Science 14Feb2020

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ACKNOWLEDGMENTS

We thank our laboratory members for lively discussions, our

colleagues C. Morrison and G. Schlosser for comments on the

manuscript, and the NIH Intramural Sequencing Center (NISC) for

generating the sequence data. All flow cytometry and imaging

cytometry analyses were performed in the Flow Cytometry Core

Facility at NUI Galway.Funding:U.F. is a Wellcome Trust

Investigator in Science (grant no. 210722/Z/18/Z, co-funded by

the SFI-HRB-Wellcome Biomedical Research Partnership). This

work was also funded by a Science Foundation Ireland Investigator

Award to U.F. (grant no. 11/PI/1020); by CURAM, SFI Centre for

Research in Medical Devices (to U.F.); and by the Intramural

Research Program of the National Human Genome Research

Institute, National Institutes of Health to A.D.B. (ZIA HG000140).

T.Q.D. was an EMBO Long-Term Fellow (grant no. ALTF 68-2016).

S.G.G. was a Marie Curie Incoming International Fellow (project

623748) and was also supported by a Science Foundation Ireland

SIRG award (grant no. 13/SIRG/2125). F. is a Hardiman Scholar

and is also supported by Thomas Crawford Hayes Research Grant.

Funding in support of imaging cytometry was received from

Science Foundation Ireland under research infrastructure grant

no. 16/RI/3760 and from the European Regional Development Fund.

Author contributions:T.Q.D. and U.F. conceptualized this project.

T.Q.D. collected all RNA samples, generated stable transgenic

animals, created CRISPR-Cas9 mutants, conducted short hairpin

experiments, and performed all microinjections, IF, and in situ

hybridization experiments. T.B. and T.Q.D. performed mutant screens.

E.C., S.H., and T.Q.D. performed the FACS experiments. C.E.S., S.N.B.,

P.G., S.G.G., and A.D.B. analyzed all RNA-seq data and performed

the computational analysis. E.T.M. generated the Piwi1 antibody.

F. designed and tested the shRNAs. J.M.G. cloned theb-tubulin

regulatory regions. T.Q.D. and U.F. wrote the paper.Competing

interests:The authors declare no competing interests.Data and

materials availability:The raw reads utilized to generate the tissue-

and cell-specific differential expression analyses (table S1 and dataset

S1) are available through the National Center for Biotechnology

Information (NCBI) Sequence Read Archive (SRA) at http://www.ncbi.nlm.

nih.gov/sra. Tissue-specific reads fromHydractinia echinataare

available as accession numbers SRR9332370 to SRR9332387. Bulk

cell reads fromH. symbiolongicarpusare available under the

accession numbers SRR9331388 to SRR9331403. Detailed descriptions

of each dataset can be found in the SRA Data tab of table S1 and

dataset S1. Transcriptomes generated for this manuscript and draft

genomes for both species are available for download at theHydractinia

Genome Portal (https://research.nhgri.nih.gov/hydractinia/).

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/367/6479/757/suppl/DC1

Materials and Methods

Figs. S1 to S12

Table S1

References ( 41 – 63 )

Movies S1 and S2

Datasets S1 and S2

View/request a protocol for this paper fromBio-protocol.

10 July 2019; accepted 6 December 2019

10.1126/science.aay6782

DuBucet al.,Science 367 , 757–762 (2020) 14 February 2020 6of6

Wnt3::Tfap2-GFP

regional

germ

cell

somatic cells

β-Tubulin::Tfap2-GFP

germ

cell

germ cell

Tfap2

GFP

Merge

Piwi1::Tfap2-GFP

A B

C

D

EI

F

H2B3/4

20 μm

100 μm

20 μm

i-cells

K

i-cell

J

Cnidarian

egg lectin

H

G

Fig. 5. Ectopic expression ofTfap2in i-cells induces germ fate.(A)Wnt3
promoter-driven Tfap2-GFP. Transgene expression is restricted to the oral end and
causes no visible phenotype. (B)b-tubulinpromoter-driven Tfap2-GFP. Transgene is
expressed in somatic cells and causes no visible phenotype. (CtoK)Piwi1promoter-
driven Tfap2-GFP. Transgene is expressed only in i-cells, transforming them to
germ cells. (C) Ectopic early oocyte in the gastrodermis of a 48-hour-old larva,
identified by morphology. (D) Sperm progenitors in the gastrodermis of a 48-hour-old

larva, identified byH2B3/4-expression. (E and F) Ectopic oocytes in the gastrodermis

of a mosaic transgenic feeding polyp expressing Tfap2-GFP under thePiwi1promoter,

where the transgene had been suppressed for 2 weeks by a shRNA. (G to I) Double

mRNA fluorescence in situ hybridization showing colocalization ofGFPandCelin a

mosaic transgenic feeding polyp treated as in (E). (G)GFPmRNA. (H)CelmRNA.

(I) Merge. (J) Schematic illustrating the localization of (G) to (I) in the polyp. (K)

Tfap2 expression in i-cells converts them to germ cells.

RESEARCH | RESEARCH ARTICLE