Science 14Feb2020

They were then genotyped by PCR and se-
quencing to check forTfap2mutations. Sev-
eral types of mutations were identified in
these injected animals, including small or
large deletions and insertions, frameshifts,
and base substitutions (Fig. 3A and fig. S8). The
mutants displayed various defects in sexual
development that included too few (but other-
wise normal) sexual polyps, deformed sexual
polyps, and rudimentary sexual polyps that
never matured and contained no germ cells
(Fig. 3, C to G, and fig. S8). G 0 mutants were
mosaics with multiple alleles, including wild-
type alleles in several cases. Crossing a G 0
mosaic mutant with aTfap2wild-type animal,
we found that G 1 heterozygote mutant i-cells
(i.e.,Tfap2+/−) could still commit to germ fate,

though less effectively (Fig. 3B), generating fer-

tile, mutant gametes. This allowed us to breed

these animals to homozygosity (i.e.,Tfap2−/−;

Fig. 3, C, F, and G).Tfap2−/−animals were

sterile, with no detectable germ cells and only

rudimentary sexual polyps (Fig. 3, C, F, and G),

which implies that Tfap2 is required for both

germ cell induction and gonad development.

The mutants had normal distributions of

i-cells, and their growth and regenerative

ability was not compromised (fig. S9).

Tfap2 acts non–cell autonomously to induce

sexual polyp development

To identify a possible non–cell autonomous role

for Tfap2 in sexual development, we trans-

planted cells from an animal with a wild-type

Tfap2gene into a sterileTfap2mutant (Fig. 4A).

To facilitate the tracking ofTfap2wild-type

cells in theTfap2mutant’s tissues, we gener-

ated a transgenic fluorescent reporter animal

as a cell donor that carried two reporter trans-

genes: aPiwi1reporter that expressed GFP in

i-cells and germ cells ( 15 )andab-tubulinre-

porter that expressed mScarlet in all other cell

types, except for i-cells (fig. S10). Hence, all cells

in the donor animal were fluorescent (Fig. 4B

and fig. S10) and, therefore, visible after trans-

plantation in the background of the nonfluores-

cent and sterileTfap2mutant that lacked germ

cells and mature gonads (Fig. 4 and movie S2).

Donor animals were fertile (Fig. 4E), having a

wild-typeTfap2genotype, and were geneti-

cally histocompatible ( 29 ) with the mutant.

DuBucet al.,Science 367 , 757–762 (2020) 14 February 2020 3of6

Tfap2Tfap2 Piwi1Piwi1 MergeMerge

20 μm 20 μm 20 μm

S

e

xu

a

l

H

e

a

eF d

e

di

n

g

H

e

a

d

Tfap2

Dvr-1

FoxO-like

Integrin beta-2

FoxQ2-like

Otx homeobox

Collier

Cnidarian egg lectin

Cnox4 - homeobox

Tbx3 - homeobox

Antennapedia-like

Myb

Doublesex

homeobox

Bye1

Net-like

Meis homeobox

Feeding Polyp Tissue Sexual Polyp Tissue

Orthopedia

Kielin/Chordin

Bmp2/4-Like

TgfB

Astacin

Chymotrypsin

Ash2

Wnt2

Chordin-like

Six6

Pax E

Pax C

POU homeobox

Posterior-like hox

Nkx 6.3

Dickkopf-3

Tolloid-like

POU homeobox

Posterior-like hox

Nkx 6.3

Dickkopf-3

Tolloid-like

head

body

All feeding polyp tissue

head

body

All sexual polyp tissue

A B

Tfap2::GFPTfap2::GFP

C

Male Body Male Body

Female Body Female Body

D

SchematicSchematic

Tfap2+Tfap2+

Piwi1+Piwi1+

epidermis

gastrodermis

Tfap2+Tfap2+ Piwi1+Piwi1+

Fig. 2. Sexually up-regulated genes inHydractinia.(A) Schematic of tissue
sampling strategy. (B) Differentially expressed genes in different tissue
compartments. (C) Live image of aTfap2transgenic female reporter animal
expressing GFP in the germinal zone (green arrowhead). (D) mRNA fluorescence

double in situ hybridization ofPiwi1andTfap2in a male sexual polyp. Arrowhead

points to double-positive cell in the epidermis. Asterisks denote the oral pole.

In the schematic, pink cells arePiwi1positive, yellow cells arePiwi1andTfap2

positive, and green cells areTfap2positive.

RESEARCH | RESEARCH ARTICLE