RESEARCH ARTICLE
◥GERM CELL BIOLOGY
Transcription factor AP2 controls cnidarian germ
cell induction
Timothy Q. DuBuc^1 *, Christine E. Schnitzler2,3, Eleni Chrysostomou^1 ,EmmaT.McMahon^1 , Febrimarsa^1 ,
James M. Gahan^1 †, Tara Buggie^1 , Sebastian G. Gornik^1 ‡, Shirley Hanley^4 , Sofia N. Barreira^5 ,
Paul Gonzalez^5 , Andreas D. Baxevanis^5 , Uri Frank^1 §
Clonal animals do not sequester a germ line during embryogenesis. Instead, they have adult stem
cells that contribute to somatic tissues or gametes. How germ fate is induced in these animals, and
whether this process is related to bilaterian embryonic germline induction, is unknown. We show that
transcription factor AP2 (Tfap2), a regulator of mammalian germ lines, acts to commit adult stem
cells, known as i-cells, to the germ cell fate in the clonal cnidarianHydractinia symbiolongicarpus.
Tfap2mutants lacked germ cells and gonads. Transplanted wild-type cells rescued gonad development
but not germ cell induction inTfap2mutants. Forced expression ofTfap2in i-cells converted them
to germ cells. Therefore, Tfap2 is a regulator of germ cell commitment across germ line–sequestering
and germ line–nonsequestering animals.
S
egregation of germ cells from somatic
fate is an irreversible, once-in-a-lifetime
event that is induced during embryonic
development by maternal or zygotic fac-
tors in many bilaterians ( 1 ). The intro-
duced barrier between soma and germ line
(known as the Weismann barrier) prohibits
somatic cells from contributing to gamete pro-
duction, and vice versa, thereby preventing the
transmission of somatic mutations to future
generations. By contrast, clonal animals, such
as sponges and some cnidarians, do not se-
quester a germ line ( 2 – 4 ). Instead, these ani-
mals maintain a population of adult stem
cells throughout life that retain the ability to
differentiate into somatic cells and into gametes
(Fig. 1A). Other animals—such as sea urchins,
snails, and annelids—specify their germ cells
after embryogenesis, but it is unknown whether
this process occurs only once or multiple times,
as in clonal animals ( 5 ).
The molecular mechanisms that induce germ
cell commitment are understood in a few germ
line–sequestering animals ( 6 – 9 ), but the genes
that induce germ cell fate in clonal species
remain unknown. This raises the question of
whether the differences in the timing of animal
germ cell specification are temporally distinct
manifestations of a shared molecular program
or have independent evolutionary origins.
We find that a single gene,transcription
factor AP2(Tfap2), is sufficient to induce germ
fate when expressed in adult stem cells in
the clonal cnidarianHydractinia.Tfap2isalso
required non–cell autonomously for proper gonad
development. A homologous gene,Tfap2C,isa
major regulator of mammalian germ cell in-
duction, which is consistent with this gene
being an ancient regulator of animal germ cells.Hydractiniaas a model for germ cell induction
in clonal animals
Hydractinia symbiolongicarpusis a clonal, co-
lonial hydrozoan cnidarian [see ( 3 ) for a
definition of coloniality]. Adult stem cells in
hydrozoans, known as i-cells ( 10 ), generate
progenitors to somatic lineages and to ga-
metes ( 11 ). Commitment to germ cell fate in
Hydractiniaoccurs continuously after reach-
ing sexual maturation in an anatomically de-
fined location ( 12 , 13 ) (Fig. 1B), making the
animal an accessible and attractive model sys-
tem to study this alternative, continuous mode
of germ cell specification.Hydractiniacolonies
are composed of genetically identical (clonal)
modular units called polyps that arise by asex-
ual budding from a single sexually produced
individual (fig. S1A). All polyps in a colony are
connected by stolonal tissue, allowing i-cell
migration throughout the colony. A newly formed
colony consists exclusively of nonreproductive
feeding polyps. Sexual polyps, which are mor-
phologically distinct (Fig. 1B and fig. S1, B and
C), appear approximately 2 months after meta-
morphosis. The body columns of both polyp
types are composed of outer epidermal and inner
gastrodermal tissues (Fig. 1B). The animal’sstemcells (the i-cells) are located exclusively in the
interstitial spaces between epithelial cells in
the epidermis and are marked by germline
multipotency program (GMP) gene expression
( 14 ); this includes, e.g.,Piwi1(Fig. 1C and figs.
S1 and S2),Vasa,andPl10( 15 ). In sexual polyps,
i-cells can acquire germ cell fate and become
gamete progenitors (Fig. 1C and fig. S1C). Early
germ cells concentrate in a narrow tissue stripe
at the neck of the sexual polyp, referred to as the
germinal zone ( 12 , 13 ), from which they migrate
into the sporosacs and mature. Germ cells ex-
press GMP genes, similar to i-cells from which
they were derived, making them the only GMP+
gastrodermal cells inHydractiniacolonies
and, therefore, easy to recognize (Fig. 1, B and
C).Hydractiniais gonochoristic, and the sexual
polyp is the exclusive site of gametogenesis,
making it functionally equivalent to gonads in
bilaterians. Early stages of male and female
sexual polyp development are morphologically
indistinguishable (fig. S1C).Tfap2 is expressed in male and female
germ cells
To identify candidate regulators of germ cell
commitment inHydractinia, we compared
gene expression between feeding and sexual
polyps. A previous study ( 16 ) compared the
transcriptomes of differentHydractiniapolyp
types using pooled male and female samples.
Analyzing these data, we found that some
genes reported to be up-regulated in sexual
polyps are primarily female specific (fig. S3)
and are probably involved in oogenesis rather
than in the earlier-occurring germ cell induc-
tion, which is likely shared by males and fe-
males ( 17 , 18 ). Therefore, we repeated this
experiment but generated separate male and
female RNA sequencing (RNA-seq) libraries
from the heads and bodies of sexual and feed-
ing polyps. This enabled us to identify genes
that are commonly up-regulated in both sexes
during sexual development and allowed us to
test whether they are differentially expressed
between the polyps’oral and aboral regions
(Fig. 2, A and B, and table S1).
Tfap2emerged as a potential candidate gene
for germ cell induction, given the known role of
one of its homologs (Tfap2C) in mammalian germ
cell specification ( 8 , 19 – 23 )andgiventhatitisnot
sex-specific inHydractinia(Fig. 2, B to D, and
table S1).Tfap2genes are found across the Meta-
zoa, including the four nonbilaterian phyla: Cte-
nophora, Porifera, Placozoa, and Cnidaria (fig. S4).
TheHydractiniagenome encodes twoTfap2-
like genes (Tfap2aandTfap2b), with phyloge-
netic analyses suggesting that they are paralogs
(fig. S4).Tfap2bmRNA could not be detected
by either reverse transcription polymerase
chain reaction (RT-PCR) or in situ hybridiza-
tion (fig. S4), making it a likely pseudogene.
On the other hand,Tfap2a(henceforthTfap2)
was exclusively expressed in the germinal zoneRESEARCH
DuBucet al.,Science 367 , 757–762 (2020) 14 February 2020 1of6
(^1) Centre for Chromosome Biology, School of Natural Sciences,
National University of Ireland Galway, Galway, Ireland.^2 Whitney
Laboratory for Marine Bioscience, University of Florida,
St. Augustine, FL 32080, USA.^3 Department of Biology,
University of Florida, Gainesville, FL 32611, USA.^4 National
Centre for Biomedical Engineering Science, National University
of Ireland Galway, Galway, Ireland.^5 Computational and
Statistical Genomics Branch, Division of Intramural Research,
National Human Genome Research Institute, National
Institutes of Health, Bethesda, MD 20892, USA.
*Present address: Biology Department, Swarthmore College,
Swarthmore, PA 19081, USA.†Present address: Sars International Centre
for Marine Molecular Biology, University of Bergen, Thormøhlensgt
55, 5008 Bergen, Norway.‡Present address: Centre for Organismal
Studies, Heidelberg University, Heidelberg 69120, Germany.
§Corresponding author. Email: [email protected]