nanos function is essential for development and regeneration of planarian germ cells

Abstract

Germ cells are required for the successful propagation of sexually reproducing species. Understanding the mechanisms by which these cells are specified and how their totipotency is established and maintained has important biomedical and evolutionary implications. Freshwater planarians serve as fascinating models for studying these questions. They can regenerate germ cells from fragments of adult tissues that lack reproductive structures, suggesting that inductive signaling is involved in planarian germ cell specification. To study the development and regeneration of planarian germ cells, we have functionally characterized an ortholog of nanos, a gene required for germ cell development in diverse organisms, from Schmidtea mediterranea. In the hermaphroditic strain of this species, Smed-nanos mRNA is detected in developing, regenerating, and mature ovaries and testes. However, it is not detected in the vast majority of newly hatched planarians or in small tissue fragments that will ultimately regenerate germ cells, consistent with an epigenetic origin of germ cells. We show that Smed-nanos RNA interference (RNAi) results in failure to develop, regenerate, or maintain gonads in sexual planarians. Unexpectedly, Smed-nanos mRNA is also detected in presumptive testes primordia of asexual individuals that reproduce strictly by fission. These presumptive germ cells are lost after Smed-nanos RNAi, suggesting that asexual planarians specify germ cells, but their differentiation is blocked downstream of Smed-nanos function. Our results reveal a conserved function of nanos in germ cell development in planarians and suggest that these animals will serve as useful models for dissecting the molecular basis of epigenetic germ cell specification.

Fig. 1.

Smed-nanos is expressed in the testes and the ovaries of juvenile and mature S. mediterranea hermaphrodites but not in young hatchlings. (A) Diagram of S. mediterranea hermaphrodite illustrating the reproductive organs. o, ovaries; t, testes; od, oviducts; ca, copulatory apparatus. (B) Whole-mount in situ hybridization showing nanos expression in testes (arrowheads); expression is not detected in newly hatched animals. From Top to Bottom: hatchling (34 of 38 lacked detectable Smed-nanos mRNA), juvenile (n = 4), smaller (n = 5), and larger mature worms (n = 11) (dorsal views). (C) Ventral views of the worms in B, showing nanos expression in the ovaries (arrowheads). (Scale bars, 1 mm.)

Fig. 2.

Smed-nanos expression in regenerating head fragments amputated anterior to the ovaries. (A) Differential interference contrast microscopic images of regenerating heads fixed 7, 14, or 21 days after amputation (animals were ≥1.2 cm when amputated). The numbers of animals in which nanos mRNA was detected were 7 of 14 at 7 days, 8 of 8 at 14 days, and 8 of 9 at 21 days. (Scale bars, 250 μm.) (B) Confocal projections corresponding to the boxed regions in A showing nanos mRNA detected by FISH. (Scale bars, 100 μm.) Arrows indicate nanos-positive cells shown at higher magnification in the Insets. (Inset scale bars, 10 μm.)

Fig. 3.

Testes regeneration in S. mediterranea hermaphrodites. (A) Expression pattern of a T-plastin homolog (DN311193) in intact and regenerating animals (n = 3 per stage). Days after amputation are indicated below each animal. (Scale bar, 1 mm.) (B) Expression pattern of Smed-nanos in intact (n = 2) and regenerating planarians (n = 4 per stage). (Scale bar, 1 mm.) (C) Confocal projections showing FISH to detect nanos mRNA in intact (n = 4) and regenerating planarians (n = 4 per stage). Arrows indicate the central portion of testes lobes. (Scale bar, 100 μm.)

Fig. 4.

Smed-nanos RNAi worms fail to regenerate testes or ovaries after amputation. (A and B) DAPI staining of animals fixed 3 months after amputation, showing normal testes and copulatory apparatus (asterisk) in controls (A; n = 7) but not in nanos RNAi animals (B; n = 8). (Scale bars, 1 mm.) (C–H) Immunofluorescent images of the boxed regions in A and B. (C and D) Anti-tubulin labels different stages of spermatogenesis (arrowhead) within testes of control animals (C) but not in RNAi animals (D). This antibody also labels ciliated excretory ducts (arrow in C). (E and F) Anti-H3-S10P labels individual mitotic figures (arrows) and germ cell cysts (arrowhead) in the testes of control animals (E), but germ cell cysts were not observed in RNAi animals (F). (G) Overlay of C and E. and (H) Overlay of D and F. (Scale bars, 100 μm.) (I–N) In situ hybridization for gonad markers. Animals were fixed 2 months after amputation. (I and J) T-plastin is expressed in the testes of controls (n = 10) but not in RNAi animals (n = 10). (K and L) germinal H4 is expressed in testes of controls (n = 12) but not in RNAi animals (n = 12). (Scale bars, 1 mm.) (M and N) germinal H4 is expressed in the ovaries (arrowheads) of control animal (M); expression is absent from RNAi animal (N). (Scale bars, 500 μm.)

Fig. 5.

Smed-nanos RNAi knockdown animals do not regenerate testes primordia. Animals were fixed 14 days after amputation posterior to the ovaries and processed to detect germinal H4 mRNA by FISH. (A–D) Control animals. (E and F) nanos RNAi animals. (B, D, and F) Confocal images corresponding to the postpharyngeal regions of the planarians shown in A, C, and E. Control animals regenerated germinal H4-positive testes primordia (n = 10). (A and B) Well developed testes lobes (arrow in B) were observed in the largest of these specimens. (C and D) The remaining animals developed smaller clusters of germinal H4-positive testes primordia (arrow in D). (E and F) germinal H4-positive dorsal clusters were not detected in nanos RNAi animals (n = 11); only somatic neoblasts were observed. ph, pharynx. [Scale bars, 500 μm (A, C, and E); 50 μm (B, D, and F).]

Fig. 6.

Smed-nanos expression in the asexual strain of S. mediterranea and the effect of Smed-nanos RNAi in asexual planarians. (A) Whole-mount in situ hybridization to nanos labels clusters of dorsolateral cells reminiscent of the testes pattern in sexual planarians (n = 34). (B) Cells observed in A are undetectable 3 days after γ-irradiation (30 Gy) (n = 22). (C) germinal H4 is expressed in the neoblasts and clusters of cells similar to nanos-positive cells (n = 18). (D) γ-irradiation (30 Gy) eliminates germinal H4 expression (n = 15). (E and F) Control and RNAi animals fixed 1 month after amputation. (E) Animals fed control bacteria have germinal H4 expression similar to untreated planarians (n = 8 heads and 12 trunk pieces). (F) nanos RNAi planarians lose expression of germinal H4 expression from the dorsal cell clusters, whereas neoblast staining is unaffected (n = 9 heads and 11 trunk pieces). All animals were imaged dorsally by using differential interference contrast microscopy. (Scale bars, 200 μm.) (Insets) Higher magnification views of the boxed areas. (Inset scale bars, 100 μm.)