Planarian Embryogenesis Crash Course

▻ Schmidtea mediterranea reproduction

▻ Sphere formation and Embryo Architecture

Schmidtea mediterranea reproduction

Top: Cartoon depicting the reproductive system of a sexually mature *Smed* hermaphrodite. Ventral view. O: ovary. OD: oviduct. SD: sperm duct. P: penis papilla. G: gonopore. Genital atrium: purple region immediately posterior to the pharynx. Bottom: Brightfield image of a live *Smed* hermaphrodite. Anterior: right. Dorsal view. Scale bar: 100 µm. White asterisk: pharynx.

Sexually reproducing Smed are cross-fertilizing hermaphrodites that contain a pair of ovaries, situated immediately posterior to the brain and adjacent to the ventral nerve cords, and numerous testes located along the dorsolateral flanks of the animal. Oocytes are fertilized internally by sperm from a partner as they enter the oviducts. Smed embryos are ectolecithal: yolk is not contained within oocytes, but rather is produced by somatic vitellaria (yolk glands) arrayed ventrolaterally beneath the testes (Chong et al., 2011; Steiner et al., 2016; Stevens, 1904). One or more zygotes are packaged, along with yolk cells, into an egg capsule in the genital atrium (Chong et al., 2011; Hyman, 1951; Newmark et al., 2008; Stevens, 1904). Egg capsules are laid through the gonopore. Smed embryos gestate in egg capsules for approximately two weeks at 20˚C prior to hatching.

For more information on Smed hermaphrodite anatomy and gametogenesis, please visit the Newmark and Rouhana lab websites.

Direct Development

Brightfield image of a live *Smed* newborn hatchling (Stage 8, 14 days post-egg capsule deposition). Anterior: right. Dorsal view. Scale bar: 100 µm.

Smedflatworms are direct developers: newborn hatchlings grow and mature into adult worms without an intervening larval stage (Sánchez Alvarado, 2003). At hatching, juveniles are sexually immature but otherwise possess a body plan grossly similar to the adult hermaphrodite (Sánchez Alvarado, 2003; Wang et al., 2007).

Dispersed Cleavage

Smedembryos undergo an evolutionarily divergent mode of development that bears little resemblance to the ancestral Spiralian cleavage programs utilized by many Lophotrochozoans. In contrast to the synchronous, oriented blastomere cleavage patterns of Spiralian embryos (Lambert, 2010), blastomeres in freshwater planarian embryos undergo dispersed cleavage among yolk cells: they divide asynchronously and are not in direct contact with one another (Bardeen, 1902; Cardona et al., 2005; Hallez, 1887; Ijima, 1884; Le Moigne, 1963; Metschnikoff, 1883; Vara et al., 2008).

Sphere formation and Embryo Architecture

Stage 2 protosphere stained with riboprobes complementary to *EF1a-like-1* (blue). Scale: 100 µm.

During sphere formation, some blastomeres differentiate into temporary embryonic cell types that provide form and function to the embryo, including the primitive ectoderm, temporary embryonic pharynx and primitive gut (Cardona et al., 2005; Hallez, 1887).

Stage 3 spherical embryo stained with riboprobes complementary to *EF1a-like-1* (blue). Cyan arrowheads: primitive ectoderm cells. Cyan arrows: undifferentiated blastomeres. Red arrowhead: temporary embryonic pharynx Red arrows: primitive gut cells. Scale: 100 µm.

Rich Media File 1: S3 embryo architecture. SPIM reconstructed S3 embryo costained with EF1a-like-1 (red) and sytox green nuclear counterstain. EF1a-like-1 is a pan-embryonic cell marker that stains primitive ectoderm cells, the temporary embryonic pharynx and undifferentiated blastomeres in the embryonic wall. EF1a-like-1 staining is absent from yolk cells in the embryonic wall and gut cavity.

Primitive ectoderm cells are the first to differentiate, forming a single cell layer bounding the sphere (Hallez, 1887; Ijima, 1884; Le Moigne, 1963; Metschnikoff, 1883). The temporary embryonic pharynx is an innervated pump containing neurons, radial muscle and associated epithelial cells that ingests yolk into an inner gut cavity. The primitive gut consists of the inner gut cavity and phagocytic cells associated with the temporary embryonic pharynx. Temporary embryonic tissues are not thought to contribute to the juvenile body plan; they are thought to degenerate as the definitive organs form and morphogenesis proceeds (Cardona et al., 2005; Le Moigne, 1963; Vara et al., 2008).

Paraffin embedded S3 embryo sectioned and stained with hematoxylin and eosin (left) or *piwi-1* riboprobes (blue) and eosin (pink, right). Black brackets denote embryonic wall. Yellow arrowhead: temporary embryonic pharynx. GC: yolk-filled gut cavity. Cyan arrows: *piwi-1+* undifferentiated blastomeres. Scale bars: 100 µm. Inset (right): magnified view of a *piwi-1+* cell. Inset scale bar: 25 µm.

piwi-1 is expressed in all undifferentiated blastomeres of S3 embryos. SPIM reconstructed S3 embryo costained with piwi-1 (red) and EF1a-like-1 (green). piwi-1 is expressed in all undifferentiated blastomeres in the embryonic wall (piwi-1+, EF1a-like-1+ cells). piwi-1 is not expressed in differentiated tissues marked by EF1a-like-1 alone, including the primitive ectoderm and temporary embryonic pharynx (green). Several fluorescent beads used for 3-dimensional reconstruction are visible (red).

A population of undifferentiated blastomeres and yolk cells remain in the embryonic wall, the parenchymal space between the primitive ectoderm and endoderm, in nascent spheres (Hyman, 1951; Sánchez Alvarado, 2003). These undifferentiated blastomeres are thought to give rise to all definitive tissues found in juvenile worms (Hallez, 1887; Hyman, 1951; Le Moigne, 1963; Sánchez Alvarado, 2003; Stevens, 1904). Definitive organ formation and morphogenesis occur during the second week of embryogenesis.

References

Bardeen, C.R. (1902). Embryonic and regenerative development in planarians. Biol Bull 3, 262-288.

Cardona, A., Hartenstein, V., and Romero, R. (2005). The embryonic development of the triclad Schmidtea polychroa. Dev Genes Evol 215, 109-131.

Chong, T., Stary, J.M., Wang, Y., and Newmark, P.A. (2011). Molecular markers to characterize the hermaphroditic reproductive system of the planarian Schmidtea mediterranea. BMC Dev Biol 11, 69.

Hallez, P. (1887). Embryogénie des Dendrocoeles d’eau douce, Vol 16.

Hyman, L.H. (1951). The invertebrates: Platyhelminthes and Rhynchocoela. The Acoelomate Bilateria. (New York, McGraw Hill).

Ijima, I. (1884). Untersuchungeniiber den Bau und die Entwicklungsgeschichte der stisswasser-Dendrocoelen(Tricladen). Vol Bd. 40.

Lambert, J.D. (2010). Developmental Patterns in Spiralian Embryos. Current Biology 20, R72-77.

Le Moigne, A. (1963). Etude du developpement embryonnaire de Polycelis nigra (Turbellarie - Triclade). Bulletin de la Societe Zoologique de France 88, 403-422.

Metschnikoff, E. (1883). Die embryologie von Planaria polychroa. Zeitschrift fur wissenschaftliche Zoologie 38, 331-354.

Newmark, P.A., Wang, Y., and Chong, T. (2008). Germ cell specification and regeneration in planarians. Cold Spring Harb Symp Quant Biol 73, 573-581.

Sánchez Alvarado, A. (2003). The freshwater planarian Schmidtea mediterranea: embryogenesis, stem cells and regeneration. Curr Opin Genet Dev 13, 438-444.

Steiner, J.K., Tasaki, J., and Rouhana, L. (2016). Germline Defects Caused by Smed-boule RNA-Interference Reveal That Egg Capsule Deposition Occurs Independently of Fertilization, Ovulation, Mating, or the Presence of Gametes in Planarian Flatworms. PLoS Genet 12, e1006030.

Stevens, N.M. (1904). On the germ cells and the embryology of Planaria simplicissima. Proc Acad Nat Sci Philadelphia 56, 208-220.

Vara, D.C., Leal-Zanchet, A.M., and Lizardo-Daudt, H. (2008). Embryonic development of Girardia tigrina (Girard, 1850) (Platyhelminthes, Tricladida, Paludicola). Braz J Biol 68, 889-895.

Wang, Y., Zayas, R.M., Guo, T., and Newmark, P.A. (2007). nanos function is essential for development and regeneration of planarian germ cells. Proc Natl Acad Sci U S A 104, 5901-5906.

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