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A Krüppel-like factor is required for development and regeneration of germline and yolk cells from somatic stem cells in planarians [1]

['Melanie Issigonis', 'Morgridge Institute For Research', 'Madison', 'Wisconsin', 'United States Of America', 'Akshada B. Redkar', 'Tania Rozario', 'Umair W. Khan', 'Program In Cellular', 'Molecular Biology']

Date: 2022-07

Sexually reproducing animals segregate their germline from their soma. In addition to gamete-producing gonads, planarian and parasitic flatworm reproduction relies on yolk cell–generating accessory reproductive organs (vitellaria) supporting development of yolkless oocytes. Despite the importance of vitellaria for flatworm reproduction (and parasite transmission), little is known about this unique evolutionary innovation. Here, we examine reproductive system development in the planarian Schmidtea mediterranea, in which pluripotent stem cells generate both somatic and germ cell lineages. We show that a homolog of the pluripotency factor Klf4 is expressed in primordial germ cells (PGCs), presumptive germline stem cells (GSCs), and yolk cell progenitors. Knockdown of this klf4-like (klf4l) gene results in animals that fail to specify or maintain germ cells; surprisingly, they also fail to maintain yolk cells. We find that yolk cells display germ cell–like attributes and that vitellaria are structurally analogous to gonads. In addition to identifying a new proliferative cell population in planarians (yolk cell progenitors) and defining its niche, our work provides evidence supporting the hypothesis that flatworm germ cells and yolk cells share a common evolutionary origin.

Funding: This work was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (R01 HD043403) to PAN. MI was a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2135-12). PAN and PWR are Investigators of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

We show that a homolog of the conserved transcription factor Krüppel-like factor 4 (klf4), a critical inducer of pluripotency in mammals [ 49 ], is expressed in male and female presumptive germline stem cells (GSCs) in the planarian Schmidtea mediterranea, as well as in a newly discovered population of mitotically competent yolk cell progenitors. We demonstrate that klf4-like (klf4l) is required for germ cell specification and that klf4l knockdown leads to the loss of both germ cell and yolk cell lineages. We provide evidence that yolk cell–producing organs in planarians consist of 2 distinct cell types: a yolk cell lineage, which is characterized by several germ cell–like attributes, and support cells, which sustain yolk cell maintenance and differentiation. Our data show that planarian vitellaria are structurally analogous to gonads and that yolk cells share several important features with both somatic neoblasts and germ cells.

Here, we investigate how new germ cells are specified from neoblasts throughout postembryonic development and during regeneration in planarians. We also examine another critical aspect of the planarian reproductive system: the development of an extensive network of accessory organs known as vitellaria. Unique among animals, eggs of most flatworms are ectolecithal: Yolk is not present within oocytes themselves, but rather is made by vitellaria that produce specialized yolk cells (vitelline cells or vitellocytes). Planarians and all parasitic flatworms are characterized by ectolecithality. However, despite the importance of vitellaria in the life cycle and transmission of these parasites [ 47 , 48 ], little is known about the development of vitellaria or production of yolk cells.

Planarian flatworms can regenerate an entire body from small tissue fragments. Intensive efforts have been devoted to understanding the mechanisms underlying this regenerative prowess. Planarian regeneration is driven by pluripotent stem cells called neoblasts that are distributed throughout the body [ 25 – 27 ]. Planarians can also inform our understanding of germ cell biology: The neoblasts that give rise to all somatic lineages also give rise to new germ cells [ 28 – 31 ]. Interestingly, neoblasts and germ cells express a shared set of conserved “germline genes,” including piwi, vasa, pumilio, and tudor [ 32 , 33 ], which play important roles in neoblast function [ 34 – 44 ]. Like mammals, planarians undergo inductive germ cell specification [ 28 – 31 , 45 , 46 ]. However, the mechanistic basis underlying germ cell specification from “somatic” neoblasts and the factors involved in adopting somatic versus germ cell fate remain obscure.

Irrespective of the mode of germ cell specification, an important commonality exists: Once formed, germ cells are set aside from the soma. The developmental decision to segregate the germ cell lineage from somatic cells is essential for species continuity; unlike the soma, which expires each generation, “immortal” germ cells pass on genetic information and serve as a perpetual link between generations. Many animals (e.g., Drosophila, C. elegans, and mice) specify their germ cells (and segregate them from their soma) only once during embryonic development [ 1 – 4 ]. However, some animals retain the ability to specify new germ cells throughout their lifetime. Sponges and cnidarians maintain into adulthood multipotent stem cells that fuel the continuous production of new germ cells while also giving rise to somatic cell lineages [ 18 – 24 ]. How do these stem cells decide between somatic and germ cell fates?

Sexually reproducing animals consist of 2 main cell types: germ cells that produce gametes (eggs and sperm) and somatic cells that make up the remainder of the body. Animal germ cells are typically specified in either of 2 ways: by determinate or inductive specification [ 1 – 4 ]. Determinate specification results from the segregation of specialized maternal determinants (germ plasm) at the onset of embryogenesis; those cells receiving germ plasm acquire germ cell fate. In contrast, inductive specification occurs later in embryogenesis when extrinsic signals from surrounding tissues instruct competent cells to form germ cells. Determinate specification has been studied extensively in traditional laboratory models, including Drosophila, Caenorhabditis elegans, zebrafish, and frogs [ 5 – 17 ]. Inductive specification, although intensively investigated in mammals, has been less well characterized mechanistically across phylogeny, even though it is the basal and most common mode of germ cell specification in the animal kingdom [ 1 – 3 ].

Results

klf4l is expressed in planarian gonads and yolk-producing accessory organs In the search for regulators of germ cell fate in planarians, we focused on the conserved transcription factor KLF4, a key pluripotency factor in mammals [49]. Sexual S. mediterranea are cross-fertilizing, simultaneous hermaphrodites. Using fluorescent RNA in situ hybridization (FISH) on sexually mature adults, we found that one of the 5 klf genes present in the S. mediterranea genome, klf4l (S1A and S1B Fig), is expressed at high levels within the ventrally situated ovaries, as well as in cells that are distributed along the medial posterior region of each lobe of the cephalic ganglia and appear to be arranged in a field anterior to each ovary (Fig 1A and 1B). Sparse klf4l+ cells are also located dorsolaterally, where the testes reside (Fig 1A and 1B). Additionally, klf4l+ cells are scattered ventrolaterally throughout the parenchyma (the tissue surrounding the planarian’s internal organs), in a pattern reminiscent of vitellaria, the yolk-producing organs essential for reproduction (Fig 1A and 1B). Thus, this pluripotency-associated transcription factor is expressed in areas associated with male and female reproductive tissues. PPT PowerPoint slide

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TIFF original image Download: Fig 1. klf4l is expressed in gonads and vitellaria and is restricted to a subset of nanos+ germ cells in planarian ovaries and testes. (A) Schematics depicting the dorsal (left) and ventral (right) views of landmark structures and various reproductive organs in adult sexual S. mediterranea. (B) Maximum intensity projections of confocal sections showing FISH of klf4l (green) in ventral head region (top), ventral tail region (middle), and dorsal tail region (bottom). (C) Maximum intensity projection of confocal sections showing dFISH of klf4l (green) and germline marker nanos (magenta) in ventral head region. klf4l- and nanos-expressing cells are detected surrounding the tuba (tu) at the base of each ovary (ov), along the periphery of the ovaries, and in anterior ovarian fields (of) situated mediolaterally along the brain. (D) Single confocal section of a planarian ovary located posterior to the brain (br) showing klf4l (green) and nanos (magenta) dFISH. klf4l- and nanos-expressing cells are found at the ovary-tuba junction, along the periphery of the ovary, and in germ cells anterior to the ovary. Dashed line denotes ovary (white) and tuba (yellow) boundary. (E) Confocal section of klf4l (green) and nanos (magenta) dFISH showing klf4l/nanos double-positive and nanos single-positive cells along the periphery of the testis. Dashed line denotes testis boundary. (B–E) Nuclei are counterstained with DAPI (gray). Scale bars, 200 μm (B), 100 μm (C), and 50 μm (D, E). dFISH, double FISH; FISH, fluorescent RNA in situ hybridization; klf4l, klf4-like. https://doi.org/10.1371/journal.pbio.3001472.g001

klf4l-expressing germ cells in ovaries and testes are mitotically active In many animals, the production of gametes in adulthood is enabled by GSCs. Our findings raise the possibility that klf4l-expressing cells are GSCs representing the top of oogonial and spermatogonial lineages. All GSCs have the ability to undergo self-renewing divisions, which give rise to differentiating daughter cells while maintaining the stem cell pool. By combining phospho-Histone H3 (pHH3) immunostaining with klf4l and nanos dFISH, we examined the mitotic profiles of cells within the germ cell hierarchy and sought to ascertain whether klf4l+/nanos+ cells are competent to divide and, therefore, fulfill a basic criterion of GSC behavior. We found that klf4l+/nanos+ germ cells within the ovarian fields and the outer periphery of the ovaries are mitotically active (0.3%, n = 3409 klf4l+/nanos+ cells) (Fig 3A and 3B). We also detected proliferation of klf4l–/nanos+ oogonia in the ovaries, whereas nanos− oogonia within the ovaries do not divide mitotically. Thus, female germ cells are specified and proliferate within the ovarian field and/or the ovary periphery, and as oogonia turn off nanos expression, they cease to divide mitotically and differentiate into oocytes. PPT PowerPoint slide

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TIFF original image Download: Fig 3. klf4l+ germ cells in planarian ovaries and testes are mitotically active. (A–C) Confocal sections showing dFISH of klf4l (green) and nanos (magenta) and immunostaining of mitotic marker pHH3 (cyan) in the ovarian field (of) located anterior to the ovary (ov) and proximal to the brain (br; boundary denoted by yellow dashed line) (A), the ovary, which is anterior to the tuba (tu) (B), and the testis (boundary denoted by gray dashed line) (C). Side panels are high magnification views of klf4l/nanos/pHH3 triple-positive cells (yellow arrowheads). (D) Confocal sections (top 4 panels) and maximum intensity projections (bottom 2 panels) showing dFISH of klf4l (green) and nanos (magenta) and immunostaining of pHH3 (cyan) in testes (boundary denoted by gray dashed line). Yellow numbers denote pHH3+ germ cells dividing throughout spermatogenesis: single nanos+ cell; single nanos− cell; 2-, 4-, 8-cell spermatogonial cysts; and 16- and 32-cell spermatocyte cysts. (A–D) Nuclei are counterstained with DAPI (gray). Scale bars, 50 μm for whole-gonad images, 20 μm for side panels. klf4l, klf4-like; pHH3, phospho-Histone H3. https://doi.org/10.1371/journal.pbio.3001472.g003 Male germ cells actively divide throughout spermatogenesis; spermatogonia undergo 3 rounds of synchronous mitotic divisions with incomplete cytokinesis to produce 2-, 4-, and 8-cell spermatogonial cysts connected by intercellular bridges, whose cells differentiate into primary spermatocytes and divide meiotically to generate 32 spermatids that ultimately transform into mature sperm [31,52]. We detected pHH3+/klf4l+/nanos+ triple-positive cells in testes of both hatchlings (1%, n = 773 klf4l+/nanos+ cells) and adults (0.2%, n = 2,436 klf4l+/nanos+ cells). In mature sexuals, mitotic, single-cell spermatogonia and mitotic doublets were observed in nanos+ germ cells (including klf4l+/nanos+ cells) along the outermost periphery of the testis (Fig 3C and 3D). We also observed nanos–/pHH3+ singlets and doublets, which might represent mitotic nanos− single-cells or 2-cell spermatogonia (Fig 3D). We never detected nanos expression in pHH3+ 4- or 8-cell premeiotic spermatogonial cysts, or in 16- or 32-cell meiotic cysts (Fig 3D). All our observations thus far support a model in which the spermatogonial lineage consists of klf4l+/nanos+ germ cells at the top of the hierarchy, giving rise to klf4l–/nanos+ and subsequently klf4l–/nanos− single-cell spermatogonia and that germ cells cease expressing nanos once spermatogonial cystogenic divisions have occurred.

Yolk cells share features with neoblasts and germ cells Our results suggest that klf4l marks the top of both germ cell and yolk cell lineages. Yolk cells are technically somatic since they do not generate gametes, yet it has long been postulated that flatworm yolk cells may share an evolutionary origin with oocytes (the female germline) [55]. One hypothesis is that yolk cells were derived from germ cells in the course of evolution and that a split/divergence between these 2 cell types may have occurred in the common ancestor of all ectolecithal flatworms [60–62]. As we found that both yolk cells and the germline share klf4l and nanos expression, we wondered whether yolk cells share other germ cell characteristics, such as expression of piwi-1 and germinal histone H4 (gH4) (S9A Fig), 2 transcripts thought to be expressed exclusively in neoblasts and germ cells [30,57,63–66]. By dFISH, we found that the vast majority of klf4l+ cells in the vitellaria are also piwi-1+ (94%, n = 789 klf4l+ cells) and gH4+ (98%, n = 399 klf4l+ cells) (Figs 6A, 6E, S9B, and S9C). Unlike other somatic tissues, in which piwi-1 mRNA is degraded during differentiation [36], piwi-1 expression perdures during yolk cell differentiation and is still detected in most CPEB1+ cells (80%, n = 2,801 CPEB1+ cells) and surfactant b+ cells (55%, n = 2,136 surfactant b+ cells), but not in MX1+ cells (0%, n = 284 MX1+ cells) (Fig 6B–6D). Similarly, gH4 is coexpressed in most surfactant b+ cells (64%, n = 4,410 surfactant b+ cells) (Figs 6F and S9D). Thus, similar to the germ cell lineages in testes and ovaries, piwi-1 and gH4 expression persist in differentiating yolk cells. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Yolk cells share features with neoblasts and germ cells. (A–D) Single confocal sections showing dFISH of neoblast and germ cell marker piwi-1 (green) and klf4l (A), CPEB1 (B), surfactant b (C), and MX1 (D) (magenta). Side panels are high-magnification views of outlined areas showing piwi-1 double-positive cells (yellow arrowheads). (E, F) Single confocal sections showing dFISH of neoblast and germ cell marker gH4 (green) and klf4l (E) and surfactant b (F) (magenta). Side panels are high-magnification views of outlined areas showing gH4 double-positive cells (yellow arrowheads). (G) Maximum intensity projections of confocal sections (5-μm thick) imaged from the ventral posterior region of sexually mature planarians showing klf4l (green) and nanos (magenta) dFISH with pHH3 (cyan) immunostaining in vitellaria. Mitotically active klf4l+/nanos+ vitellocytes with high (top panels) and low levels (middle panels) of klf4l expression are shown (yellow arrowheads). Nondividing klf4l+/nanos+ cells are also present (white arrowheads). klf4l–/nanos+ yolk cell progenitors are able to divide (bottom panels; yellow arrowheads). (A–G) Nuclei are counterstained with DAPI (gray). Scale bars, 50 μm for overview images, 20 μm for side panels (A–F), 20 μm (G). Underlying data can be found in S1 Data. dFISH, double FISH. https://doi.org/10.1371/journal.pbio.3001472.g006 In addition to the retention of germ cell features in yolk cells, these cells are mitotically active. We detected pHH3 staining in klf4l+/nanos+ as well as klf4l–/nanos+ yolk cells (Fig 6G). Taken together, these results show that even though yolk cells do not give rise to gametes (and are therefore not germ cells), they do exhibit several germ cell characteristics, including expression of the germline markers nanos, piwi-1, and gH4, and the capacity to proliferate.

Vitellaria contain distinct cell types: A yolk cell lineage and nonyolk support cells Gonads are not composed solely of germ cells: They also contain somatic support cells (or niche cells) that govern germ cell behavior. Thus, we asked whether vitellaria contain nonyolk vitelline support cells and whether they could play a niche-like role in maintaining the klf4l+/nanos+ stem/progenitor population for sustaining the yolk cell lineage. Previously, expression of the orphan G-protein–coupled receptor ophis, a somatic gonadal cell marker, was detected in the vitellaria, but its role there was not characterized [53]. We found that in mature sexual planarians, the vitellaria are arranged in an extensively branched network containing 2 populations of ophis-expressing cells: ophishigh cells, which express ophis predominantly in the nucleus, and ophislow cells with weak signal throughout the cell (Fig 7A). ophis+ cells are interspersed throughout the vitellaria, similar to klf4l+ cells (S10A–S10D Fig). klf4l+ cells are tightly juxtaposed with ophishigh cells; however, they never coexpress high levels of ophis (0% klf4l+ cells are ophishigh, n = 368 klf4l+ cells) (Fig 7A and 7E). On the other hand, a large fraction of klf4l+ cells are ophislow (60% klf4l+ cells are ophislow, n = 368 klf4l+ cells). These results led us to hypothesize that ophislow versus ophishigh cells represent 2 distinct classes of cells in the vitellaria: ophislow cells constitute the yolk cell lineage proper and ophishigh cells are support cells that closely associate with the yolk cells and comprise the remaining structure of the vitellaria. PPT PowerPoint slide

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TIFF original image Download: Fig 7. Vitellaria contain distinct cell types: Yolk cells and nonyolk support cells. (A–D, F–G) Single confocal sections showing dFISH. Side panels are high-magnification views of outlined areas. (A) dFISH of klf4l (magenta) and vitellaria marker ophis (green). ophishigh cells do not coexpress klf4l (filled white arrowhead) but ophislow cells do (unfilled white arrowhead). (B-D) dFISH of ophis (green) and CPEB1 (B), surfactant b (C), and MX1 (D) (magenta). ophislow cells (unfilled white arrowheads), but not ophishigh cells (filled white arrowheads), express yolk cell lineage differentiation markers. (E) proportion of cells in the vitellaria that coexpress low levels (left) versus high levels (right) of ophis. ophislow cells predominantly coexpress markers of the yolk cell lineage. Conversely, most ophishigh cells coexpress LamA but do not express yolk cell markers. (F) dFISH of LamA (magenta) and ophis (green). ophishigh cells coexpress LamA (filled white arrowhead) whereas ophislow cells do not (unfilled white arrowhead). (G) dFISH of LamA (magenta) and klf4l (green). LamA and klf4l are never coexpressed in the same cells. (A–D, F–G) Nuclei are counterstained with DAPI (gray). Scale bars, 50 μm for overview images, 20 μm for side panels. (H) Schematic depicting genes expressed during developmental progression of ophislow yolk cells and associated ophishigh support cells. Underlying data can be found in S1 Data. dFISH, double FISH; klf4l, klf4-like. https://doi.org/10.1371/journal.pbio.3001472.g007 If the ophislow population represents the yolk cell lineage of which klf4l+ cells are the precursors, then we would expect klf4l–/ophislow cells to express markers of progressive yolk cell differentiation. Consistent with this idea, almost all CPEB1+, surfactant b+, and MX1+ cells coexpress low levels of nuclear and cytoplasmic ophis mRNA (98%, n = 2,914 CPEB1+ cells; 100% n = 1,760 surfactant b+ cells; 96%, n = 256 MX1+ cells) (Fig 7B–7E). On the other hand, high levels of nuclear ophis were rare in CPEB1+, surfactant b+, and MX1+ cells (2%, n = 2,914 CPEB1+ cells; 0%, n = 1,760 surfactant b+ cells; 1%, n = 256 MX1+ cells). These results indicate that ophislow expression emerges in a subset of klf4l+ yolk cell progenitors and subsequently persists as these cells differentiate (Fig 7H), whereas ophishigh expression defines a distinct cell type in the vitellaria. In agreement with the model that ophishigh cells constitute a separate cell lineage, the majority of these cells do not express yolk cell markers (0%, n = 784 ophishigh cells are klf4l+; 12%, n = 519 ophishigh cells are CPEB1+; 0%, n = 721 ophishigh cells are surfactant b+; 1%, n = 521 ophishigh cells are MX1+). Instead, most ophishigh cells express Laminin A (LamA) (81%, n = 440 ophishigh cells are LamA+) (Fig 7F), a gene expressed in the vitellaria (S10E and S10F Fig) as well as in somatic gonadal cells in the testes and ovaries (S10G Fig). This result corroborates the finding that ophishigh expression marks support cells within the vitellaria. Notably, klf4l and LamA are never coexpressed within the vitellaria (0%, n = 540 klf4l+ cells, 0%, n = 867 LamA+ cells) (Fig 7G). Taken together, our data suggest that 2 cell lineages exist in the vitellaria: the yolk cell lineage (ophislow), which includes klf4l+ cells, and a second population made up of ophishigh/LamA+ cells. It was previously reported that ophis transcript was expressed in the somatic gonadal cells of the ovary [53]. In addition to this expression pattern, we detect low levels of ophis expression in the oogonial lineage, similar to yolk cells (S10H Fig). The dichotomy between ophislow versus ophishigh expression in the germline and somatic lineages of the ovary is reminiscent of what we observed in the 2 vitellarial lineages.

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