Available online at www.sciencedirect.com
ScienceDirect
Colonial ascidians as model organisms for the study of
germ cells, fertility, whole body regeneration, vascular
biology and aging
Susannah H Kassmer, Delany Rodriguez and
Anthony W De Tomaso
Colonial ascidians are marine invertebrate chordates that are and vertebrates. The larval stage of the colonial ascidian
the closest invertebrate relative to the vertebrates. Colonies of B. schlosseri shows characteristic chordate features, includ-
Botryllus schlosseri undergo a continuous asexual ing a notochord, dorsal hollow nerve tube, post-anal tail,
reproduction process that involves the regeneration of entire striated musculature and pharynx with gill slits. During
new bodies, which include all somatic and germline tissues. metamorphosis into the sessile adult, these features are
This adult regenerative process depends on signaling lost. B. schlosseri colonies consist of independent, filter
pathways known to regulate the formation of tissues and feeding individuals (known as zooids) that each contain
organs during embryonic development. The formation of their own incurrent and excurrent siphons, heart, gastroin-
gonads within regenerated bodies depends on migration and testinal tract, nervous system, musculature and germline.
homing of germ cell precursors to niches within the developing Individual zooids within the colony are interconnected by
bodies, and Botryllus colonies can cycle between fertile and a shared, extracorporeal vasculature, and are embedded in
infertile states. The vasculature that connects all individuals an extracellular matrix known as the tunic (Figure 1a). The
within the colony is highly regenerative, and is a valuable tool vascular system of Botryllus consists of two major parts: an
for the study of angiogenesis in adult blood vessels. The internal plot of sinuses and lacunae that surround the major
tremendous regenerative capacity of the vasculature even organs of individual zooids, and a shared extracorporeal
results in regeneration of entire new bodies solely from vasculature consisting of ramified vessels. These vessels
fragments of blood vessels upon surgical removal of all bodies. connect the zooids to a common, extracorporeal blood
The mechanism underlying this regeneration of whole bodies is supply, and form terminal protrusions called ampullae
not well understood, but appears to depend on proliferation of (Figure 1a). Ampullae are involved in multiple processes,
circulating, blood borne cells. Because of all of these features, including adherence of the colony to the substrata.
colonial ascidians are ideal model organisms for the study of
germ cell migration, fertility, vascular biology and regeneration.
B. schlosseri colonies increase in size via a continuous
Address asexual reproduction process known as ‘budding’, during
Molecular Cellular and Developmental Biology, University of California which entire new bodies are regenerated, including all
Santa Barbara, Santa Barbara, CA 93106, USA somatic and germline tissues. The adult bodies are con-
nected to two generations of developing buds, termed
Corresponding author: De Tomaso, Anthony W
primary and secondary buds, which grow from the body
wall of the primary buds (Figure 1a). When the primary
buds complete their development, the adult zooids die
Current Opinion in Genetics & Development 2016, 39:101–106 and are resorbed, and the primary buds take their place,
This review comes from a themed issue on Developmental becoming the new adult zooids, in a process known as
mechanisms, patterning and evolution takeover. During takeover, the secondary buds become
Edited by Detlev Arendt and Cassandra Extavour the new primary buds, and begin forming the next gen-
eration of secondary buds, beginning as small protrusions
and eventually forming a closed vesicle. Through inva-
ginations and tissue differentiation, the buds complete
http://dx.doi.org/10.1016/j.gde.2016.06.001 their development into the adult form (Figure 1a). Take-
over is autonomous and the adult zooid only has a 7-day
0959-437/# 2016 Published by Elsevier Ltd.
lifespan, thus regeneration of one bud/zooid is required
just to maintain colony size, and growth occurs when
multiple (up to 4) buds develop per generation.
The source of the germ line in each asexually derived
Introduction: the basic biology of the colonial generation is a population of mobile germ cell precursors,
ascidian Botryllus schlosseri which migrate to new germline niches within the second-
Ascidians are marine invertebrate chordates that represent ary bud. These germ cell precursors then develop into
an evolutionary link between invertebrate deuterostomes functional gonads as the primary bud matures into an
www.sciencedirect.com Current Opinion in Genetics & Development 2016, 39:101–106
102 Developmental mechanisms, patterning and evolution
Figure 1
(a) (b) (c)
(d) (e) (f)
(g) (h) (i)
(j) (k) (l)
Current Opinion in Genetics & Development
Botryllus schlosseri as a model organism for the study of fertility, vascular biology and regeneration. (a) Brightfield image showing the morphology
of a Botryllus colony. Individual zooids (z) and their developing buds (b) are interconnected by a common vasculature that forms peripheral
protrusions termed ampulle (a). (d) Infertile colony without gonads. (g) Fertile colony with gonads. Arrows indicate maturing oocytes and
arrowheads indicate mature testes. (j) Migration of germ cell progenitors from primary bud to secondary bud (red arrow). Progenitors = light blue,
maturing oocyte = pink and maturing testes = dark blue. (b, e, h, and k) Vessel regeneration. Black dashed circle on e = ampullaectomy. (h and k)
After surgical removal of ampullae, regeneration occurs within 24–48 hours. (c, f, i and l) Whole body regeneration. After surgical removal of
zooids and buds (black dashed circle in f), the vasculature undergoes extensive tissue rearrangement. Within 3–7 days after surgery, a new body
develops from the remaining vascular tissue (i). This new body then undergoes asexual reproduction to form a complete new colony
(l). Zooids = green. Blood vessels and ampullae = light blue. Scale bars indicate 1 mm for bright field images.
Current Opinion in Genetics & Development 2016, 39:101–106 www.sciencedirect.com
Colonial ascidians as model organisms for germ cells Kassmer, Rodriguez and De Tomaso 103
adult zooid (Figure 1g). Ascidians are hermaphrodites, Gonad differentiation is an essential function for all
and adult zooids can develop both testes and eggs sexually reproducing species, and many aspects of these
(Figure 1g). Therefore, sexual and asexual reproduction developmental processes are highly conserved among the
occur simultaneously within the same colony, and these metazoa. B. schlosseri offers a unique trait that can be
two processes are linked together in a precisely timed and utilized to characterize the genes underlying gonad de-
synchronized fashion [1 ]. velopment: colonies show variable fertility, and in the lab
cycle in and out of reproductive (fertile) and non-repro-
Germ cells and fertility ductive (infertile) states (Figure 1d). Rodriguez et al. used
In ascidians, the germline is segregated early in devel- this trait to generate a transcriptome database of genes
opment, and the specification of primordial germ cells regulating fertility in Botryllus [6 ] by analyzing the
(PGC) is due to maternally localized determinants. transcriptomes of both fertile and infertile colonies
Maternally deposited vasa mRNA segregates early in throughout blastogenesis. Genes that were identified as
development to a posterior lineage of cells, suggesting regulating fertility were expressed by follicle cells sur-
that germline formation is determinative in colonial rounding both testis and oocyte precursors as well as in
ascidians. In adults, vasa expression was observed in developing gonads. The current state of research on
the gonads, as well as in a population of mobile cells gonad development and fertility in Botryllus has been
scattered throughout the open circulatory system [2]. In recently reviewed elsewhere [1 ].
addition to vasa, one study identified ddx1, gamma
H2ax and cadherin as markers expressed in PGC-like Asexual reproduction and whole body
cells, but no double staining was performed to confirm regeneration
that the cells expressing these markers are indeed germ In most adult animals, regeneration is restricted to resto-
cells [3]. ration of specific tissues and organs in response to normal
growth, aging and injury. However, the potential to
Germline stem cells migrate to developing niches in the regenerate missing tissues varies greatly between species,
newly developing bodies during every single asexual and the cellular identity and molecular mechanisms that
reproductive cycle (Figure 1j). This coordinated and control sources of regeneration in many organisms and
synchronized migration at a defined time point during tissues are not well understood. Colonial ascidians are
the blastogenic cycle provides a unique platform to study ideal model organisms to study the principles of adult
the signaling mechanisms guiding germ cells to their regeneration, as two separate adult regenerative processes
niches. It is possible to characterize and measure this occur in these animals: asexual reproduction is a continu-
homing process in vivo in response to manipulation of ous and synchronized process that leads to regeneration of
signaling pathways with small molecule inhibitors [4 ] or new bodies in the process termed ‘budding’. In addition,
RNAi. It has recently become possible to prospectively B. schlosseri and other botryllid tunicates are the only
isolate germ cells from Botryllus, using Integrin-alpha-6 chordates able to regenerate entire new bodies solely
as a surface marker, and to assess their response to from fragments of vascular tissue (whole body regenera-
chemotactic agents in vitro, using transwell migration tion) (Figure 1c,f,i,l). The ascidian embryo follows a
assays [4 ]. This technology has enabled the identifica- determinative, or mosaic development, with cell fate
tion of sphingosine 1 phosphate as a major pathway specification determined by inheritance of cytoplasmic
directing germ cell chemotaxis in Botryllus. Tracking factors. The ability of the adult animals to regenerate
and measuring the dynamics of the migratory activity complete new bodies suggests that colonial ascidians
of fluorescently labeled germ cells in vivo is currently in possess several independent developmental programs
development. Single cell transcriptomics of Integrin- that can generate an identical adult body plan in
alpha-6-positive germ cells isolated at different stages completely different contexts.
of the blastogenic cycle will reveal subpopulations of
germ cells and associated marker genes, as well as path- To understand the molecular basis of this regenerative
ways regulating migration, differentiation and self re- ability, a recent study assessed the role of the canonical
newal of germline stem cells. One study found that cells Wnt pathway during the early stages of asexual budding.
within in the so-called ‘cell islands’ — which are aggre- Three Wnt ligands (Wnt2B, Wnt5A, and Wnt9A) are
gates of circulatory cells that line the adult endostyle — expressed during bud development, and chemical manip-
expressed germ cell markers and gave rise to germline ulation of the pathway resulted in atypical budding due to
and soma upon transplantation [5]. This is in line with the duplication of the A/P axes and loss of the overall cell
other studies showing that large numbers of cells expres- apical-basal polarity. These data show that Wnt signaling is
sing germ cell markers can be found anywhere in the used for equivalent developmental processes both during
circulation, including the blood vessels, ampullae and embryogenesis and asexual development in colonial asci-
lacunae of the adult zooid [2,4 ]. Whether or not any of dians [7 ]. The Pitx pathway is involved in the formation of
these locations function as a germ cell niche remains to multiple tissues during ascidian embryogenesis, including
be tested. the stomodeum, pituitary gland, and determination of
www.sciencedirect.com Current Opinion in Genetics & Development 2016, 39:101–106
104 Developmental mechanisms, patterning and evolution
left-right asymmetry. Spatial-temporal expression stud- Likewise, it is unknown at this time whether somatic
ies during asexual budding in Botryllus suggest that Pitx and germline tissues of the regenerated body originate
has similar roles during this developmental program [8]. from separate cellular sources. Future experiments will
Knockdown of Pitx at distinct stages of asexual budding be aimed at addressing these questions.
results in phenotypes that correspond to the expression
patterns of Pitx and can block new secondary bud Vascular biology and angiogenesis
initiation from the primary bud. Thus, Pitx may play The process of blood vessel formation is essential for a
a role on the earliest stages of blastogenesis [9]. The wide variety of physiological and pathological process-
transcriptome database of the entire blastogenic cycle es, including vertebrate embryogenesis and tumour
[6 ] will be a valuable tool for future studies of gene vascularization, and is difficult to study in vivo in most
expression dynamics throughout the process asexual organisms. The large and experimentally accessible
reproduction. vasculature, natural parabiosis that occurs between his-
tocompatible colonies, and vast regeneration potential
The ability of the adult animal to regenerate entire of the vasculature makes Botryllus a unique model for
bodies from fragments of vasculature suggests the pres- studies of vascular biology in vivo. When the peripheral
ence of lineage restricted or multipotent stem cells. circulatory system of a colony is surgically removed,
Several studies support the idea that circulating, blood it can completely regenerate within 24–48 hours
borne stem cells are at least partially involved in this (Figure 1b,e,h,k). The regeneration of these differenti-
regeneration process. Brown et al. observed that the ated vascular tissues has been shown to occur through a
early stages of regeneration are characterized by an sprouting mechanism with the participation of angio-
outpocketing of the vascular epithelium and a cluster- genic factors: VEGF, FGF-2, and EGF [14,15]. In
ing of hemocytes [10]. This is followed by expansion of addition, the homolog of VEGR-2 is necessary for
epithelium into a vesicle, and formation of hemocyte the regeneration exhibited by vascular tissue. siRNA-
aggregates next to vesicle epithelium. Piwi antibody mediated knockdown of the VEGFR blocked vascular
staining was detected in circulating cells and in cells regeneration, and interfered with vascular homeostasis
associated with the epithelial layer of the vesicle [10]. [15]. Braden et al. [16 ] used a novel lineage tracing
In Botrylloides leachi, proliferating cells are present in technique to trace and isolate cells that participate in
aggregated foci of hemocytes in regeneration niches, the regeneration of the vasculature and de novo forma-
followed by the formation of a single layered epithelial tion of ampullae. Regeneration of vascular tissue is
sphere. In this species, piwi positive cells appear at induced by removing the entire peripheral vasculature,
2 days post surgery, lining the vascular epithelium [11]. consisting of marginal vessels and ampullae, in a pro-
piwi is expressed in cell aggregates close to the epithe- cedure known as an ampullaectomy. Within the first
lium. SiRNA mediated knockdown of piwi expression 24 hours post ampullaectomy, the majority of vascular
resulted in regeneration arrest [5,11]. Retinoic acid regeneration, including vascular remodeling and ampu-
receptor is expressed by in blood cells within regener- lae budding, takes place. During this process, the
ating niches and also in later stages of regenerating number of actively dividing cells within the remaining
buds. The addition of retinoic acid (RA) inhibitors as vessels increases significantly [16 ]. A novel vascular
well as RNA interference knockdown experiments cell lineage tracing technique utilized both the endo-
resulted in WBR arrest and bud malformations, sug- cytic properties of the Botryllus vasculature as well as
gesting that RA signaling is required for the regenera- the pH-stability of mCherry protein, fluorescein labeled
tion process [12]. Besides somatic tissues, the bodies dextran, and Alexa-Fluor conjugated bovine serum
arising from WBR also acquire a new germline. Kawa- albumin. This punctate fluorescent labeling of the
mura et al. investigated the regeneration of gonads in vasculature is selective and stable, and is maintained
buds growing from the vasculature of Botryllus primi- in the vascular tissue for about 15 days. Using this
genus [13]. In this species, vascular buds arise as an technique, the study showed that pre-existing vascular
aggregate of undifferentiated coelomic cells beneath cells were the source of newly regenerated blood ves-
the vascular epithelium. vasa-positive cells from the sels and ampullae, and that immobile, resident vascular
common vasculature enter into these vascular buds, cells regenerate new vessels and have the ability to
suggesting that circulatory germ cell precursors are reversibly switch between vessel and ampullae cell
the source of new gonads in regenerating vascular buds. fates during vascular remodeling events [16 ]. A recent
study developed a methodology to quantitatively ana-
To date, the precise nature of the cellular source of lyze the effect of injected solutes on angiogenesis on
regenerated body remains unresolved. It remains to be Botryllus [17 ]. Human recombinant VEGF or EGF
tested whether the clustering cells associated with the was injected into colonies after ampullaectomy, which
epithelial layer are true stem cells, whether they are significantly enhanced vascular regeneration. Together,
lineage committed or pluripotent, what is their origin, these studies establish Botryllus as a model for in vivo
and what are the signals regulating their behavior. studies of angiogenesis and vascular regeneration.
Current Opinion in Genetics & Development 2016, 39:101–106 www.sciencedirect.com
Colonial ascidians as model organisms for germ cells Kassmer, Rodriguez and De Tomaso 105
Allorecognition and controlled vessel The analysis of spatial and temporal expression of genes
anastomoses in situ has remained challenging in Botryllus until recently.
Botryllus undergoes a natural allorecognition reaction Langenbacher et al. [26 ] developed a robust protocol for
when two individuals grow into proximity. The first fluorescent in situ hybridization that enables the simul-
tissues to come into contact are the ampullae, which taneous detection of multiple transcripts in whole mount
subsequently will either anastomose, parabiosing the Botryllus colonies.
two individuals (called fusion), or reject each other, an
inflammatory reaction that results in a blocking of vessel Manni et al. [27] have constructed the first ontology for
fusion. Fusion or rejection is controlled by a single, highly anatomy and asexual development of B. schlosseri. The B.
polymorphic locus called the fuhc (for fusion/histocom- schlosseri Ontology of Development and Anatomy
patibility). Individuals that share one or both fuhc alleles (BODA) is based on studies that investigate the anatomy,
will fuse, those that share neither will reject. This is blastogenesis and regeneration of this organism and will
analogous to the ‘missing self’ recognition responsible facilitate comparisons between sexual and asexual devel-
for vertebrate Natural Killer cell function, and two recep- opment. BODA allows for the search and identification of
tor families control this reaction [18,19]. Like most im- anatomical structures, definition of developmental stages,
mune responses, allorecognition in Botryllus appears to and to following the morphogenetic events of a tissue
depend on the integration of positive and negative sig- and/or organ of interest throughout asexual development.
naling pathways. In future studies, analysis of FACS BODA can be downloaded as an OBO file from OBO
purified populations of vascular cells at both the genetic Foundry portal, from the Ontology Bioportal (http://
and protein levels will identify the proteins and mecha- bioportal.bioontology.org/ontologies/BODA) or from the
nisms which underlie this highly specific allorecognition Tunicate Portal (http://www.tunicate-portal.org/
reaction. wordpress/?page_id=145).
Aging and senescence Conclusions and future directions
Botryllus has a number of traits that make it an excellent The unique biology of colonial ascidians allows for the
model for studies on aging. The lifespans of Botryllus investigation of processes that cannot be easily studied in
colonies can be classified into three groups, short (7–9 other species, such as regeneration of whole bodies,
months), intermediate (10–14 months) and long (over changes in fertility, germ cell migration, vascular regen-
1.5 years), and this variable lifespan can be utilized to eration and vascular aging. At the same time, the genetic
dissect the basis of aging at the molecular and cellular toolkit underlying these processes is fundamentally simi-
level. The mechanisms of aging are not fully understood. lar to the genetic toolkit operating in vertebrate develop-
One theory attributes aging to progressive deterioration in ment. Current studies are investigating the cellular
the molecular and cellular machinery and disruption of sources and molecular mechanisms underlying whole
physiological homeostasis [20,21]. The second suggests body regeneration from vascular tissue as well as the
that life span is genetically programmed, and aging may regulation of germline stem cell self renewal, migration
be derived from intrinsic processes, which enforce a non- and differentiation during asexual reproduction. Another
random, terminal time interval for the survivability of the main area of research is focused on vascular biology,
organism [22,23]. In B. schlosseri, aging appears to be investigating the molecular mechanisms involved in ves-
related to both processes. About 45% of both short and sel regeneration and aging of the vasculature. Current
long-lived genotypes show a non-random profile of or- efforts are aimed at generating transgenic animals to
ganismal senescence [24]. Because of the ongoing process facilitate the analysis of cell fate during regenerative
of asexual reproduction and replacement of zooids, the processes. Achieving gene knockout using tools such as
only long-lived organ in the colony is the vasculature, CRISPR/Cas9 would greatly enhance the possibilities of
which undergoes a number of age-related functional and assessing gene function.
morphological changes. Current experiments are investi-
gating the hypothesis that aging is due to changes in the Acknowledgement
structure and functionality of vascular cells, and are This work was supported by the NIH [grant number AG037966] and [grant
number AI041588].
assessing the effect of parabiosis between young and
old individuals on vascular cell functionality.
References and recommended reading
Papers of particular interest, published within the period of review,
Recently developed resources and have been highlighted as:
techniques:
of special interest
The recent publication of a draft genome sequence for B.
of outstanding interest
schlosseri has identified homologs of vertebrate genes
known for their function in lymphoid-mediated immuni- 1. Rodriguez D, Kassmer SH, De Tomaso AW: Gonad
development and hermaphroditism in the ascidian Botryllus
ty as well as eye, ear and heart function [25 ]: http://
schlosseri. Mol Reprod Dev 2016 http://dx.doi.org/10.1002/
botryllus.stanford.edu/botryllusgenome/ mrd.22661. [in press].
www.sciencedirect.com Current Opinion in Genetics & Development 2016, 39:101–106
106 Developmental mechanisms, patterning and evolution
Comprehensive review on germline biology, fertility and gonad differen- compound ascidian is similar to vertebrates. Evol Dev 2008,
tiation in colonial ascidians. 10:591-605.
2. Brown FD, Tiozzo S, Roux MM, Ishizuka K, Swalla BJ, De 15. Tiozzo S, Voskoboynik A, Brown FD, De Tomaso AW: A
Tomaso AW: Early lineage specification of long-lived germline conserved role of the VEGF pathway in angiogenesis of an
precursors in the colonial ascidian Botryllus schlosseri. ectodermally-derived vasculature. Dev Biol 2008, 315:243-255.
Development 2009, 136:3485-3494.
16. Braden BP, Taketa DA, Pierce JD, Kassmer S, Lewis DD, De
3. Rosner A, Moiseeva E, Rabinowitz C, Rinkevich B: Germ lineage Tomaso AW: Vascular regeneration in a basal chordate is due
properties in the urochordate Botryllus schlosseri — from to the presence of immobile, bi-functional cells. PLOS ONE
markers to temporal niches. Dev Biol 2013, 384:356-374. 2014, 9:e95460.
Use a novel vascular cell lineage tracing methodology to assess vascular
4. Kassmer SHRD, Langenbacher AD, Bui C, De Tomaso AW: regeneration and demonstrate that pre-existing vascular resident cells
Migration of germline progenitor cells is directed by give rise to regenerating vessels.
sphingosine-1-phosphate signalling in a basal chordate. Nat
17. Gasparini F, Caicci F, Rigon F, Zaniolo G, Manni L: Testing an
Commun 2015, 6.
unusual in vivo vessel network model: a method to study
First study identifying the bioactive lipid sphingosine-1-posphate as a
angiogenesis in the colonial tunicate Botryllus schlosseri. Sci
major signaling molecule regulating germ cell migration and homing.
Rep 2014, 4:6460.
5. Rinkevich Y, Voskoboynik A, Rosner A, Rabinowitz C, Paz G, Establish the vasculature of Botryllus as a useful in vivo vessel network
Oren M, Douek J, Alfassi G, Moiseeva E, Ishizuka KJ et al.: model for testing the effects of specific injected solutes on vessel
Repeated, long-term cycling of putative stem cells between dynamics.
niches in a basal chordate. Dev Cell 2013, 24:76-88.
18. Nyholm SV, Passegue E, Ludington WB, Voskoboynik A,
6. Rodriguez D, Sanders EN, Farell K, Langenbacher AD, Taketa DA, Mitchel K, Weissman IL, De Tomaso AW: fester, a candidate
Hopper MR, Kennedy M, Gracey A, De Tomaso AW: Analysis of allorecognition receptor from a primitive chordate. Immunity
the basal chordate Botryllus schlosseri reveals a set of genes 2006, 25:163-173.
associated with fertility. BMC Genomics 2014, 15:1183.
19. McKitrick TR, Muscat CC, Pierce JD, Bhattacharya D, De
Transcriptome analysis of fertile and infertile colonies, in vivo analysis of
Tomaso AW: Allorecognition in a basal chordate consists of
differentially expressed genes, identified genes driving gonad maturation.
independent activating and inhibitory pathways. Immunity
2011, 34:616-626.
7. Di Maio A, Setar L, Tiozzo S, De Tomaso AW: Wnt affects
symmetry and morphogenesis during post-embryonic
20. Kirkwood TB: Understanding the odd science of aging. Cell
development in colonial chordates. Evodevo 2015, 6:17.
2005, 120:437-447.
Shows that Wnt signaling is used for equivalent developmental processes
both during embryogenesis and asexual development in an adult organ-
21. Hartl FU: Cellular homeostasis and aging. Annu Rev Biochem
ism. 2016.
8. Tiozzo S, Christiaen L, Deyts C, Manni L, Joly JS, Burighel P:
22. Travis JM: The evolution of programmed death in a spatially
Embryonic versus blastogenetic development in the
structured population. J Gerontol A Biol Sci Med Sci 2004,
compound ascidian Botryllus schlosseri: insights from Pitx 59:301-305.
expression patterns. Dev Dyn 2005, 232:468-478.
23. Goldsmith TC: Solving the programmed/non-programmed
9. Tiozzo S, De Tomaso AW: Functional analysis of Pitx during aging conundrum. Curr Aging Sci 2015, 8:34-40.
asexual regeneration in a basal chordate. Evol Dev 2009,
11:152-162. 24. Munday R, Rodriguez D, Di Maio A, Kassmer S, Braden B,
Taketa DA, Langenbacher A, De Tomaso A: Aging in the colonial
10. Brown FD, Keeling EL, Le AD, Swalla BJ: Whole body chordate. Invertebr Reprod Dev 2015, 59:45-50.
regeneration in a colonial ascidian, Botrylloides violaceus. J
Exp Zool B Mol Dev Evol 2009, 312:885-900. 25. Voskoboynik A, Neff NF, Sahoo D, Newman AM, Pushkarev D,
Koh W, Passarelli B, Fan HC, Mantalas GL, Palmeri KJ et al.: The
11. Rinkevich Y, Rosner A, Rabinowitz C, Lapidot Z, Moiseeva E, genome sequence of the colonial chordate, Botryllus
Rinkevich B: Piwi positive cells that line the vasculature schlosseri. Elife 2013, 2:e00569.
epithelium, underlie whole body regeneration in a basal Comparison of homologous genes between B. schlosseri and other
chordate. Dev Biol 2010, 345:94-104. diverse taxonomic groups revealed genomic events underlying the evo-
lution of vertebrates and lymphoid-mediated immunity.
12. Rinkevich Y, Paz G, Rinkevich B, Reshef R: Systemic bud
induction and retinoic acid signaling underlie whole body 26. Langenbacher AD, Rodriguez D, Di Maio A, De Tomaso AW:
regeneration in the urochordate Botrylloides leachi. PLoS Biol Whole-mount fluorescent in situ hybridization staining of the
2007, 5:e71. colonial tunicate Botryllus schlosseri. Genesis 2015, 53:
194-201.
13. Kawamura K, Sunanaga T: Role of Vasa, Piwi, and Myc- Report a robust protocol for in situ hybridization that enables the simul-
expressing coelomic cells in gonad regeneration of the taneous detection of multiple transcripts in whole adult B. schlosseri.
colonial tunicate, Botryllus primigenus. Mech Dev 2011,
128:457-470. 27. Manni L, Gasparini F, Hotta K, Ishizuka KJ, Ricci L, Tiozzo S,
Voskoboynik A, Dauga D: Ontology for the asexual development
14. Gasparini F, Burighel P, Manni L, Zaniolo G: Vascular and anatomy of the colonial chordate Botryllus schlosseri.
regeneration and angiogenic-like sprouting mechanism in a PLOS ONE 2014, 9:e96434.
Current Opinion in Genetics & Development 2016, 39:101–106 www.sciencedirect.com