Testing an unusual in vivo vessel network model: a method to study angiogenesis in the colonial tunicate Botryllus schlosseri

Abstract

Tunicates are the closest relatives to vertebrates and include the only chordate species able to reproduce both sexually and asexually. The colonial tunicate Botryllus schlosseri is embedded in a transparent extracellular matrix (the tunic) containing the colonial circulatory system (CCS). The latter is a network of vessels external to zooids, limited by a simple, flat epithelium that originated from the epidermis. The CCS propagates and regenerates by remodelling and extending the vessel network through the mechanism of sprouting, which typically characterises vertebrate angiogenesis. In exploiting the characteristics of B. schlosseri as a laboratory model, we present a new experimental and analysis method based on the ability to obtain genetically identical subclones representing paired samples for the appropriate quantitative outcome statistical analysis. The method, tested using human VEGF and EGF to induce angiogenesis, shows that the CCS provides a useful in vivo vessel network model for testing the effects of specific injected solutes on vessel dynamics. These results show the potentiality of B. schlosseri CCS as an effective complementary model for in vivo studies on angiogenesis and anticancer therapy. We discuss this potentiality, taking into consideration the origin, nature, and roles of the cellular and molecular agents involved in CCS growth.

Whole-Body Regeneration in the Colonial Tunicate Botrylloides leachii

“…1; Mukai et al 1978). Originating from the ectoderm, this vasculature has been shown to resemble that of vertebrates, and even respond to human factors that stimulate angiogenesis such as vascular endothelial growth factor (Gasparini et al 2008(Gasparini et al , 2014Tiozzo et al 2008b). B. leachii blood (haemolymph) is mostly colourless and composed of nine cell types with functions incipient to that of vertebrate blood cells, which can be classified into five cell lineages (Cima et al 2002;Blanchoud et al 2017): undifferentiated cells (haemoblasts), immunocytes (hyaline amebocytes, macrophage-like cells, granular amebocytes, morula cells), mast cell-like cells (granular cells), transport cells (compartment amebocytes, compartment cells) and storage cells (pigment cells, nephrocytes).…”

“…Thus, the study of these animals may offer a unique evolutionary perspective into a wide range of biological processes conserved within the phylum Chordata. Colonial tunicates are also established models for other important biological processes including allorecognition, chimerism, ageing and angiogenesis (Rinkevich and Weissman 1987;Saito et al 1994;Paz and Rinkevich 2002;Kürn et al 2011;Gasparini et al 2014;Voskoboynik and Weissman 2015;Kassmer et al 2016). Of particular interest, these modular organisms are the only known chordates that can undergo whole-body regeneration (WBR), whereby a fully functional adult is restored from a minute portion of their vascular system containing as little as 100-200 circulating blood cells in a short period of about 10 days (Rinkevich et al 1995(Rinkevich et al , 2007bVoskoboynik et al 2007;Brown et al 2009).…”

Transcriptome dynamics in the asexual cycle of the chordate Botryllus schlosseri

“…It reproduces both sexually and asexually, and is now considered a reference chordate for the study of asexual reproduction [ 4 , 5 ]. It is also considered a good model to analyze: i) the relationship between embryogenesis and blastogenesis, two developmental pathways producing similar individuals (e.g., oozooids and blastozooids) through different reproductive processes (starting from germ cells or somatic stem cells [ 6 ]); ii) germ cell recycling and somatic chimeras (reviewed in [ 7 , 8 ], see also [ 9 ]) and somatic cell clearance and turnover [ 10 ]; iii) natural apoptosis occurring cyclically in the colony [ 11 , 12 ] and the mechanisms underlying aging related to tissue regenerative potential and stem cell functionality [ 13 , 14 ]; iv) the allorecognition phenomenon and its molecular basis [ 15 ]; v) the strategies of immune defense [ 15 – 17 ]; vi) the potentiality of the colonial circulatory system as a model for in vivo studies of angiogenesis [ 18 , 19 ]. A draft of the B. schlosseri genome has been recently released [ 4 ] and its ontology of anatomy and development defined [ 5 ].…”

Sexual and asexual development: two distinct programs producing the same tunicate

“…Colonial tunicates are a key to solving this mystery. These marine organisms are unique among chordates: they can produce their adult body through two pathways ( Figures 1A , 1B , 2A , and S1 ), and they possess whole body regenerative capabilities ( Alié et al, 2020 ; Gasparini et al, 2014 ; Manni et al, 2019 ; Voskoboynik et al, 2007 ). A colony of the tunicate Botryllus schlosseri contains many individuals (zooids), derived by asexual reproduction from a single metamorphosed larva.…”

“…Blastogenesis differs in origin but similarly establishes body axis and organ formation ( Figure 2A ). The expression pattern of very few genes was comparatively studied in both embryogenesis and blastogenesis ( Gasparini et al, 2011 , 2014 ; Tiozzo et al, 2005 ). Therefore, we investigated comprehensively whether the convergent morphology of oozooids and blastozooids implies convergent molecular mechanisms, how organogenesis compares between sexual and asexual processes, and how the stem cells mediating these processes vary.…”