Science 14Feb2020

of female and male sexual polyps (Fig. 2, C
and D, and fig. S4). Given thatPiwi1marks
i-cells (in the epidermis) and germ cells (in
the gastrodermis; Fig. 1C), we performed double-
fluorescence in situ hybridization in sexual
polyps to detectPiwi1-andTfap2-expressing
cells. We found double-positive cells in both
the epidermis and the gastrodermis, with the
latter containing the majority (Fig. 2D). Some
Tfap2+ cells in the gastrodermis werePiwi1−.
The function of these cells is currently unknown.
We developedTfap2transgenic reporter
animals that enabled us to study the localiza-
tion ofTfap2in vivo (Fig. 2C, fig. S5, and movie
S1). The expression pattern observed in the
transgenic reporter animals and the in situ
localization ofTfap2mRNA are consistent
withTfap2being expressed not only in re-
cently induced germ cells but also in early
gametogonia and inPiwi1−cells whose func-
tion remains unknown. Notably,Tfap2is down-
regulated in late gametogonia and gametocytes,
as well as in gametes.

To gain insight into the genes acting down-

stream of induction to activate the germ cell

transcriptional program, we compared the

transcriptomes of isolated germ cells with

those of their i-cell progenitors and with so-

matic cells. For this, we dissociated sexual

polyps from male and femaleTfap2reporter

animals and feeding polyps from aPiwi1re-

porter animal ( 15 ). We established a fluorescence-

activated cell sorting (FACS) protocol to sort

GFP+cells at high purity (figs. S6 and S7). We

also collected GFPlowand GFP−cells from the

Piwi1reporter, representing all somatic lineages

(fig. S6C). Transcriptomic analysis of these cells

revealed up-regulation of conserved germ cell

genes inTfap2+cells as compared with i-cells

(fig. S6, D and E). However, many germ cell

genes, such asPiwi1/2,Nanos1/2,andPl10,were

expressed in both cellular fractions (fig. S6D

and dataset S1). This reflects the dual com-

petence of i-cells to contribute to both somatic

cells and germ cells. The long half-life of GFP

resulted in the inclusion of not only recently

induced germ cells but also of gametocytes

that no longer expressedTfap2.Thiswasmade

evident by the up-regulation of late female and

male gametogenesis genes and meiosis genes

(fig. S6E and dataset S1). These results are in

line with those of previous studies showing that

the metazoan germ cell transcriptional program

downstream of induction is partly conserved

across clades ( 24 ).

Tfap2 is essential for germ cell commitment

and gonad development

Next, we performed CRISPR-Cas9–mediated

mutagenesis experiments to study the role of

Tfap2in sexual development ( 25 – 28 ). Two sin-

gle guide RNAs (sgRNAs) were designed to

target the 5′and 3′ends of the predicted DNA

binding domain of theTfap2gene, respec-

tively (fig. S8); these were then injected into

zygotes, together with recombinant Cas9. In-

jected embryos were allowed to develop into

larvae, metamorphose, and grow to ages at

which sexual maturity is normally reached.

DuBucet al.,Science 367 , 757–762 (2020) 14 February 2020 2of6

head

body

column

feeding polyp mature sexual polyp

embryonic

cell

embryonic

cell (i-cell)

embryogenesis adult life

Hydractinia

Most Animals

germ cell

germ cell

germ

cell

i-cell

i-cell

somatic

cells

somatic

cells

A

embryonic

tissue

ectoderm

endoderm

mesoderm

B

i-cells

germinal

zone with

early germ

cells

eggs

sperm

gametes

gametes

i-cells

C

Piwi1 mRNA Piwi1 protein Piwi^1 mRNA Piwi1 protein mRNA Piwi1 protein

200 μm 50 μm

feeding polyp male sexual polyp female sexual polyp

epidermis mesoglea

gastrodermis

gastrodermis

gastrodermis

Fig. 1. Sexual development inHydractinia.(A) Timing of germ cell induction in
germ line–sequestering and germ line–nonsequestering animals. (B) Tissue
architecture and location of i-cells (pink) and germ cells (green) inHydractinia
feeding polyp and a hypothetical sexual polyp with both sexes. (C) Expression
of Piwi1 in feeding and sexual polyps. Solid blue line indicates the body’sepidermal

outline. Dashed green line indicates the basement membrane (mesoglea)

separating the epidermis and gastrodermis. Piwi1+cells in the epidermis (i-cells)

are encircled in purple. Piwi1+cells in the gastrodermis are germ cells. Asterisks

denote the oral pole. The distribution of i-cells can vary between polyps and

extends more orally in sexual polyps compared with feeding polyps.

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