Transcription factor AP2 controls cnidarian germ cell induction

Abstract

Clonal animals do not sequester a germline during embryogenesis. Instead they have adult stem cells that contribute to somatic tissues or gametes. How germ fate is induced in these animals and whether this process is related to bilaterian embryonic germline induction is unknown. We show that Transcription factor AP2 (Tfap2), a regulator of mammalian germlines, acts to commit adult stem cells, known as i-cells, to the germ cell fate in the clonal cnidarian Hydractinia symbiolongicarpus. Tfap2 mutants lacked germ cells and gonads. Transplanted wild type cells rescued gonad development but not germ cell induction in Tfap2 mutants. Forced expression of Tfap2 in i-cells converted them to germ cells. Therefore, Tfap2 is a regulator of germ cell commitment across germline-sequestering and germline-non-sequestering animals.

Segregation of germ cells from somatic fate is an irreversible, once-in-a-lifetime event that is induced during embryonic development by maternal or zygotic factors in many bilaterians (1). The introduced barrier between soma and germline (also known as the Weismann barrier) prohibits somatic cells from contributing to gamete production, and vice versa, thereby preventing transmission of somatic mutations to future generations. By contrast, clonal animals, such as sponges and some cnidarians, do not sequester a germline (2–4). Instead, these animals maintain a population of adult stem cells throughout life that retain the ability to differentiate both into somatic cells and into gametes (Fig. 1A). Other animals, such as sea urchins, snails, and annelids, specify their germ cells after embryogenesis, but it is unknown whether this process occurs only once or multiple times as in clonal animals (5).

Figure 1. Sexual development in Hydractinia.

(A) Timing of germ cell induction in germline-sequestering and germline-non-sequestering animals. (B) Tissue architecture and location of i-cells (pink) and germ cells (green) in Hydractinia 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’s epidermal outline. Dashed green line indicates the basement membrane (mesoglea) separating 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 comparing to feeding polyps.

The molecular mechanisms that induce germ cell commitment are understood in a few germline-sequestering animals (6–9), but the genes that induce germ cell fate in clonal species remain unknown. This raises the question of whether the differences in timing of animal germ cell specification are temporally distinct manifestations of a shared molecular program or have independent evolutionary origins.

We find that a single gene, Transcription factor AP2 (Tfap2), is sufficient to induce germ fate when expressed in adult stem cells in the clonal cnidarian Hydractinia. Tfap2 is also required non-cell-autonomously for proper gonad development. A homologous gene, Tfap2C, is a major regulator of mammalian germ cell induction, consistent with this gene being an ancient regulator of animal germ cells.

Hydractinia as a model for germ cell induction in clonal animals

Hydractinia symbiolongicarpus is a clonal, colonial hydrozoan cnidarian (see ref. (3) for a definition of coloniality). Adult stem cells in hydrozoans, known as i-cells (10), generate progenitors to somatic lineages and to gametes (11). Commitment to germ cell fate in Hydractinia occurs continuously after reaching sexual maturation in an anatomically defined location (12, 13) (Fig. 1B), making the animal an accessible and attractive model system to study this alternative, continuous mode of germ cell specification. Hydractinia colonies are composed of genetically identical (clonal) modular units called polyps that arise by asexual budding from a single sexually produced individual (fig. S1A). All polyps in a colony are connected by stolonal tissue, allowing i-cell migration throughout the colony. A newly formed colony consists exclusively of non-reproductive feeding polyps. Sexual polyps, which are morphologically distinct (Fig. 1B; Fig. S1B and C), appear approximately two months post metamorphosis. The body columns of both polyp types are composed of outer epidermal and inner gastrodermal tissues (Fig. 1B). The animal’s stem cells (the i-cells) are located exclusively in interstitial spaces between epithelial cells in the epidermis and are marked by germline multipotency program (GMP) gene expression (14); this includes e.g. Piwi1 (Fig. 1C, and fig. S1 and S2), Vasa, and Pl10 (15). In sexual polyps, i-cells can acquire germ cell fate and become gamete progenitors (Fig. 1C and fig. S1C). Early germ cells concentrate in a narrow tissue stripe at the neck of the sexual polyp that is referred to as the germinal zone (12, 13), from which they migrate into the sporosacs and mature. Germ cells express GMP genes similar to that of the i-cell from which they were derived, making them the only GMP⁺ gastrodermal cells in Hydractinia colonies and, therefore, easy to recognize (Fig. 1B and C). Hydractinia is gonochoristic and the sexual polyp is the exclusive site of gametogenesis, making it functionally equivalent to gonads in bilaterians. Early stages of sexual polyp development appear identical in males and females (fig. S1C).

Tfap2 is expressed in male and female germ cells

To identify candidate regulators of germ cell commitment in Hydractinia we compared gene expression between feeding and sexual polyps. A previous study (16) compared the transcriptomes of different Hydractinia polyp types using pooled male and female samples. Analyzing these data, we found that some genes reported to be upregulated in sexual polyps are primarily female-specific (fig. S3) and are probably involved in oogenesis rather than in the earlier-occurring germ cell induction that is likely shared by males and females (17, 18). Therefore, we repeated this experiment but generated separate male and female RNA-seq libraries from heads and bodies of sexual and feeding polyps, enabling us to identify genes commonly upregulated in both sexes during sexual development, as well as allowing us to test whether they are differentially expressed between the polyps’ oral and aboral regions (Fig. 2A, B, and table S1).

Figure 2. Sexually upregulated genes in Hydractinia.

(A) Schematic of tissue sampling strategy. (B) Differentially expressed genes in different tissue compartments. (C) Live image of a Tfap2 transgenic female reporter animal expressing GFP in the germinal zone. (D) mRNA fluorescence double in situ hybridization of Piwi1 and Tfap2 in a male sexual polyp. Arrowhead points to double-positive cell in the epidermis. Asterisks denote the oral pole. In the schematic, pink cells are Piwi1 positive; orange cells are Piwi1 and Tfap2 positive; green cells are Tfap2 positive.

Transcription factor activating protein 2 (Tfap2) emerged as a potential candidate gene for germ cell induction, given the known role of one of its homologs (Tfap2C) in mammalian germ cell specification (8, 19–23) and for not being sex-specific in Hydractinia (Fig. 2B to D, table S1). Tfap2 genes are found across Metazoa, including all four non-bilaterian phyla: Ctenophora, Porifera, Placozoa, and Cnidaria (fig. S4).

The Hydractinia genome encodes two Tfap2-like genes (Tfap2a and Tfap2b), with phylogenetic analyses suggesting that they are paralogs (fig. S4). Tfap2b mRNA could not be detected by either RT-PCR or in situ hybridization (fig. S4), making it a likely pseudogene. On the other hand, Tfap2a (henceforth Tfap2) was exclusively expressed in the germinal zone of female and in male sexual polyps (Fig. 2C, D, and fig. S4). Given that Piwi1 marks i-cells (in the epidermis) and germ cells (in the gastrodermis; Fig. 1C), we performed double-fluorescence in situ hybridization in sexual polyps to detect Piwi1 and Tfap2 expressing cells. We found double-positive cells both in the epidermis and in the gastrodermis, with the latter being the majority (Fig. 2D). Some Tfap2+ cells in the gastrodermis were Piwi1^-. The identity of these cells is currently unknown.

We developed a Tfap2 transgenic reporter animal that enabled us to study the localization of Tfap2 in vivo (Fig. 2C, fig. S5, and movie S1). The expression pattern observed in the transgenic reporter animal, and the in situ localization of Tfap2 mRNA, are consistent with Tfap2 being expressed not only in recently induced germ cells, but also in early gametogonia and in Piwi1^- cells whose function remains unknown. Notably, Tfap2 is downregulated in late gametogonia and gametocytes, as well as in gametes.

To gain insight into the genes acting downstream 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 somatic cells. For this, we dissociated sexual polyps from male and female Tfap2 reporter animals and feeding polyps from a Piwi1 reporter animal (15). We established a fluorescence-activated cell sorting (FACS) protocol to sort GFP⁺ cells at high purity (fig. S6 and S7). We also collected GFP^low and GFP^- cells from the Piwi1 reporter, representing all somatic lineages (fig. S6C). Transcriptomic analysis of these cells revealed upregulation of conserved germ cell genes in Tfap2⁺ cells as compared to i-cells (fig. S6D and E). However, many germ cell genes, such as Piwi1/2, Nanos1/2, and Pl10, were expressed in both cellular fractions (fig. S6D and file S1). This reflects the dual competence of i-cells to contribute both to somatic cells and to 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 expressed Tfap2. This was evident by upregulation of late female and male gametogenesis genes and of meiosis genes (fig. S6E and file S1). These results are in line with 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 Tfap2 in sexual development (25–28). Two single guide RNAs (sgRNAs) were designed to target the 5’ and 3’ ends of the predicted DNA binding domain of the Tfap2 gene, respectively (fig. S8); these were then injected into zygotes, together with recombinant Cas9. Injected embryos were allowed to develop into larvae, metamorphose, and grow to ages where sexual maturity is normally reached. They were then genotyped by PCR and sequencing to check for Tfap2 mutations. Several 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 otherwise normal) sexual polyps, deformed sexual polyps, and rudimentary sexual polyps that never matured and contained no germ cells (Fig. 3C to G, and fig. S8). G0 mutants were mosaics with multiple alleles, including wild type ones in several cases. Crossing a G0 mosaic mutant with a Tfap2 wild type animal, we found that G1 heterozygote mutant i-cells (i.e. Tfap2^+/-) could still commit to germ fate, though less effectively (Fig. 3B), generating fertile, mutant gametes, allowing us to breed these animals to homozygosity (i.e. Tfap2^-/-, Fig. 3C, F, and G). Tfap2^-/- animals were sterile, having no detectable germ cells and only rudimentary sexual polyps (Fig. 3C, F, and G), implying that Tfap2 is required both for germ cell induction and for gonad development. The mutants had normal distribution of i-cells, and their growth and regenerative ability was not compromised (fig. S9).

Figure 3. Breeding strategy for generating Tfap2^-/- knockout animals.

(A) Genomic structure of wild type and mutant alleles of Tfap2. (B) G1 generation that includes homozygote Tfap2 wild type and heterozygote mutant animals; the latter produced fewer gametes. All animals shown also carry a Piwi1 reporter transgene inherited from their Tfap2 wild type father. (C-G) The G2 generation resulting from breeding G1 siblings. (C) Overview of Tfap2^-/- homozygote mutant. Only rudimentary sexual polyps are present (arrowheads). The colony appears otherwise normal. (D, E) Tfap2 wild type rudimentary male and female sexual polyps. Early gastrodermal germ cells express GFP, driven by the Piwi1 reporter transgene. (F) Rudimentary sexual polyp of a Tfap2^-/- mutant. This animal also carries a Piwi1 reporter transgene but has no germ cells. (G) Close-up of the same polyp shown in (F), showing GFP⁺ epidermal i-cells.

Tfap2 acts non-cell-autonomously to induce sexual polyp development

To identify a possible non-cell-autonomous role for Tfap2 in sexual development, we transplanted cells from an animal with a wild type Tfap2 gene into a sterile Tfap2 mutant (Fig. 4A). To facilitate the tracking of Tfap2 wild type cells in the Tfap1 mutant’s tissues, we generated a transgenic fluorescent reporter animal as a cell donor that carried two reporter transgenes: the first was a Piwi1 reporter that expressed GFP in i-cells and germ cells (15), whereas the other was a β-tubulin reporter 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 following transplantation in the background of the non-fluorescent and sterile Tfap2 mutant that lacked germ cells and mature gonads (Fig. 4, and movie S2). Donor animals were fertile (Fig. 4E), having a wild type Tfap2 genotype, and were genetically histocompatible (29) with the mutant.

Figure 4. Transplantation of wild type, allogeneic cells into a Tfap2 mutant.

(A) Experimental set-up. Dashed red line denotes the interface between the two grafted animal colonies. (B) Female Tfap2 wild type, fluorescent donor feeding polyp. (C) Recipient mutant colony, lacking sexual polyps. (D) The interface between the grafted donor and recipient colonies (dashed red line), viewed from above. Donor-derived cells are visible in the recipient’s tissues. They are more numerous closer to the interface. (E) Fluorescent, Tfap2 wild type sexual polyp of the donor animal. Oocytes are encircled by a dashed line. (F) Immature chimeric sexual polyp composed of donor (fluorescent, Tfap2 wild type) and recipient (non-fluorescent, Tfap2 mutant) cells. (G) Mature chimeric sexual polyp. Oocytes encircled by dashed line are exclusively donor-derived. Animals were pictured live and the red and the green channels representing the β-tubulin::mScarlet and Piwi1::GFP reporter transgenes were merged and false-colored green in (B) and (D-G) for simplicity. Blue in E-G represents DNA. All 10 grafts performed resulted in induction of sexual polyps in the mutant.

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. 4D to G, and movie S2). We found that cells from the wild type animal that 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 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 rudimentary 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 bilaterians, germ cells are necessary for proper gonad development in some species (31, 32), and our results are consistent with a previously 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 express Tfap2 in three different cellular contexts using three transgenic constructs (fig. S10), generating mosaic transgenic animals expressing Tfap2-GFP in different cell types. First, we used the Wnt3 promoter to drive Tfap2-GFP expression in the oral region, where Wnt3 is normally 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. However, Wnt3 promoter-induced Tfap2 expression resulted in phenotype-free animals (Fig. 5A).

Figure 5. Ectopic expression of Tfap2 in 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) β-tubulin promoter-driven Tfap2-GFP. Transgene is expressed in somatic cells and causes no visible phenotype. (C-K) Piwi1 promoter-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 larva, identified by morphology. (D) Sperm progenitors in the gastrodermis of a 48 hour larva, identified by H2B3/4 expression. (E, F) Ectopic oocytes in the gastrodermis of a mosaic transgenic feeding polyp expressing Tfap2-GFP under the Piwi1 promoter where the transgene had been suppressed for two weeks by a shRNA. (G-I). Double mRNA FISH showing co-localization of GFP and Cel in a mosaic transgenic feeding polyp treated as in (E). (G) GFP mRNA. (H) Cel mRNA. (I) Merge. (J) Schematic illustrating the localization of (G-I) in the polyp. (K) Tfap2 expression in i-cells converts them to germ cells.

Next, we drove Tfap2-GFP expression by the β-tubulin promoter that is active in all differentiated cells but not in i-cells (fig. S10B 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 Piwi1 promoter to restrict transgene expression to i-cells (Fig. 5C to J, and fig. S10B and C). This experiment resulted in large GFP⁺ cells that resembled morphologically early stage oocytes in mosaic transgenic embryos that were probably females (Fig. 5C). Other embryos (probably males) displayed cells that expressed H2B3/4, a spermatogenesis marker (36) (Fig. 5D, and fig. S11). Normally, germ cells appear 2-3 months post-metamorphosis. Ectopic germ cells in embryos never matured and vanished post-metamorphosis; this suggests that, whereas Tfap2 was effective in inducing germ fate in embryonic Piwi1⁺ 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 post-metamorphosis. In the absence of a conditional expression system in Hydractinia, we focused on inhibiting transgene expression until after metamorphosis. For this, we co-injected zygotes with the Piwi1::Tfap2-P2A-GFP ectopic expression construct alongside a short hairpin RNA (shRNA) (37), targeting the GFP sequence in the transgene’s mRNA. Injected embryos and larvae remained GFP-free, consistent with effective transgene repression by the shRNA (fig. S12). The suppressive effect of the shRNA dissipated about a week post-metamorphosis and transgenic feeding polyps developed ectopic GFP⁺ oocytes in the gastrodermis that appeared morphologically more mature than ectopic oocytes in larvae (Fig. 5E and F). Furthermore, they expressed Cnidarian egg lectin (Cel) – an exclusive early oogenesis marker (38) – in GFP⁺ cells (Fig. 5G to J) that was undetectable in larval ectopic oocytes. Taken together, our results show that Tfap2 acts cell-autonomously and is essential and sufficient to induce germ cell fate in Piwi1⁺ i-cells but not in differentiated cells (Fig. 5K). Tfap2 acts non-cell-autonomously downstream of germ cell induction to drive sexual polyp maturation.

The evolution of bilaterian sequestered germlines

Tfap2 is a critical regulator of germ cell induction in Hydractinia, an animal that does not sequester a germline, as well as in germline-sequestering animals, such as mouse (8) and human (39). Drosophila and C. elegans germlines are specified maternally, a mechanism that is thought to be evolutionarily derived (1). In mammals, AP2γ (encoded by Tfap2C) acts in concert with other transcription factors such as Blimp1, Prdm14, PAX5, and SOX17 to induce germ cell fate in epiblast cells in a species-specific combination (7, 8, 40). Partners of Tfap2 in Hydractinia are yet unknown but, unlike in mammals, its expression in i-cells – which are similar to epiblast cells in being somatic and germ cell competent – is sufficient to induce germ cell fate. It has been suggested that a non-sequestered germline is an ancestral trait in metazoans (4). Under this hypothesis, a key event in the evolution of bilaterian sequestered germlines would have been the redeployment of the hypothesized ancestral adult germ cell induction program, which is still present in extant Hydractinia, during embryogenesis.

A sequestered germline prevents transmission of somatic mutations to future generations and was also proposed to help select for mitochondrial quality in complex bilaterians with high mutation rates (4). Why then would clonal animals not sequester a germline? We suggest that a sequestered germline in clonal animals would be detrimental, exposing them to the risk of stochastically generating new clonal individuals that lack germ cells. Therefore, maintaining uncommitted cells post-embryogenesis that can provide progenitors to any lineage of somatic cells or germ cells grants full developmental potential to new clonemates. This may represent the selective pressure that prevented the evolution of a sequestered germline in clonal animals.