

    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.

    View full text