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Early lineage specification of long-lived germline precursors in the colonial ascidian Botryllus schlosseri
Biology Department, Center for Developmental Biology, and Institute for Stem Cell and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.Development (Impact Factor: 6.46). 10/2009; 136(20):3485-94. DOI: 10.1242/dev.037754
Source: PubMed
In many taxa, germline precursors segregate from somatic lineages during embryonic development and are irreversibly committed to gametogenesis. However, in animals that can propagate asexually, germline precursors can originate in adults. Botryllus schlosseri is a colonial ascidian that grows by asexual reproduction, and on a weekly basis regenerates all somatic and germline tissues. Embryonic development in solitary ascidians is the classic example of determinative specification, and we are interested in both the origins and the persistence of stem cells responsible for asexual development in colonial ascidians. In this study, we characterized vasa as a putative marker of germline precursors. We found that maternally deposited vasa mRNA segregates early in development to a posterior lineage of cells, suggesting that germline formation is determinative in colonial ascidians. In adults, vasa expression was observed in the gonads, as well as in a population of mobile cells scattered throughout the open circulatory system, consistent with previous transplantation/reconstitution results. vasa expression was dynamic during asexual development in both fertile and infertile adults, and was also enriched in a population of stem cells. Germline precursors in juveniles could contribute to gamete formation immediately upon transplantation into fertile adults, thus vasa expression is correlated with the potential for gamete formation, which suggests that it is a marker for embryonically specified, long-lived germline progenitors. Transient vasa knockdown did not have obvious effects on germline or somatic development in adult colonies, although it did result in a profound heterochrony, suggesting that vasa might play a homeostatic role in asexual development.
Germline precursors in most metazoans segregate from somatic
lineages specified during early stages of embryonic development
and are irreversibly committed to gamete production (Santos and
Lehmann, 2004). By contrast, studies in animals that can propagate
asexually suggest that germline precursors can also originate in
adults, from either pluripotent stem cells that can give rise to somatic
or germline fate, or de- and/or transdifferentiation of cells and tissues
(Blackstone and Jasker, 2003; Extavour et al., 2005; Nieuwkoop and
Sutasurya, 1979; Nieuwkoop and Sutasurya, 1981).
We are studying germline precursors in the colonial ascidian
Botryllus schlosseri. Ascidians are sessile marine invertebrate
chordates that develop from swimming tadpole larvae with
characteristic chordate features, and they are closely related to
vertebrates (Bourlat et al., 2006; Delsuc et al., 2006).
Embryogenesis in solitary ascidians is the textbook example of
determinative or mosaic development, with most cell specification
being due to the inheritance of cytoplasmic determinants (Conklin,
1905; Imai et al., 2006). Following a free-swimming phase, the
larvae settle and metamorphose into a sessile, filter-feeding form,
called an oozooid, wherein most chordate characteristics are lost. In
most species, this initial metamorphosis is followed by growth and
sexual maturity of the solitary individual. By contrast, colonial
ascidian species grow by asexual propagation, resulting in a colony
of genetically identical individuals (Swalla, 2006). This regenerative
ability appears to be due to the presence of self-renewing progenitors
(Laird et al., 2005; Sabbadin and Zaniolo, 1979), but the nature of
these cells (e.g. pluripotent or lineage restricted), and the
mechanisms by which they contribute to asexual reproduction are
To date, germline development has been studied in solitary
ascidians, such as Ciona intestinalis, and progenitors have been
found to segregate from somatic lineages early in development,
resulting in maternally specified germ cells that migrate into the
gonad rudiments during metamorphosis, and that later mature into
gametes (Fujimura and Takamura, 2000; Shirae-Kurabayashi et al.,
2006; Takamura et al., 2002; Yamamoto and Okada, 1999).
Embryos in colonial species are brooded and lineage-tracing studies
lag behind those in the more accessible free-spawning solitary
species. Nevertheless, determinative development is likely to be a
conserved characteristic in ascidians, and we hypothesize that
colonial species retain long-lived germline progenitors specified
during embryogenesis.
B. schlosseri is a colonial ascidian with three co-existing
generations arranged spatially into a star-shaped group called a
system, containing both adults (zooids) and buds, all connected by
a common vasculature (Fig. 1). The center of each system is
occupied by the zooids, which are actively feeding and capable of
sexually reproducing. They are joined peripherally by 'primary
buds', which are completing their development of both somatic and
germline tissues. In turn, these are connected to 'secondary buds',
which are in the initial stages of development. The zooid has a
lifespan of only one week, after which it dies in a massive wave of
apoptosis called 'takeover' (Lauzon et al., 2002). Development is
coordinated throughout the colony, and during takeover the zooid
Early lineage specification of long-lived germline precursors
in the colonial ascidian Botryllus schlosseri
Federico D. Brown
, Stefano Tiozzo
*, Michelle M. Roux
*, Katherine Ishizuka
, Billie J. Swalla
Anthony W. De Tomaso
In many taxa, germline precursors segregate from somatic lineages during embryonic development and are irreversibly committed
to gametogenesis. However, in animals that can propagate asexually, germline precursors can originate in adults. Botryllus
schlosseri is a colonial ascidian that grows by asexual reproduction, and on a weekly basis regenerates all somatic and germline
tissues. Embryonic development in solitary ascidians is the classic example of determinative specification, and we are interested in
both the origins and the persistence of stem cells responsible for asexual development in colonial ascidians. In this study, we
characterized vasa as a putative marker of germline precursors. We found that maternally deposited vasa mRNA segregates early in
development to a posterior lineage of cells, suggesting that germline formation is determinative in colonial ascidians. In adults,
vasa expression was observed in the gonads, as well as in a population of mobile cells scattered throughout the open circulatory
system, consistent with previous transplantation/reconstitution results. vasa expression was dynamic during asexual development in
both fertile and infertile adults, and was also enriched in a population of stem cells. Germline precursors in juveniles could
contribute to gamete formation immediately upon transplantation into fertile adults, thus vasa expression is correlated with the
potential for gamete formation, which suggests that it is a marker for embryonically specified, long-lived germline progenitors.
Transient vasa knockdown did not have obvious effects on germline or somatic development in adult colonies, although it did result
in a profound heterochrony, suggesting that vasa might play a homeostatic role in asexual development.
KEY WORDS: Asexual reproduction, Botryllus schlosseri, Blastogenesis, Heterochrony, Regeneration, Budding, Colonial ascidian,
Coloniality, Germ cells, Germline, Stem cells, vasa
Development 136, 3485-3494 (2009) doi:10.1242/dev.037754
Biology Department, Center for Developmental Biology, and Institute for Stem Cell
and Regenerative Medicine, University of Washington, Seattle, WA 98195, USA.
Friday Harbor Laboratories, University of Washington, Friday Harbor, WA 98250,
Molecular, Cellular and Developmental Biology, UC Santa Barbara, Santa
Barbara, CA 93106, USA.
*These authors contributed equally to this work
Present address: Evolutionary Biology – Division 4, Max Planck Institute for
Developmental Biology, 72076 Tübingen, Germany
Author for correspondence (firstname.lastname@example.com)
Accepted 17 August 2009
Page 1
bodies undergo apoptosis and are removed via phagocytic cells in
the blood, the primary bud migrates into the newly vacated region
of the colony, opening its siphons and becoming a zooid, the
secondary bud becomes the primary bud, and a new secondary bud
begins to develop. Thus, the life history of Botryllus consists of a
constant succession of individual zooids, each with a three-week
lifespan (Lauzon et al., 2002).
Asexual development (blastogenesis) takes 14 days under
laboratory conditions, and can be divided into seven distinct visual
stages (Fig. 1; stages A-1 through D) (Lauzon et al., 2002). A new
generation starts as a secondary bud, first visible as a thickening of
the peribranchial epithelium of a primary bud (Fig. 1; stage A1),
which evaginates and forms a closed vesicle (Fig. 1; stage A-2
through B-2). Next, a series of epithelial invaginations and
protrusions (Fig. 1; stage C-1) differentiate into somatic tissues and
organs (Fig. 1; stage C-2). After seven days (Fig. 1; stage D), the
secondary bud transitions to a primary bud and continues to develop.
At day 14, the siphons open and the primary bud becomes a filter-
feeding adult zooid. Each zooid can generate multiple buds each
week, so the colony will eventually expand asexually. While
interconnected, the zooids and buds develop independently, and
individuals can be separated from the colony without disturbing their
growth (i.e. subcloning), thus multiple experiments can be done on
a single genotype.
Following metamorphosis, colonies undergo at least 8-12
developmental cycles prior to the first appearance of gametes
(sexual maturity). In addition, populations show seasonal fertility,
and in the lab cycle in and out of reproductive (fertile) and non-
reproductive (infertile) states. However when the colony is fertile
development of the gametes is synchronized with somatic
development (Mukai, 1977; Mukai and Watanabe, 1976; Sabbadin
and Zaniolo, 1979). The first appearance of gonads occurs in the
secondary bud (stage B), when mobile progenitors in the blood
migrate to a region between the inner epithelium and the epidermis
and begin to proliferate. Concurrently, oocytes at various stages of
development also appear (Fig. 1). Over the next 10 days, the medial
region of the blastema will differentiate into the lobular testis, while
the lateral region will become the ovary (Sabbadin and Zaniolo,
1979). For the latter, one or several oocytes will become fixed on the
epithelia of the peribranchial chamber, and an oviduct will form
from the outer follicular layer. Upon transition to the adult zooid,
mature eggs will immediately ovulate into the peribranchial
chamber, be fertilized by exogenous sperm, and develop in situ.
Several hours to days later the testes will complete development, and
sperm will be released into the peribranchial chamber and flushed
into the water column, fertilizing neighboring colonies (Johnson and
Yund, 2004). The time lag between ovulation and sperm release
(protogyny) prevents self-fertilization.
Given this plasticity, it appears that germline precursors are
mobile, and can migrate to a niche in the secondary bud then expand
and differentiate into gametes. Moreover, at least for oocytes,
intermediate stages migrate between each successive asexual
generation until maturity, where they become fixed and contribute
to sexual reproduction in that generation (Izzard, 1968; Mukai,
1977; Sabbadin and Zaniolo, 1979). In summary, each asexual
generation probably initiates gametogenesis of both male and female
gametes, as well as harboring intermediate stages of oocytes.
The mobility of germline precursors has also been demonstrated
independently. B. schlosseri undergoes a natural transplantation
reaction that can result in vascular fusion, uniting the circulation of
two individuals. In these chimeras, both long-lived germline and
somatic chimerism has been observed, even months after surgical
RESEARCH ARTICLE Development 136 (20)
Fig. 1. Outline of asexual development (blastogenesis) in Botryllus schlosseri. Animals were staged according to Lauzon et al. (Lauzon et al.,
2002). Each stage (A-D) represents 1 day under laboratory conditions. Drawings represent dorsal views of zooids (green frame), primary buds
(yellow frame) and secondary buds (red frame). For simplification, each adult zooid carries only one set of buds. A secondary bud appears as a
thickening of the epidermis and the peribranchial chamber leaflet of the primary bud (stage A1), which evaginates into a closed vesicle (stage B2),
followed by organogenesis (stages C1-D). Gonadogenesis occurs in the secondary bud from mobile precursors (blue; stages B1-C2). During
takeover (stages C2-D), the secondary bud becomes the primary bud and a new blastogenic cycle begins for the next secondary bud. After the
second takeover event, the primary bud opens its siphons and becomes a functional adult (zooid). In fertile colonies (as illustrated here), the
hermaphroditic gonad fully matures on both sides of the zooid.
Page 2
separation of the two individuals (Sabbadin and Zaniolo, 1979).
Moreover, when stem cells of two genotypes mix, the populations will
compete, and stem cells from one genotype will often replace the
germline and somatic tissues of another, in a process called 'stem cell
parasitism' (Buss, 1982; Stoner and Weissman, 1996). Germline and
somatic precursors can be prospectively enriched in B. schlosseri and
show long-term reconstitution ability upon transplantation. In
addition, limiting dilution experiments show that transplantation of
single cells results in either somatic or germline chimerism, but not
both, which suggests that both germline-committed and somatic-
committed stem cell populations exist in adults (Laird et al., 2005).
Our long-term goal is to isolate and study these stem cells in order to
understand the cellular and molecular basis of asexual development
(regeneration) and stem cell parasitism.
In this study, we characterized the expression and function of
vasa as a putative lineage-specific marker for germline
progenitors, and tested for the presence of functional germline
progenitors in juveniles. vasa encodes an ATP-dependent RNA
helicase, and its expression is restricted to primordial germ cells
(PGCs) in most phyla studied to date (Sengoku et al., 2006).
However, in some metazoans, vasa has also been implicated in
aspects of somatic development (Dill and Seaver, 2008; Extavour
et al., 2005; Mochizuki et al., 2001). In Botryllus, our results
suggest that vasa labels long-lived, functional germline
progenitors specified during embryogenesis that are also found in
enriched stem cell populations, and these results correlate with
functional reconstitution following transplantation (Laird et al.,
2005). By contrast, transient siRNA-mediated knockdown of vasa
had no effect on germline formation during blastogenesis, but did
produce heterochronic shifts of somatic growth in the colony,
suggesting that vasa-positive (vasa+) cells play a role in somatic
regeneration as well.
Botryllus schlosseri colonies were raised, staged, crossed and screened at
18-20°C according to Boyd et al. (Boyd et al., 1986). Embryos were isolated
and prepared for in situ hybridization as previously described (Brown and
Swalla, 2007). Following metamorphosis, colonies are considered juveniles
until the first appearance of gametes (sexual maturity). Following this time,
colonies with gametes are defined as fertile, those without are referred to as
Quantitative PCR
cDNA was synthesized from embryonic and adult stages as described
(Tiozzo and De Tomaso, 2009). Quantitative PCR (qPCR) was carried out
(Tiozzo et al., 2008) with the following set of Botryllus specific vasa
primers: 5⬘-GGCGGATTTAGCGATGATGAG-3⬘ and 5⬘-TTCCCCCATA -
GC GACTGTTAGAC-3⬘. Analysis of q-PCR was performed using 2
according to Livak and Schmittgen (Livak and Schmittgen, 2001). Fold
change of mRNA was normalized to either actin (ActinF, 5⬘-CTA -
TACGCTTCCGGCAGAAC-3⬘; ActinR, 5⬘-CAAGAGCGACATAGC -
ACAGC-3⬘) or elongation factor 1 alpha (EFF, 5⬘-CGTGGTC ATT -
CCGGGAGCGTCGATG-3⬘) as specified. Individual experiments were
then normalized to experiment-specific reference mRNA quantity (see
Results). Each experiment was repeated at least three times from two
different genotypes of every stage analyzed, with samples run in triplicate.
Error bars were calculated using s.d. and statistical significance using paired
t-tests (P<0.05).
Cell staining and sorting
Botryllus cells were labeled for sorting as outlined in Laird et al. (Laird et
al., 2005). Following incubation, cells were washed, resuspended in
Botryllus buffer containing 25 mM Verapamil and 1 μg/ml propidium iodine
(Molecular Probes) to assay viability. FACS was carried out using a
FACSAria cell sorter (BD Biosciences, San Jose, CA, USA). Samples were
compensated prior to sorting and dead cells were excluded by gating-out
high propidium iodine signals. Analysis was performed using FlowJo
software (Tree Star, San Carlos, CA, USA). Sorted cells were stained with
0.05% Trypan Blue to verify quantity and viability. SSC
and SSC
were pooled for qPCR analysis.
In situ hybridization and histological analysis
In situ hybridization (ISH) was performed as described (Brown and Swalla,
2007). The sequence used for probes is shown in Fig. S1 in the
supplementary material. Prehybridization proteinase K concentrations used
were: 500 μg/ml for oozoid and adult whole mounts, 100 μg/ml for tadpole
embryos and larvae, and 1 μg/ml for sections. Results are from at least three
independent whole-mount ISH replicates, and at least five different colonies
were sectioned for independent ISH replicates. Positive cells identified with
either sense or antisense probes were compared in five independent ISH
replicates (see Fig. S2 in the supplementary material) (Brown and Swalla
(2007). Sectioning and image capture were performed as described
previously (Tiozzo and De Tomaso, 2009).
siRNA delivery
siRNAs to vasa were generated as described (Tiozzo and De Tomaso, 2009;
Tiozzo et al., 2008). Sequences targeted for siRNA are shown in Fig. S1 in
the supplementary material. Samples were observed and collected for
histology and RNA isolation, and results were correlated with vasa
expression levels. Seven independent experimental replicates were done
using different genotypes. For each experiment, a colony was separated into
two subclones and vasa siRNA or control (GFP) siRNA treatment was
initiated at the same stage in the blastogenic cycle. In all experiments, the
level of vasa transcripts declined in the first 3 days and remained low during
the treatment, as demonstrated by qPCR and RT-PCR (see Fig. S3 in the
supplementary material).
Juvenile/adult transplantation and analysis of germline
Genetically distinguishable genotypes were crossed, and progeny were fused
into naïve subclones of the parents. We used six different genotypes in three
independent crosses. At each time point, multiple testes were dissected from
the colony and DNA isolated (Laird et al., 2005). Testes were tested for the
presence of three PCR-based genetic markers (e18sp6, L19sscp and H9hdx),
as previously described (De Tomaso and Weissman, 2003; De Tomaso and
Weissman, 2004). Results were equivalent for each marker.
Spatiotemporal expression of vasa during
embryogenesis, metamorphosis and in the adult
A B. schlosseri vasa cDNA clone was isolated, sequenced and
annotated (see Fig. S1 in the supplementary material; GenBank
Accession number FJ890989). Probes were designed to unique
regions of the gene and expression analyzed in eggs and during
embryogenesis (Fig. 2A-O). vasa mRNA concentrates in granular
structures of the fertilized B. schlosseri egg cortex (Fig. 2A).
During the first cleavage, vasa mRNA localized to the vegetal
pole of the embryo (Fig. 2B); after the third cleavage, vasa
segregated in the cleavage furrow of the posterior-most B4.1 pair
of cells of the eight-cell embryo (Fig. 2C), and in the descendant
B5.1 pair of cells of the 16-cell embryo (Fig. 2D). During
gastrulation (Fig. 2E), vasa mRNA was expressed in a pair of cells
found posterior to the blastopore that are likely to correspond to
the precursor germline B7.6 pair (Fig. 2F) (Shirae-Kurabayashi
et al., 2006).
vasa localization was then characterized during metamorphosis,
in oozooids, and in zooids derived after several rounds of asexual
development, but prior to sexual maturity. During initiation of
3487RESEARCH ARTICLEvasa in Botryllus schlosseri
Page 3
metamorphosis, zygotic expression of single vasa+ cells was
scattered throughout the larval head (Fig. 2G). Immediately
following metamorphosis, vasa+ cells were present in the mantle
of the oozooid and within the peripheral vasculature of the tunic
(Fig. 2H). In fertile colonies, vasa expression was observed in
oocytes labeled with an antisense probe (Fig. 2I), but not in those
labeled with a sense control (Fig. 2J). Histological analysis (Fig.
2L-O) showed vasa+ cells in the periphery of the lobules in
developing testes (Fig. 2M), in single hemocytes in ampullae,
throughout the peripheral vasculature (Fig. 2N), and in a region of
mesenchyme surrounding the sinuses and lacunae of the zooid (Fig.
ISH in adult colonies is difficult due to the presence of the tunic,
which shows medium to high non-specific background, but was
necessary to comprehensively characterize vasa localization.
Background was normalized as described (Brown and Swalla,
2007), and results were verified by qPCR. Expression was
quantified in tadpoles, in post-metamorphosis oozooids and in first-
generation asexually derived zooids. Tadpoles contained the lowest
levels of vasa mRNA expression, oozooids showed a slightly higher
expression, and zooids showed ≥threefold higher levels, regardless
of developmental stage (Fig. 2K).
Additionally, we confirmed that vasa is expressed in the
extracorporeal circulation as well as within the zooid bodies (Fig.
3D). Colonies were dissected and vasa expression was quantified
and compared between the extracorporeal vasculature and the
zooid and bud tissues in fertile colonies (Fig. 3D). vasa+ cells
were observed in the circulation both inside and outside of the
zooid body; however, there were differences in the number of
vasa+ cells in ampullae and in the zooid tissues between fertile
and infertile colonies, most likely due to vasa expression in the
RESEARCH ARTICLE Development 136 (20)
Fig. 2. vasa expression during embryogenesis, metamorphosis and in adult colonies as shown by ISH. (A) Fertilized egg with polar bodies
in the animal pole and granule-like aggregation of maternal vasa (arrowheads) in the cortex. (B) A two-cell-stage embryo with vasa mRNA
(arrowheads) aggregated on one side of the cleavage furrow. (C) An eight-cell-stage embryo with two proximate aggregates of vasa (arrowheads)
at the posterior cortical region of the B4.1 blastomeres. (D) Sixteen-cell-stage embryo with vasa mRNAs (arrowheads) at the posteriormost cortical
region of the B5.1 blastomeres. (E) Gastrula- and neural-plate-stage embryos (past the 110-cell stage) show invagination and blastopore formation
on the vegetal side of the embryo. (F) Gastrula- and neural-plate-stage embryos with a single aggregate of vasa (arrowheads) in the posterior region
of the embryos. (G) Detail of a larval head during metamorphosis. Anterior adhesive papillae (p) can be observed on the right; vasa+ cells
(arrowheads) are scattered throughout the head (h). (H) vasa expression in a newly settled colony is seen in individual cells (arrowheads) in the
oozooid (oz), and within the extracorporeal vasculature. (I) In a sexually mature adult, vasa expression is seen in small oocytes (o) at the site of the
gonads of the zooid (z) and in individual cells (arrowheads) within the ampullae (a). (J) A sexually fertile adult shows general background levels of
autofluorescence in blood and tunic cells, as shown by the sense probe negative control (cf. H and I; antisense probe). (K) Quantitative PCR analysis
of vasa mRNA levels during metamorphosis. Larvae show the lowest mRNA levels, oozooids show a slight increase, and first-generation asexually
derived zooids show a further increase after ten days of settlement. (L) Illustration of a colony (dorsal view) showing location of the testis, ampullae
and zooid body (framed), corresponding to histological sections in M-O. (M) Fluorescence in situ hybridization shows two vasa expressing cells (red;
arrowheads) at the periphery of a mature testis containing highly packed nuclei of sperm precursors (blue). (N) vasa expressing cells (red,
arrowheads) in the ampullae. vasa+ cells are scattered throughout the vasculature and are surrounded by other hemocytes (blue, nuclei
counterstained with DAPI). (O) Cells expressing vasa (red, arrowheads) are also seen in the sinuses and lacunae surrounding the epithelial folds of
the stomach (s) in an adult zooid of the colony. a, ampullae; h, head; o, oocyte; oz, oozoid; p, papillae; s, stomach; t, testis; tu, tunic; z, zooid. Scale
bars: 100 μm.
Page 4
Quantitative analysis of vasa expression during
Asexual somatic development occurs independently of
gametogenesis, and adults will often fluctuate between fertile and
infertile states in both laboratory-reared and natural populations.
vasa expression was quantified and compared between fertile and
infertile adult colonies across one complete cycle of asexual
development (Fig. 3A-C). In fertile colonies, vasa expression was
highest in stage A1 and fluctuated throughout the asexual cycle (Fig.
3A). Statistically significant changes (*) occurred at the transition
from A1 to A2 (P=0.002), B1 to B2 (P=0.007) and D to A1
(P=0.04). Variability was highest in stage C1, the point in the cycle
when germline formation is being initiated in secondary buds (Fig.
1, Fig. 3A). In infertile colonies (Fig. 3B), vasa expression was
highest at stage A1 and fluctuated across successive stages of
asexual development, with statistically significant changes detected
between A1 and A2 (P=0.011), and D and A1 (P=0.0005). Fertile
colonies were then compared with infertile colonies (Fig. 3C). In all
stages, fertile colonies had higher levels of vasa expression (Fig.
3C). Changes in vasa expression most likely reflect cell proliferation
owing to an increase in colony size following takeover in both fertile
and infertile adults, as well as during gamete development in fertile
vasa enrichment in BAAA+ stem cell populations
Previous studies have enriched self-renewing germline and somatic
progenitors 10-fold versus total cells based on ALDH activity using
the reagent BAAA. We tested whether vasa expression was enriched
in BAAA+ versus total cells from both fertile and infertile colonies
by qPCR (Fig. 4). Botryllus cells were analyzed by FACS,
comparing the unstained population (not treated with BAAA; Fig.
4A) with BAAA+ populations consisting of both side scatter (SSC)
high and low populations (Fig. 4B). Both the BAAA+/SSC
the BAAA+/SSC
population were sorted, pooled and used for
qPCR analysis of vasa gene expression. vasa expression was
normalized first to the Elongation factor 1 alpha (EF1α) expression
level, then to pooled cDNA consisting of total mRNA from each
stage (Stage Mix), to determine average vasa expression during
blastogenesis for both states of fertility (dashed threshold line). In
BAAA+ cells from infertile colonies, vasa was highly enriched in
3489RESEARCH ARTICLEvasa in Botryllus schlosseri
Fig. 3. qPCR analysis of vasa expression levels. vasa
expression levels were analyzed during asexual development
(A-C) and in isolated tissues (D). (A) Fertile colonies (testes,
oocytes and embryos present). Values were normalized as
described in the Materials and methods. Resulting
differences in gene expression are given in fold change with
respect to the threshold (stage A1 normalized to self,
dashed line). (B) Infertile colonies. Values were normalized
and presented as in A. Asterisks indicate statistical
significance (P-value displayed) with respect to the
preceding developmental stage (see Results).
(C) Comparative analysis of vasa expression in fertile versus
infertile colonies. (D) vasa expression compared between
the peripheral vasculature and zooid bodies in both fertile
and infertile colonies.
Fig. 4. vasa gene expression in BAAA+ cells from
fertile and infertile colonies. FACS plots showing
Botryllus cell populations based on Side Scatter (SSC-A,
y-axis) and FITC-A emission (BAAA+, x-axis). (A)No
treatment control showing autoflurescence only.
(B) Cells treated with BAAA+ and PI for viability. Gates
indicate populations sorted for qPCR analysis:
BAAA+/SSC high and low (blue boxes) with total
percentage indicated (⫻100). (C,D) qPCR analysis of
vasa gene expression in BAAA+ populations from (C)
fertile and (D) infertile colonies. Asterisk indicates
statistically significant differences.
Page 5
comparison with the average expression (P=0.006; Fig. 4D). By
contrast, BAAA+ cells from fertile colonies showed lower than
average vasa expression (Fig. 4C). Results from experiments shown
in Figs 2-4 suggest that the increased vasa expression in fertile
colonies is likely to be due to differentiating gametes, which are not
found in the BAAA+ pool, indicating that at least a proportion of the
vasa+ cells in the BAAA+ population are long-lived progenitors.
Rapid and long-term germline chimerism induced
by juvenile progenitors
The identification of vasa+ cells during embryogenesis and in
juvenile colonies suggested that germline progenitors are specified
early in development. We independently tested for the presence of
functional progenitors by assessing germline chimerism following
the natural transplantation of cells between juveniles and fertile
adults. We crossed two genetically distinct colonies and fused F1
oozooids back into naïve subclones of the parents (Table 1, Fig. 5).
Three to four weeks following transplantation, we tested for the
presence of donor markers in the testes of the recipient. As germline
formation is initiated in the secondary bud at stage B1 (Fig. 1), this
time point is the earliest in which chimerism in the testes could be
detected. Eggs were not tested because they are surrounded by a
somatic follicular layer, and because the time to maturation is not
well defined (Sabbadin and Zaniolo, 1979).
When fertile, each zooid had one or two testes dissected and
motile sperm isolated, free of somatic contamination (Laird et al.,
2005; Stoner et al., 1999). Testes from individual zooids in the
recipient were isolated (6-9 testes/experiment) and chimerism was
assessed using several previously characterized genetic markers (De
Tomaso and Weissman, 2003). In each cross, we observed that one
or two testes from a group of 6-9 contained donor markers, whereas
the rest were derived from the recipient (Table 1, Fig. 5). Control
progeny from the same crosses did not become sexually mature until
10-12 weeks following metamorphosis, which is the normal time to
sexual maturity under laboratory conditions.
On average, a B. schlosseri genotype has a lifespan of 3-9 months,
whereas some laboratory-reared strains can live to be over two years
(De Tomaso et al., 1998). To test whether these germline progenitors
were long lived, we re-analyzed subclones of the same chimeric
individuals at later time points. In all cases, chimerism was detected
in the germline (Table 1, Fig. 5). In summary, sexually immature
juveniles contain long-lived functional germline progenitors that can
contribute to gamete formation immediately upon transplantation
into a fertile colony, and that show long-term self-renewal.
Functional analysis of vasa during blastogenesis
Functional analysis of vasa was performed using siRNA-mediated
genetic knockdown. It takes about 3 days to knockdown >95% of
the mRNA for multiple genes (see Fig. S3 in the supplementary
material), therefore in the first set of experiments siRNA treatment
of fertile colonies began in stage C2, so that new buds would initiate
and develop under near complete knockdown conditions, allowing
us to follow an asexual generation throughout the life cycle.
In control experiments, subclones were unaffected, and takeover
and the transition of asexual generations occurred normally (Fig.
6A-D). Experimental colonies also underwent takeover normally
(Fig. 6F); however, vasa knockdown severely affected the timing of
this process, extending it from 24 hours to around 4 days.
Histological analysis showed that despite this delay, there were no
abnormalities in the process (see Fig. S4 in the supplementary
material). This delay can be easily visualized: adult zooids shrink
and the primary buds grow much slower (compare Fig. 6G with 6C):
However, the buds developed normally (Fig. 6H; see also Fig. S5 in
the supplementary material). These experiments were repeated four
times on different genotypes with no deviation.
vasa knockdown had no effect on the development of the
germline in fertile colonies. Eggs and testes of the primary buds
continued normal development (Fig. S6 in the supplementary
material), and, in secondary buds, oocyte and gonad cell recruitment
and blastema formation occurred similar to in untreated colonies
(see Fig. S6E,F in the supplementary material). The adult gonads
(egg and testis) presented no obvious defects. However, the germline
developed in context of the delayed resorption during takeover and
was delayed by four days (see Fig. S6A-D in the supplementary
To further dissect the effect of vasa on the timing of blastogenesis,
siRNA treatment was initiated at stage B2 or C1 and continued for
two consecutive blastogenic cycles. vasa knockdown caused
colonies to undergo an early takeover event, which began 24 hours
following the initiation of siRNA treatment (about 24-48 hours
before in the control). The phenotype was equivalent to that in the
previous experiment: takeover was delayed, with resorbtion of the
zooids and growth of the primary buds, as well as their transition to
RESEARCH ARTICLE Development 136 (20)
Table 1. Genetic analysis of chimeric testes
Donor Recipient Time (weeks) Number testes sampled Number testes donor Number testes recipient
1025f 1025c 3 8 2 6
12 6 2 4
32 12 8 4
Yw1519 921a 3 6 2 4
12 5 1 4
Sc6a-b 3143j 3 9 1 8
12 14 6 8
112 8 8 0
Donor and recipient (D and R in Fig. 5) genotypes were crossed and a single F1 individual was fused to a naïve subclone of the recipient parent. Three to four weeks later,
individual testes were removed from the recipient and tested for the presence of donor markers.
Fig. 5. Genetic analysis of chimeric testes. F1 chimerism results from
genotypes 1025f and 1025c (see also Table 1). 1025f was homozygous
for a dominant marker (e18sp6, see Materials and methods). Donor
and recipient genotypes were crossed and a single F1 individual was
fused to a naïve subclone of the recipient parent. Four weeks later,
eight testes were isolated from individual zooids throughout the
subclone and tested for the presence of the donor marker. R, recipient
(1025c); D, donor (1025f); 1-8 are individual testes from the donor
subclone, 4 weeks following fusion, two show the presence of donor
markers. Independent markers gave equivalent results.
Page 6
an adult delayed by about 48 hours (Fig. 7). Interestingly, forcing the
colonies into early takeover via vasa knockdown coupled to a slower
primary bud to zooid transition caused the transition to occur
simultaneously in control and experimental subclones, and the two
were re-synchronized at the same stage of development five days
later (stage A; Fig. 7E,J). However, once vasa siRNA-treated
subclones had completed this first takeover process (day 5; Fig. 7J),
they immediately entered another takeover phase, around 6-7 days
before the control (Fig. 7K). The zooids began to shrivel with the
siphons closing, but in this case the takeover was further delayed,
and the process took about 5-6 days (the entire cycle) to be
completed (Fig. 7K-O). Despite this further delay, the process was
again completed and the second generation (i.e. the secondary buds
that were developing when siRNA treatment began) became adults,
about 10 days from the start of the experiment, with no apparent
defects (Fig. 7O). At this point, the colony immediately went into
takeover for a third time, but rapidly became disorganized and died
within two days. This was probably due to the fact that the early
takeover phenotype caused the siphons to remain closed, preventing
feeding for about two weeks. A schematic is shown in Fig. 7P. The
same phenotype was observed regardless of the developmental stage
of the colony at the onset of the siRNA treatment (day 0). Thus, it
appears that knockdown of vasa causes the adult generation to
undergo takeover within 24 hours, desynchronizing development
between the zooids and buds. However, development of the primary
and the secondary buds was not delayed, and the colonies re-
synchronized at stage A, in step with the control subclones (Fig.
We next tested the reversibility of the observed phenotypes, as
well as the potential effects of knockdown on succeeding
blastogenic generations. vasa knockdown was initiated at stage B
and the colony underwent early takeover (Fig. 8A,B). Six days later,
vasa siRNA delivery was stopped and the colonies were allowed to
continue development (Fig. 8C). Seven days later, vasa knockdown
colonies had re-established normal coordination between takeover
of the adult zooid and development of the bud, concurrent with vasa
re-expression (Fig. 8D).
In summary, acute vasa knockdown in adults had no effect on
germline development, but caused the colony to go into early
takeover, and in addition the takeover event itself was delayed. The
period of delay was not constant, but was proportional to when
siRNA treatment began. By contrast, development of both primary
and secondary buds was not affected and occurred with normal
timing. Although we do not have the ability to carry out rescue with
transgenic expression, experiments in which siRNA treatment was
stopped revealed no long-term effects of knockdown on somatic or
germline development. Thus, vasa appears to be functionally
involved in the timing of takeover.
B. schlosseri is a basal chordate with the ability to regenerate all
organs and tissues on a weekly basis, but the origins and persistence
of the progenitors responsible are not understood. Previous studies had
revealed three properties of germline progenitors in adults. First,
Sabbadin and Zaniolo demonstrated that progenitors are mobile, and
can naturally transplant between parabiosed individuals resulting in
long-term germline chimerism and parasitism (Sabbadin and Zaniolo,
1979). Second, adults in both natural and laboratory-reared
populations cycle between fertile and infertile states, demonstrating
that germline progenitors that can be quiescent exist within an
individual (Boyd et al., 1986; Sabbadin, 1953). Finally, prospective
isolation studies suggested that both germline and somatic progenitors
could be enriched about 10 times based on ALDH activity (Laird et
al., 2005). These same studies also suggested that distinct germline
and somatic lineages exist. First, single cell transplants from ALDH
(BAAA+) populations resulted in germline or somatic chimerism,
3491RESEARCH ARTICLEvasa in Botryllus schlosseri
Fig. 6. Effects of vasa knockdown on asexual
development. (A-D) Development of control colony,
treated every day (d0-5) with GFP siRNA (d0; stage C).
(E-H) Development of knockdown colony over a 5-day
period. Development is delayed by 2 days compared with
the control colony (compare G and B; H and C).
(I,J) Schematic representation of zooid development in
control and treated colonies: vasa knockdown causes a
desynchronization in development between adult and
buds, as illustrated on the black timeline. Asterisks
indicate when the treated samples lost synchrony and the
zooids underwent an early takeover. cz, contracted zooid;
nz, new zooid; pb, primary bud; rz, regressing zooid;
z, zooid. Scale bar: 1 mm.
Page 7
never both (Laird et al., 2005). In addition, following natural
transplantation, parasitism of the germline is repeatable, hierarchical
and appears to be genetically controlled. By contrast, somatic
chimerism is random and does not correlate with germline results. In
fact, following the fusion of two individuals, the resulting chimera is
often composed of soma from one genotype, and germline from the
other (Sabbadin and Zaniolo, 1979; Stoner et al., 1999), thus germline
and somatic development seem to occur from independent
populations of cells. The results presented here show that vasa
expression is strongly correlated with germline precursors.
Early germline specification of long-lived
germline progenitors
The constant development and turnover of zooids in colonial
ascidians, coupled to the determinative development characteristic
of the Tunicata, suggest that long-lived progenitors are specified
during embryogenesis that contribute to development throughout
life, but the nature of these cells (pluripotent or lineage-restricted) is
not well understood. Our spatiotemporal localization of vasa in B.
schlosseri embryos revealed expression patterns nearly equivalent
to those found in solitary ascidians (Brown and Swalla, 2007;
Shirae-Kurabayashi et al., 2006; Tanaka et al., 2000), with
maternally deposited vasa segregating into a pair of posterior
blastomeres, suggesting an early specification of germline-restricted
progenitors. At later time points, vasa+ cells are seen scattered in
the head of the tadpole, a region equivalent to the circulatory
distribution in the adult body plan following metamorphosis.
Independently, we found that cells in newly metamorphosed
individuals are competent to reconstitute the germline following
transplantation into fertile colonies, and that this chimerism is
maintained for the life of the adult recipient (up to 112 weeks in one
pairing). In both fertile and infertile adults, vasa is expressed in cells
in the extracorporeal vasculature, as well as near lacunae
surrounding the zooid body. Circulatory vasa+ cells have been
observed in all botryllid species examined to date (Brown and
Swalla, 2007; Rosner et al., 2009; Sunanaga et al., 2006; Sunanaga
et al., 2008). vasa expression is also enriched in BAAA+ cells,
which can reconstitute the germline (Laird et al., 2005).
Finally, in recent studies, we have analyzed the contribution of
individual genotypes to germline and somatic tissues in chimeras
made from juvenile individuals immediately following
metamorphosis. We found that when these chimeras reached sexual
maturity, patterns of reconstitution matched those of chimeras made
from adults, and included complete germline parasitism and a lack
of correlation between somatic and germline outcomes. Thus, even
if chimeras were made months prior to sexual maturity, germline
progenitors acted independently of somatic progenitors (M. C.
Carpenter and A.W.D., unpublished).
Therefore, our data support the hypothesis that self-renewing,
lineage-restricted germline progenitors are specified during
embryogenesis that become functional immediately following
metamorphosis and contribute to gametogenesis throughout the life
RESEARCH ARTICLE Development 136 (20)
Fig. 7. vasa knockdown during two consecutive blastogenic cycles. (A-E) Development of control colony treated every day (d0-11) with GFP
siRNA (d0; stage B). (F-O) Development of knockdown colony over a two week period. Control colony develops and initiates takeover (stage D)
normally (D; day 4), and the process is completed in about 24 hours (E, day 5). By contrast, the vasa knockdown colony goes into takeover within
24 hours (G), and disrupted development continues over the next 3 days (H,I), but terminates in a transient recovery of developmental synchrony
(compare J with E). Although the control colony continues development, the vasa knockdown colony immediately goes back into takeover (K),
which lasted the entire asexual cycle, but terminated development synchronously for a second time (compare O with E) in early stage A.
(P) Schematic of blastogenic development during vasa knockdown showing the shifts in synchrony (shifts in black timeline) that occur between the
zooids and buds. The asterisk indicates when the treated samples lost synchrony and the zooids underwent an early takeover, but synchrony was
briefly reestablished after both takeover events. cz, contracted zooid; nz, new zooid; pb, primary bud; rz, regressing zooid; z, zooid. Scale bar:
500 μm.
Page 8
of the colony. This suggests that fertility is based on developmental
cues that cause these progenitors to move from the circulation to
regions within the secondary bud and initiate germline formation.
vasa expression and enrichment of germline
We compared the enrichment of vasa in BAAA+ cells isolated from
fertile and infertile adults to independently correlate vasa expression
with long-lived progenitors. We found dramatic changes between the
two. In infertile colonies, vasa expression was highly enriched in
BAAA+ cells. By contrast, fertile colonies had lower vasa expression
in BAAA+ versus total cells. This suggests that vasa expression is
upregulated in cells of the developing gonads that are not part of the
BAAA+ population. The BAAA+ populations from both fertile and
infertile adults contain an enriched pool of stem cells (Laird et al.,
2005), so our results provide strong evidence that a proportion of
vasa+ cells in the BAAA+ population are germline progenitors.
However, these data do not rule out the existence of vasa-negative
(vasa–) germline progenitors, as studies in two related species have
revealed that surgical ablation of regions containing vasa+ cells did
not affect germline development, and that new vasa+ cells appeared
(Takamura et al., 2002; Sunanaga et at., 2006). These reports suggest
that vasa– progenitors exist that become vasa+ at later stages of
development. This would be analogous to the situation in mice,
where PGCs are specified at day E6, but vasa expression does not
turn on until day E9, when PGCs are migrating to the genital ridge
(Lacham-Kaplam, 2004; Tanaka et al., 2000). However, in B.
schlosseri, vasa+ cells are present at all times, as are functional
germline progenitors.
Previous studies suggested that vasa+ somatic cells exist in
ascidians. During Ciona embryogenesis, high vasa expression is
detected in the head; however, the fate of these cells following
metamorphosis is unknown, as in the adult vasa expression is
restricted to the gonads (Takamura et al., 2002). Although vasa+
circulating somatic stem cells could exist in B. schlosseri, the only
tissue outside of these circulatory cells in which vasa expression was
seen is the gonad. In summary, our study cannot discount the
possibility of vasa– germline progenitors nor of vasa+ somatic
and/or pluripotent progenitors. However, the lack of congruity
between somatic and germline chimerism following transplantation
(either natural or experimental) suggests that a germline-committed
population exists, and the distribution of vasa+ cells correlates
exactly with previous limiting-dilution studies (Laird et al., 2005).
Effects of vasa knockdown in asexual
Transient knockdown of vasa resulted in no obvious morphological
defects in development of the germline but showed an effect on the
synchrony of asexual development in the colony. One possible
explanation for the absence of specific germline phenotypes is that
only short-term knockdowns (2-3 weeks) were possible before the
colonies degenerated. Testes require at least two weeks to develop,
and oocytes can mature over several generations, therefore our
treatment might not be long enough to assess the potential role of
vasa in germline development. Conversely, vasa knockdown in
different species shows disparate phenotypes, ranging from no
defects [zebrafish (Braat et al., 2001) and flatworms (Pfister et al.,
2008)] to sex-specific defects on male gamete formation, but not
female [mice (Tanaka et al., 2000)], to defects in migration but not
in the identity of the germ cell precursors [medaka (Li et al., 2009)].
Thus, it would not be surprising if vasa was shown not to be required
for germline formation; however, its expression can still be used to
identify precursors, as has been shown in other animals.
Given that vasa+ cells were scattered throughout the circulation,
the global knockdown phenotype was unexpected. However, a
somatic role for vasa would not be surprising. vasa is expressed in
somatic cells and tissues of embryos and adults in species of different
phyla, such as in the head of Ciona ascidian larvae (Fujimura and
Takamura, 2000; Shirae-Kurabayashi et al., 2006), in the somatic
stem cells of planarians and cnidarians (Shibata et al., 1999; Extavour
et al., 2005; Rebscher et al., 2007), and in the embryonic mesodermal
posterior growth zone and multiple somatic tissues of polychaetes
(Rebscher et al., 2007; Dill and Seaver, 2008). Although ultimately
there was no affect on somatic development, we speculate that the
colony might regulate the number of progenitors as it asexually
expands, and that vasa knockdown affects homeostasis. In previous
experiments, it was found that the zooids appear to be monitoring
growth of the buds, and, if delayed, they will undergo early takeover,
presumably to transfer resources to the next generation (Tiozzo et al.,
2008; Lauzon et al., 2002), so vasa+ cells might be involved in this
process. This might also explain the dynamic expression of vasa in
both fertile and infertile colonies, which in general resembles
previous results that characterize telomerase activity during the
budding cycle (Laird and Weissman, 2004).
In conclusion, given the experimental considerations mentioned
above, we cannot yet rule out any germline defects. However, we
did consistently observe a novel and robust phenotype of vasa
knockdown: the breakdown of synchrony between zooid and buds,
but the role vasa plays in these processes is not clear. Whether vasa
is directly involved in regulating short- or long-range
communication between cells and tissues (autocrine or endocrine)
of the colony to trigger takeover, or whether vasa+ cell-specific
defects, e.g. migration or determination defects, indirectly affect
takeover remain open questions for future studies.
We would like to thank Karla Palmeri, Tanya McKitrick and Randy Will for their
help and assistance. We thank Dr A. Sabbadin and Dr P. Burighel for valuable
discussions for this manuscript. F.D.B. was supported by a predoctoral grant
3493RESEARCH ARTICLEvasa in Botryllus schlosseri
Fig. 8. Release of vasa siRNA treatment has no effect on succeeding asexual generations. (A) vasa expression analyzed by RT-PCR at
different timepoints in control and knockdown colonies; siRNA treatment was discontinued at day 6. (B) Colony at the onset of the vasa siRNA
treatment; stage B of blastogenesis; (C) on day 4, zooids are in takeover (stage D), showing the typical vasa knockdown phenotype; (D) on day 13,
zooids contain palleal buds that are synchronized with the zooids, showing that release of vasa siRNA treatment results in the re-synchronization of
asexual development. nz, new zooid; pb, primary bud; rz, regressing zooid; z, zooid. Scale bars: 200 μm.
Page 9
from the American Heart Association, by the Lerner Gray Fund for Marine
Research from the American Museum of Natural History, and by the University
of Washington Biology Department. S.T. was supported by a Stanford Dean's
Fellowship. M.M.R. was supported by the NIH, individual NRSA
(1F32GM086018-01). Part of this research was funded by a University of
Washington Royalty Research Grant to B.J.S. This work was supported by the
NIH (RO1A104588/R01DK405762) and the Ellison Medical Foundation to
A.W.D. Deposited in PMC for release after 12 months.
Supplementary material
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RESEARCH ARTICLE Development 136 (20)
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- "Where blastemas can be characterized by a mass of cells clustering in the area of the wound after closure, leading to growth and regeneration into new organs or new body parts, it has been shown that the histological nature of this mass of cells is different in different regenerative structures (Shibata et al., 1999; Cebrià et al., 2002; Bosch et al., 2008; Kragl et al., 2009; Wenemoser and Reddien, 2010; Aboobaker, 2011; Reddien, 2013; Bely et al., 2014). Additionally in some epimorphic regenerative events, i.e., where local stimulation of cell proliferation precedes the development of the new part, there is a lack of structures that resemble a blastema, such as during the Ciona intestinalis siphon regeneration (Auger et al., 2010) or the whole body regeneration in colonial ascidians (Brown et al., 2009b). To track evolutionary trajectories of regenerative events it may be more convenient to dissect the conserved chronological event that occurs during the restoration of a particular structure, i.e., wound healing, nature and mobilization of the precursors, and morphogenesis, and compare them in clades that at least come from the same phylum, or that belong to an even more restrained taxonomical clade, e.g., class, order or family. "
Dataset: Tiozzo15(regenerationREV)
- "Where blastemas can be characterized by a mass of cells clustering in the area of the wound after closure, leading to growth and regeneration into new organs or new body parts, it has been shown that the histological nature of this mass of cells is different in different regenerative structures (Shibata et al., 1999; Cebrià et al., 2002; Bosch et al., 2008; Kragl et al., 2009; Wenemoser and Reddien, 2010; Aboobaker, 2011; Reddien, 2013; Bely et al., 2014). Additionally in some epimorphic regenerative events, i.e., where local stimulation of cell proliferation precedes the development of the new part, there is a lack of structures that resemble a blastema, such as during the Ciona intestinalis siphon regeneration (Auger et al., 2010) or the whole body regeneration in colonial ascidians (Brown et al., 2009b). To track evolutionary trajectories of regenerative events it may be more convenient to dissect the conserved chronological event that occurs during the restoration of a particular structure, i.e., wound healing, nature and mobilization of the precursors, and morphogenesis, and compare them in clades that at least come from the same phylum, or that belong to an even more restrained taxonomical clade, e.g., class, order or family. "
Dataset: Tiozzo15(regenerationREV)
[Show abstract] [Hide abstract] ABSTRACT: Regeneration of body structures is an ability widely but unevenly distributed amongst the animal kingdom. Understanding regenerative biology in metazoans means understanding the multiplicity of the cellular and molecular mechanisms that lead to the differentiation, morphogenesis and ultimately the development of a particular regenerating unit. In this manuscript we critically assess the evolutionary considerations suggesting that regeneration is an ancestral trait rather than a mechanism independently evolved in different taxa. In order to test different phylogenetic hypotheses among different taxa we propose mechanistically dissecting the regenerative processes according to its conserved chronological steps: wound healing, mobilization of cell precursors and morphogenesis. We then suggest interpreting regenerative biology from an evo-devo perspective, proposing a possible theoretical framework and experimental approaches without necessarily invoking a common origin or only multiple losses of regenerative capabilities.
- "Additionally in some epimorphic regenerative events, i.e. where local stimulation of cell 536 proliferation precedes the development of the new part, there is a lack of structures that 537 resemble a blastema, such as during the Ciona intestinalis siphon regeneration (Auger et al., 538 2010) or the whole body regeneration in colonial ascidians (Brown et al., 2009b). "


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Good Shepherd Medical Center (GSMC) held a "Welcome Breakfast and White Coat Reception" for the 2016 graduating class of Internal Medicine Residents. The event featured orations from hospital officials and a representative from UT Health-Northeast; introduction of the new class of interns, the presentation of White Coats, the 2012-2013 Avicenna Award presentation, 2012-2013 Intern of …
Tag Archive: UT Tyler
Permanent link to this article: http://www.easttexasreview.com/gsmc-welcomes-interns/
May 28 2013
PTISD names new principal
The Pine Tree ISD School Board named Vanessa Robinson as principal of Pine Tree Junior High School in a special board meeting, May 20, 2013. Robinson, is a native of Longview and completed her Bachelor of Science in Interdisciplinary Studies and Master of Education in Leadership from University of Texas at Tyler. "I am honored …
Permanent link to this article: http://www.easttexasreview.com/ptisd-names-new-principal/
Feb 27 2013
AT&T, UT Tyler partner to provide funding to students
AT&T and The University of Texas at Tyler will provide a financial boost to a program aimed at increasing the number of highly qualified math and science teachers. AT&T will contribute $20,000, which will be matched by UT Tyler, to the UTEACH Scholars Program, according to a university news release. The program, which started at …
Permanent link to this article: http://www.easttexasreview.com/att-ut-tyler-partner-to-provide-funding-to-students/
Feb 19 2013
UT Tyler Nursing Graduate Ranks Best
The University of Texas at Tyler graduate nursing program has been ranked as one of the best by the U.S. News and World Report's Best Online Nursing Programs, President Rodney H. Mabry announced. The UT Tyler master's and doctorate programs are ranked 11th out of the 101 online nursing programs surveyed. This is the second …
Permanent link to this article: http://www.easttexasreview.com/ut-tyler-nursing-graduate-ranks-best/
Feb 15 2013
UT Tyler Campaign Exceeds Goal
The University of Texas at Tyler raised $30.6 million in the Inspiring Excellence comprehensive fundraising campaign, President Rodney H. Mabry announced. The campaign goal was $30 million. "This is the first campaign of its size for UT Tyler, and the primary interest was to raise funds to increase our endowed student scholarships, increase the number …
Permanent link to this article: http://www.easttexasreview.com/ut-tyler-campaign-exceeds-goal/
Dec 26 2012
Judson Middle School Choir students were recently selected as members of All-Region Choir in Region 21. They performed in concert at UT Tyler. Cathy Cole is the director. Pictured are: front row (L to R): Leslie online pharmacy viagra Campos, Triniti Jones, Yasmin Benitez, Kelsey Pentland, Kyerra Bagby, Haley Boyd, Jazmin Dennis 2nd row: Daija Garrett, …
Permanent link to this article: http://www.easttexasreview.com/superior-singing/
Jul 21 2012
UT Tyler Patriot Classic sets record
The University of Texas at Tyler Patriot Classic raised $177, 205.55 to benefit the university's scholarship program, President Rodney H. Mabry announced. This is the largest amount raised in the 26-year history of the fundraiser, which includes the annual UT Tyler Suddenlink Million Dollar Hole-in-One Contest and the Patriot Golf Classic. Organizers presented UT Tyler …
Permanent link to this article: http://www.easttexasreview.com/ut-tyler-patriot-classic-sets-record/
Jul 21 2012
Innovation Academy comes to town
The University of Texas at Tyler and Longview Chamber of Commerce officials recently held a ribbon cutting ceremony for the university's charter school in Longview. The UT Tyler Innovation Academy, a university charter school with campuses in Tyler, Longview and Palestine, will focus on developing, implementing and disseminating new and promising practices in education. The …
Permanent link to this article: http://www.easttexasreview.com/innovation-academy-comes-to-town/

subset: commoncrawl


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UN's World Food Programme says heavy fighting has left it unable to deliver food to hardest-hit places in war-torn Syria
The United Nations warned on Tuesday it was unable to deliver food to up to 1 million hungry and desperate Syrians because of spiralling violence across the country and a lack of fuel.
The UN World Food Programme (WFP) said nearly 2.5 million people - most of them internally displaced by the fighting - needed emergency food aid. But WFP is only able to reach 1.5 million of them, as the situation on the ground worsens, it said.
"Food needs are growing in Syria," Elisabeth Brys, a WFP spokeswoman, said. She added that it was increasingly difficult "to reach the hardest-hit places", after almost two years of continuous fighting, upheaval and civil war.[view whole blog post ]
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African Union troops in Bangui (file photo): The UN has expressed alarm...
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Asamoah Gyan of Ghana (file photo): The Super Eagles have boosted their...


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The implementation of the Affordable Care Act began in earnest on Oct. 1. There will be a sense of relief, anger, confusion and I assume, other emotions regarding the registration for health care. Some of the language describing those emotions will be unprintable in the newspaper.
Opponents of the bill have had several years to use the legislative process to rectify the parts of the bill they found objectionable. Several years to find common ground for the common good of the American people they were elected to represent. Instead, they have indulged in fear-mongering, name-calling, lies about death panels and other dirty tricks to demonize what is basically a Republican plan set forth by a Republican governor (Mitt Romney) when he held office in a heavily Democratic Party state (Massachusetts).
There will be registration problems for sure, but they could have been lessened if certain members of both parties had the true courage of their convictions instead of those bought and paid for by special interests. I am grateful for my health care and want every citizen to have the care they need.
Randy Cate


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Premarital Counseling
All too many couples get caught up in spending time planning their weddings without developing a plan for their marriage. The early stages of a relationship, when the feelings you have for your partner are usually positive, is the ideal time to become more conscious and intentional in the ways you interact and work together. Counseling can give you insights into where your particular relationship dynamics might be heading in the wrong direction.
- What Does Premarital Counseling Do?
- Recent Historical Changes in Marriage Structure
- Post-Marriage Depression
- Case Examples
What Does Premarital Counseling Do?
Couples considering committing to one another in a long-term union such as marriage may benefit from exploring their relationship issues (or potential issues) prior to making a long-term commitment. Premarital counseling is an opportunity to explore the dynamics of your partnership and set yourselves up for a satisfying future together. There is often an educational aspect to premarital counseling, so that you can learn better communication skills and how to negotiate conflict so that you can resolve your differences in a way that actually strengthens your connection. Also, there is often a coaching aspect to premarital counseling where couples can practice, with the therapist's help, the new perspectives and new skills that they are learning.
Find a Therapist
Premarital counseling is as varying and unique as the personality and background of the therapist providing the counseling. Finding someone that you can trust, and who honors your unique situation, is essential. The good news is that new models for developing strong, healthy relationships have been emerging, giving us maps for how to find our way in this new paradigm. Premarital counseling uses these models to help a couple build a strong, conscious foundation for their marriage. Remember that relationships are created; they are dynamic and perennially evolving. The relationship that you yearn for is not the one you start out with but the one you co-create with your partner over time. This takes commitment, trust, and the willingness to look more closely at your own process rather than that of your partner.
We come together from different families of origin. We have learned and have been imprinted, for better or worse, by these early experiences of connection. We come into a marriage with different personalities and temperaments, differing values and needs, and baggage from previous intimate connections. We come into the co-creating of a life together without a training manual or toolkit for how to manage the sometimes treacherous waters of our differences. Premarital counseling can provide this guidance so that each person can mature to their fullest potential within the boundaries of a vital, fulfilling relationship. Through premarital counseling, a couple can explore their dreams, their fears, their differences, and come to a greater understanding of what is sourcing their choices and behaviors.
Recent Historical Changes in Marriage Structure
Intimate relationships have experienced a major paradigm shift over the past forty years. We as a society are reshaping our core dynamics from a role-based model to a partnership model, but not without consequences. We have a divorce rate in the United States where more than half of marriages end in divorce. What is happening here?
Marriages used to serve family and society and the quality of the man/woman connection was secondary. Community pressure would hold a marriage together. Now, for the first time in history, couples are on their own. We have shifted from a community (or village) to a nuclear family so that we have one person, rather than many, to meet our needs. We have also shifted from a survival mode to one of self-actualization--we are demanding deeper levels of intimacy which puts pressure on each partner to fulfill more needs. We live in a youth addicted culture that promotes romantic ideals of love rather than images of couples who have matured after years of finding their way together. We also live in a throw-away society that reinforces the belief that we can have what we want by giving up what we have for the promise of something better. The divorce rate for second marriages is even more sobering at 67%, and 74% for third marriages, according to the Forest Institute of Professional Psychology.
Post-Marriage Depression
Another important aspect of premarital counseling is to normalize what can happen in the early years of a marriage. Many of us have been seduced by the fairytale of the prince and his princess finding their perfect love. But the story really begins when they move into the castle together. Sometimes a newly married person experiences what could be called a post-marriage depression. The commitment can feel like a weight that is taking them down. They have said "yes" to this particular individual but "no" to all the other potential partners--this may result in grief. But ironically, the pain of this condition can deepen one's ability to connect because by working through the impulses to run, by overcoming the belief that the grass is greener over there, and by making the grass greener here, this individual can find the freedom and joy that is possible in a long-term, committed relationship.
Case Examples
- Afraid of commitment: Sam and Pat, in their early thirties, are stuck in the dating phase of their relationship because even though Sam feels a strong love for Pat, he suffers from an even stronger fear of commitment. In premarital counseling, Sam discovers that the root of his feeling entrapped is related to a belief that he has to give up what he wants and needs in order for Pat to be happy. His behavior becomes defensive and he stays away from a deeper connection with his partner by "doing his own thing." During therapy, he discovers that Pat is open to working with him and to giving him what he wants, when Pat knows more clearly what it is that he wants. He finds that when he opens up emotionally, Pat feels more connected and becomes less demanding. This enhances Sam's ability and desire to give Pat more of what Pat needs, knowing that this does not mean sacrificing what he needs. Through this therapeutic work Sam comes to experience the freedom that exists inside a commitment.
- Communication style differences: Julie and Peter are about to be married and come into premarital therapy to learn how to communicate more effectively. They have a pattern where each partner's communication style triggers the other. Julie grew up in a family that met conflict head on. Her family would often become very loud and passionate in their arguments. By contrast, Peter's family did not talk about important things and stayed more distant in their interactions. What attracted Peter to Julie was her warmth and ability to engage with him, but as the wedding approaches, her emotionality has become too much to handle and he has started to withdraw and cut off conversation. Julie is feeling a sense of panic and tries even harder to engage Peter, who then feels even more overwhelmed and withdraws further. In therapy, this dance step of approach/avoidance becomes more clearly defined. The therapist facilitates a process so that each partner plays the role of the other. Both partners gain a greater understanding and compassion for how to meet on common ground. Peter learns how to stay present and feel safe when a conversation gets heated. Julie learns to trust Peter's need to withdraw, and trusts that he will return to engage with her when his system has calmed down.
~ Page content provided by Lynne Foote, www.LynneFoote.com
Last updated: 03-21-2016
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Underbelly of the U.S.'s Most Economically Segregated City
In order to foster the political will that is necessary to enact changes in policy, Austinites must first learn to acknowledge the suffering they usually do not see in their midst.Read more
The Aspen Institute Announces LBJ School Professor Chris King as Ascend Fellow
The Aspen Institute announced yesterday that Christopher King, director of the Ray Marshall Center for the Study of Human Resources at The University of Texas at Austin, will be in the inaugural class of its Ascend Fellowship program. The Ascend Fellows are a select group of 20 leaders from across the country who are pioneering two-generation approaches to move families beyond poverty.Read more
Being Poor Can Suppress Children's Genetic Potentials
Growing up poor can suppress a child's genetic potential to excel cognitively even before the age of 2, according to research from psychologists at The University of Texas at Austin.Read more
Alumnus Edward Zigler has become psychology pioneer
Growing up the child of immigrant parents in America, Edward Zigler (doctor's degree in psychology, 1958) didn't speak English until he participated in a program similar to today's Head Start program.Read more
Professors discuss violence in Latin America in Q&A
In anticipation of its upcoming conference titled "Republics of Fear: Understanding Endemic Violence in Latin America Today," the Teresa Lozano Long Institute of Latin American Studies asked faculty members to share their thoughts on the threat of violence in Latin America. The conference takes place March 4-5 in the Santa Rita Room of the Texas Union.Read more


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Los Angeles, California has long been one of the central hubs of the music industry. The City of Angels is home to the Recording Academy, the Grammy Awards (happening this Sunday, January 26th), and some of the biggest record companies in the world. It also possesses a thriving indie music scene, something we certainly appreciate here in Austin.
With today's Four on the Floor, we're highlighting four SXSW 2014 Showcasing Artists who have become a big part of that particular group. Four bands doesn't even begin to put a dent in the amount of SXSW 2014 Showcasing Artists who will be traveling from LA to perform here at the SXSW Music Festival - so please check the online schedule for more Los Angeles-based artists. Enjoy!
Originally, Avi Buffalo was the solo project of Avigdor Zahner-Isenberg, a guitarist based in LA who for years spent the majority of his days practicing and recording what he could on his laptop. Avi Buffalo later blossomed into a full band, catching the ears of LA music-lovers and, eventually, Sub Pop Records with their lo-fi pop-rock tunes.
RIYL: The Shins, Real Estate
Starting out as the producer for the late rapper Charizma in the early 90s, Peanut Butter Wolf has continued to crank out funky turntable jams to this day. The hip hop DJ started Stones Throw Records - the label responsible for releasing the classic J Dilla album, Donuts - back in 1996. These days, Peanut Butter Wolf is busy traveling the world pursuing his career as a DJ.
RIYL: Madlib, DJ Premier
The sounds created by Ghost Loft, the project of LA producer Danny Choi, could be summed up as euphoric, breathy, sexy, and captivating electronic music. Ghost Loft, who has remixed tracks for jj, Icky Bottoms, and Little Daylight, as well as toured with The Neighbourhood last year. Choi will be bringing his chill beats to SXSW for the first time this March.
RIYL: James Blake, Toro y Moi
Gavin Turek started out with little to no intention of releasing her music to public. The pop singer's sister pushed her into the limelight by posting her music online, unbeknownst to Gavin. She has since collaborated with many high-profile electronic artists, including Paper Diamond and TOKiMONSTA.
RIYL: Santigold, Basecamp
Keep up with the list of showcasing artists here.
Photo credits:
Avi Buffalo photo by Eydie McConnell
Peanut Butter Wolf photo courtesy of the artist
Ghost Loft photo courtesy of the artist
Gavin Turek photo courtesy of the artist


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Dan Harrison
Dan Harrison is Health and Indigenous Affairs Correspondent for Fairfax Media. He is based in Canberra.
Proposal for government loans to fund court cases
Dan Harrison Litigants unable to afford lawyers should be able to accrue HECS-style debts.
Health academic says $140m could be saved by following drug advice
Dan Harrison The Abbott government could save more than $140 million over the next eight months simply by adhering to a recommendation from the expert body that advises it on medicines, a health expert says.
UN climate report warns permanent change has already begun
Tom Arup, Lisa Cox, Dan Harrison Climate change is being felt in all corners of the globe, and some parts of the natural world may already be undergoing irreversible changes, an assessment by the UN Intergovernmental Panel on...
Western Australia's Senate election: The will or the swill of the people?
Dan Harrison A preference system that can be gamed and an electoral administration that loses ballot boxes.
Missing Malaysia Airlines plane: Australian ship in search of MH370 black box, debris
Dan Harrison, Tom Allard Equipment to recover the ''black box'' flight recorder of Malaysia Airlines flight MH370 is being loaded on to an Australian naval vessel in Perth, Prime Minister Tony Abbott has told reporters.
Same-sex couples can marry in UK consulates from June
Dan Harrison Same-sex couples will be able to use British laws to marry in Australian cities from June.
Medibank Private customers to miss out when insurer is privatised
Mark Kenny, Gareth Hutchens and Dan Harrison None of Medibank Private's almost 4 million policyholders nor its 4000 staff will receive preferential treatment such as share allocations when the government-owned insurer is privatised next year.
Alastair Furnival strategy to avoid conflict of interest stays secret
Dan Harrison, Amy Corderoy The federal Health Department has refused a request for documents setting out how former ministerial staffer Alastair Furnival would avoid a conflict of interest between his business affairs and his...
Majority of Australians think nation has problem with alcohol
Dan Harrison And more than three quarters believe Australia's battle with the bottle will not get better over the next decade, according to new polling.
Government healthcare spending figures may bolster case for a GP co-payment
Dan Harrison Federal spending per person on primary healthcare services, including visits to GPs, grew by 50 per cent over the past decade, according to new figures that may bolster the case for new fees to visit...
OECD report: Poverty engulfing many Australians
Dan Harrison Child poverty is on the rise and fewer of us are donating to charity, according to a report that brings into question Australia's self-perception as the land of the fair go.
Australian child poverty up, generosity takes a beating
Dan Harrison Child poverty is on the rise and fewer of us are donating to charity, according to a report that calls into question Australia's self-perception as the land of the fair go.
Journalist Peter Greste's parents 'rocked' by courtroom cage images
Dan Harrison The parents of jailed Australian journalist Peter Greste say they are haunted by images of him in a courtroom cage, as a spokesman for Tony Abbott said the Prime Minister was ''deeply concerned''...
Peter Greste: PM Tony Abbott 'deeply concerned' by charges
Dan Harrison The Prime Minister is ''deeply concerned'' about Australian journalist Peter Greste, now jailed in Egypt, and the government has made representations for his release, a spokesman said, as images of...
Health system could save a billion a year
Dan Harrison Governments could save a billion dollars a year by cutting waste in public hospitals.
Report slams TV alcohol ads
Dan Harrison Current rules on alcohol advertising are failing to protect children and must be tightened, according to the government agency charged with preventing disease.
Exemption on alcohol ads on TV sports programs exposes young
Dan Harrison Existing rules on alcohol advertising are failing to protect children and must be tightened, the government agency charged with preventing disease says.
Doctors fear overcrowding in emergency departments if $6 GP fee adopted
Kate Hagan and Dan Harrison A minimum $6 fee to see a GP would lead more patients to seek treatment in overcrowded emergency departments including those with conditions that had deteriorated because of deferred care, doctors...
Raising Medicare levy the solution to health costs, says architect
Dan Harrison One of the architects of Medicare has called for an increase in the Medicare levy, warning Australians will not accept any change that restricts access to healthcare to those who can pay for it.
Cashed-up private medical insurers in rude good health
Dan Harrison Head of the agency charged with advising the government on private health insurance premium rises says the industry is in 'pretty good nick', despite Abbott government recently signing off on the...


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Claim you profile
Zara Whiley
Kidderminster, England, UK
Training Ex-Racehorses
I have successfully re-trained 2 ex-racehorses and have recently aquired my third who is doing very well and proving to be a good all rounder. I have competed my ex-racer in showjumping, dressage, ridden showing and have done fun rides with him aswell.
I do basic flat, pole and lateral work with every horse I re-train and also jumping depending on the horse's conformation, behaviour and tendons, this is so that they have the basics to do any discipline.
I have worked in a race yard, working race horses on the flat, up gallops and some jumping aswell. This experience has enabled me to understand how they are worked when racing and therefore makes it less stressfull for the horse and sometimes easier for me when re-training them.
I have a lot of experience clipping horses/ponies and I have my own battery powered clippers. I have done all sorts of clips from a bib, neck and belly upto hunter/full clip, I have also clipped designs onto my own horses and have had possitive comments from people when out competing and hacking.
Show Jumping
I have competed in unaffiliated showjumping 2'0" - 3'3", competed in MIDARC championships, have done team and pairs jumping. I have successfully tought young horses and ex-racehorses to jump.
I have competed in unaffiliated dressage but I have ridden experienced dressage horses. I have a natural light seat, leg position, soft hands and good balance. I have had good feed back from judges when competing.
Flat Work
I do a lot of flat work with any horse I ride, I have found that it improves the overall rideability of the horse and can also improve how the horse jumps.
I have schooled a variety of horses and ponies on the flat, from 2/3 year olds upto 28 year olds and everything inbetween this includes cobs, hunters, show ponies, dressage, showjumpers, ex-racehorses, race horses, hacks, inexperienced/green horses ect.
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Monday, 11 Nov 2013 03:12 PM
About 49,100 people have enrolled in Obamacare plans through 12 state-run insurance exchanges, according to a consulting company that's providing a hint on the data congressional Republicans sought during hearings in the past two weeks.
Enrollment through Nov. 10 represents 3 percent of the 1.4 million people projected to sign up in those states by the end of 2014, Washington-based Avalere Health said in a statement. The data doesn't include California, the most populous U.S. state, Massachusetts or Oregon. It also doesn't account for those enrolled through the federal website serving 36 states.
A nationwide tally for October will be released this week, according to congressional testimony by Obama administration officials, who have said they expect enrollment to be low initially. Republican lawmakers have been pressuring the government to reveal just how much sign-ups have been affected by website outages and other flaws plaguing the federal website.
"Enrollment in new programs begins slowly and often takes several months to build momentum," said Dan Mendelson, the chief executive officer of Avalere Health. "With aggressive marketing there is still time for awareness of the program to grow and participation to begin."
In Avalere's tally, New York's exchange reported 13,300 people enrolled in plans from Oct. 1 through Nov. 10, the highest number of enrollees amongst the 11 states plus Washington, D.C., that have shared data. Washington state had the second-highest enrollment at 7,300.
800,000 Target
The exchanges were created so people can shop for private health insurance with the help of government subsidies, a core part of the Patient Protection and Affordable Care Act of 2010. The administration had a target of 800,000 sign-ups nationwide for the first two months, and the Congressional Budget Office has projected that 7 million people would enroll through 2014.
Memos from administration staff meetings showed that the exchanges had enrolled 248 people in their first two days as software errors shut out customers. Kathleen Sebelius, the secretary of the U.S. Department of Health and Human Services, said the federal exchanges should be fully functioning by the end of this month.
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