Science 14Feb2020

The grafting procedure (Fig. 4A) allowed
i-cells and progeny to migrate between the
partners, generating chimeras whose cellular
origin could be directly observed in vivo by
fluorescence microscopy ( 30 ) (Fig. 4, D to G,
and movie S2). We found that cells from the
wild-type animal, which had migrated into
the mutant, induced development of sexual
polyps that consisted somatically of mutant
and wild-type cells (Fig. 4F). However, the
gametes produced by the chimeric sexual
polyps were exclusively fluorescent and, thus,
donor derived; no mutant-derived gametes
were obtained (Fig. 4G). Given that non-
chimeric mutant animals only produced ru-
dimentary sexual polyps, we conclude that
Tfap2 expressed in donor-derived cells acted
non–cell autonomously to promote sexual
polyp development in the mutant but could
not induce mutant i-cells to germ fate. In bi-
laterians, germ cells are necessary for proper
gonad development in some species ( 31 , 32 ),
and our results are consistent with a previ-
ously described phenomenon in animals.

Tfap2 acts cell autonomously to induce germ

fate in i-cells

To investigate a cell-autonomous role of Tfap2,

we used a random-integration transgenesis

approach to ectopically expressTfap2in three

different cellular contexts using three trans-

genic constructs (fig. S10). This generated

mosaic transgenic animals expressing Tfap2-

GFP in different cell types. First, we used the

Wnt3promoter to drive Tfap2-GFP expres-

sion in the oral region, whereWnt3is normal-

ly expressed ( 33 – 35 ) (fig. S10A). As i-cells are

normally not present in the oral pole ( 15 ), we

expected to observe the consequences of Tfap2

expression in differentiated head cells. How-

ever,Wnt3promoter-induced Tfap2 expression

resulted in phenotype-free animals (Fig. 5A).

Next, we drove Tfap2-GFP expression by the

b-tubulinpromoter that is active in all differ-

entiated cells but not in i-cells (fig. S10, B and

C). This approach also resulted in no visible

phenotype (Fig. 5B), suggesting that Tfap2 can

induce neither germ cells nor gonads when

expressed in somatic cells.

Finally, we expressed Tfap2-GFP under the

Piwi1promoter to restrict transgene expres-

sion to i-cells (Fig. 5, C to J, and fig. S10, B and

C). This experiment resulted in large GFP+

cells that morphologically resembled early

stage oocytes in mosaic transgenic embryos

that were probably females (Fig. 5C). Other

embryos (probably males) displayed cells that

expressedH2B3/4, a spermatogenesis marker

( 36 ) (Fig. 5D and fig. S11). Normally, germ cells

appear 2 to 3 months after metamorphosis.

However, ectopic germ cells in embryos never

matured and vanished after metamorphosis.

This suggests that, whereas Tfap2 was effec-

tive in inducing germ fate in embryonicPiwi1+

cells, the larval tissue microenvironment could

not support gametogenesis downstream of

germ cell induction.

We hypothesized that ectopic oocytes would

develop to a later stage if Tfap2 expression

commenced only after metamorphosis. In the

absence of a conditional expression system in

Hydractinia, we focused on inhibiting trans-

gene expression until after metamorphosis.

DuBucet al.,Science 367 , 757–762 (2020) 14 February 2020 4of6

Piwi1:: Mutant 4

+

+

+

A

X

Tfap2+/+ Tfap2+/- Tfap2+/- Tfap2+/+

X

Tfap2-/- Tfap2+/+ Tfap2-/-

Insertion mutation and frameshift

Tfap2 alleles Tfap2 alleles (mosaic animal)

exon1 exon2 exon3 exon1 exon2 exon3

B

C DE F G

GFP

Fig. 3. Breeding strategy for generatingTfap2−/−knockout animals.
(A) Genomic structure of wild-type and mutant alleles ofTfap2.(B)G 1 generation
that includes homozygoteTfap2wild-type and heterozygote mutant animals; the
latter produced fewer gametes. All animals shown also carry aPiwi1reporter
transgene inherited from theirTfap2wild-type father. (CtoG) The G 2 generation
resulting from breeding G 1 siblings. (C) Overview ofTfap2−/−homozygote

mutant. Only rudimentary sexual polyps are present (arrowheads); the colony

appears otherwise normal. (D and E)Tfap2wild-type rudimentary male and

female sexual polyps. Early gastrodermal germ cells express GFP, driven by the

Piwi1reporter transgene. (F) Rudimentary sexual polyp of aTfap2−/−mutant.

This animal also carries aPiwi1reporter transgene but has no germ cells. (G)

Close-up of the same polyp in (F), showing GFP+epidermal i-cells.

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