Science 14Feb2020

For this, we coinjected zygotes with thePiwi1::
Tfap2-P2A-GFPectopic expression construct
alongside a short hairpin RNA (shRNA) ( 37 ),
targeting the GFP sequence in the transgene’s
mRNA.Injectedembryosandlarvaeremained
GFP-free, consistent with effective transgene
repression by the shRNA (fig. S12). The suppres-
sive effect of the shRNA dissipated ~1 week after
metamorphosis, and transgenic feeding polyps
developed ectopic GFP+oocytes in the gastro-
dermis that appeared morphologically more
mature than ectopic oocytes in larvae (Fig. 5, E
and F). Furthermore, they expressedCnidarian
egg lectin(Cel)—an exclusive early oogenesis
marker ( 38 )—in GFP+cells(Fig.5,GtoJ),which
was undetectable in larval ectopic oocytes. Taken
together, our results show that Tfap2 acts cell
autonomously and is essential and sufficient
to induce germ cell fate inPiwi1+i-cells but not
in differentiated cells (Fig. 5K). Tfap2 acts non–
cell autonomously downstream of germ cell
induction to drive sexual polyp maturation.

The evolution of bilaterian sequestered

germ lines

Tfap2 is a critical regulator of germ cell in-

duction inHydractinia,ananimalthatdoes

not sequester a germ line, as well as in germ

line–sequestering animals, such as mice ( 8 )and

humans ( 39 ).DrosophilaandC. elegansgerm

lines are specified maternally, a mechanism

that is thought to be evolutionarily derived

( 1 ). In mammals, AP2g(encoded byTfap2C)

acts in concert with other transcription factors,

such as Blimp1, Prdm14, PAX5, and SOX17, to

induce germ cell fate in epiblast cells in a

species-specific combination ( 7 , 8 , 40 ). Partners

of Tfap2 inHydractiniaare yet unknown, but,

unlike in mammals, its expression in i-cells—

which are similar to epiblast cells in being

somatic and germ cell–competent—is sufficient

to induce germ cell fate. It has been suggested

that a nonsequestered germ line is an ances-

tral trait in metazoans ( 4 ). Under this hypoth-

esis, a key event in the evolution of bilaterian

sequestered germ lines would have been the

redeployment of the hypothesized ancestral

adult germ cell induction program, which is

still present in extantHydractinia, during

embryogenesis.

A sequestered germ line prevents transmis-

sion of somatic mutations to future genera-

tionsandwasalsoproposedtohelpselectfor

mitochondrial quality in complex bilaterians

with high mutation rates ( 4 ). Why, then, would

clonal animals not sequester a germ line? We

suggest that a sequestered germ line in clonal

animals would be detrimental, exposing them

to the risk of stochastically generating new

clonal individuals that lack germ cells. There-

fore, maintaining uncommitted cells after em-

bryogenesis, which can provide progenitors to

any lineage of somatic cells or germ cells, grants

full developmental potential to new clonemates.

Thismayrepresenttheselectivepressurethat

prevented the evolution of a sequestered germ

line in clonal animals.

DuBucet al.,Science 367 , 757–762 (2020) 14 February 2020 5of6

Fig. 4. Transplantation
of wild-type, alloge-
neic cells into aTfap2
mutant.(A) Experimen-
tal setup. Dashed red
line denotes the interface
between the two
grafted animal colonies.
(B) FemaleTfap2
wild-type, fluorescent
donor feeding polyp.
(C) Recipient mutant
colony, lacking sexual
polyps. (D) The interface
between the grafted
donor and recipient col-
onies (dashed red line),
viewed from above.
Donor-derived cells are
visible in the recipient’s
tissues. They are more
numerous closer to the
interface. (E)Fluorescent,
Tfap2wild-type sexual
polyp of the donor animal.
Oocytes are encircled
by a dashed white line.
(F) Immature chimeric
sexual polyp composed
of donor (fluorescent,
Tfap2wild type) and
recipient (nonfluorescent,
Tfap2mutant) cells.
(G) Mature chimeric
sexual polyp. Oocytes
encircled by dashed line
are exclusively donor-
derived. Animals were pictured live and the red and the green channels representing theb-tubulin::mScarletandPiwi1::GFPreporter transgenes were merged
and false-colored green in (B) and (D) to (G) for simplicity. The blue color in (E) to (G)represents DNA. All 10 grafts that were performed resulted in
induction of sexual polyps in the mutant.

Migration of cells

Tfap2

wild type tissue

Tfap2

mutant tissue

C

D

EF G

A

Tfap2 mutant

recipient

Tfap2 wild type

fluorescent donor

migration

invading

cells

Tfap2 wild type Tfap2 mutant

Wild type fluorescent sexual polyp Young chimeric sexual polyp Mature chimeric sexual polyp

invading

cells

B

200 μm

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