A positive feedback loop between germ cells and gonads induces and maintains sexual reproduction in a cnidarian

Abstract

The fertile gonad includes cells of two distinct developmental origins: the somatic mesoderm and the germ line. How somatic and germ cells interact to develop and maintain fertility is not well understood. Here, using grafting experiments and transgenic reporter animals, we find that a specific part of the gonad-the germinal zone-acts as a sexual organizer to induce and maintain de novo germ cells and somatic gonads in the cnidarian Hydractinia symbiolongicarpus. Germ cells express a member of the transforming growth factor-β family, Gonadless (Gls), that induces gonad morphogenesis. Loss of Gls resulted in animals lacking gonads but having nonproliferative germ cells. We propose that primary germ cells drive gonad development though Gls secretion. The germinal zone in the newly formed gonad provides positive feedback to induce secondary germ cells by activating Tfap2 in resident pluripotent stem cells. The contribution of germ cell signaling to the patterning of somatic gonadal tissue may be a general animal feature.

Fig. 1.. Hydractinia sexual development.

(A) Feeding polyp morphology showing i-cell localization (green) in the epidermis of the lower part of the body column. (B) Sexual polyp morphology showing germ cells (orange) and their commitment from i-cells in the germinal zone. Once committed, germ cells become gastrodermal and start their maturation into functional gametes. The left side represents a male and the right side a female. (C) Close-up of the germinal zone [black rectangle in (B)] showing i-cells being induced to germ cell fate by Tfap2 expression.

Fig. 2.. De novo induction of germ cells and somatic gonad tissue upon heterotopic grafting of gonad tissue onto feeding polyps.

(A) Tfap2::GFP reporter animal. (B) Cartoon of the experimental grafting procedure showing different parts of sexual polyps grafted to a feeding polyp body column. WT, wild type. (C to F) Outcomes 5 days after grafting of feeding head grafting (C); sporosac region without germinal zone grafting (D); oral tip only (E); head including germinal zone graft showing induction of new germ cells (GFP⁺) in the recipient feeding polyp (F). White arrows show developing sporosacs in (F). White dashed line shows the outline of the polyp. (G) An Ef1a::GFP knock-in animal with ubiquitous GFP expression. (H) Cartoon of a chimera between a wild-type germinal zone (orange) and an Ef1a::GFP knock-in feeding polyp (green) at day 0 after grafting. (I) Confocal images of a representative chimera at day 5 after grafting. White rectangles show close-up views of germinal zone (1) and sporosac (2), both composed of a mixture of GFP⁺ and GFP⁻ cells. Scale bar, 100 μm.

Fig. 3.. Gls expression pattern.

(A) Cartoon of a sexual polyp. The black box corresponds to the position of the confocal images. (B) Maximum projection of mRNA in situ hybridization of Tfap2 and Gls in a male sexual polyp. DAPI, 4′,6-diamidino-2-phenylindole. (C) Maximum projection of mRNA in situ hybridization of Tfap2 and Gls in a female sexual polyp. Scale bar, 50 μm. Green rectangle corresponds to the close-up view shown in (D). (D) Single confocal section showing expression of Tfap2 and Gls in the cells boxed in (C). White dashed line represents the outline of the epidermis, and continuous white line represents the basement membrane (mesoglea) separating the gastrodermis from the epidermis. Scale bar, 10 μm.

Fig. 4.. Morphological and cellular characterization of Gls^−/− animals.

(A) Graphical representation of the genomic structure of wild-type and mutant alleles of Gls. (B) A Gls^−/− mutant, having only feeding polyps. (C) Maximum projection of mRNA in situ hybridization of Tfap2 in wild-type (upper) and Gls^−/− (lower) feeding polyps. Tfap2⁺ germ cells are present in the mutant but not in the wild-type feeding polyp. (D) Cartoon of a wild-type sexual polyp. The black box corresponds to the position of the confocal images shown in (E) and (F). (E and F) Maximum projection of immunostaining of Piwi1 (green) and BrdU (red) in the gastrodermis of a female (E) and a male (F). Yellow boxes represent a close-up view of a single confocal section shown below, respectively. Germ cells proliferate in wild-type animals. (G) Cartoon of a Gls^−/− feeding polyp. The black box corresponds to the position of the confocal images shown in (H) and (I). (H and I) Maximum projection of immunostaining for Piwi1 (green) and BrdU (red) in the gastrodermis of a female mutant (H) or of a male mutant polyp. Yellow boxes represent a close-up of a single confocal section showing that germ cells do not proliferate in Gls^−/− mutants. Scale bar, 100 μm. Scale bar in the boxes, 10 μm.

Fig. 5.. Heterotopic grafting of Ef1a::GFP fluorescent Gls^+/+ sexual tissue onto sterile Gls^−/− animals.

(A) Cartoon of the experimental grafting procedure. (B) Confocal images of a chimera, 5 days after grafting. Scale bar, 100 μm. White squares represent closes-up images of a newly formed sporosac (1) and the germinal zone (2), showing that they are composed of a mix of Gls^−/− (GFP⁻) and Gls+/+ (GFP⁺) cells. Scale bar, 10 μm. (C) A model for Hydractinia sexual development. Germ cells induce somatic gonad morphogenesis via Gls signaling, and the newly formed tissue, in turn, promotes secondary germ cell induction (by activating Tfap2 in pluripotent stem cells), proliferation, and maturation.