Developmental diversity in free-living flatworms

Abstract

Flatworm embryology has attracted attention since the early beginnings of comparative evolutionary biology. Considered for a long time the most basal bilaterians, the Platyhelminthes (excluding Acoelomorpha) are now robustly placed within the Spiralia. Despite having lost their relevance to explain the transition from radially to bilaterally symmetrical animals, the study of flatworm embryology is still of great importance to understand the diversification of bilaterians and of developmental mechanisms. Flatworms are acoelomate organisms generally with a simple centralized nervous system, a blind gut, and lacking a circulatory organ, a skeleton and a respiratory system other than the epidermis. Regeneration and asexual reproduction, based on a totipotent neoblast stem cell system, are broadly present among different groups of flatworms. While some more basally branching groups - such as polyclad flatworms - retain the ancestral quartet spiral cleavage pattern, most flatworms have significantly diverged from this pattern and exhibit unique strategies to specify the common adult body plan. Most free-living flatworms (i.e. Platyhelminthes excluding the parasitic Neodermata) are directly developing, whereas in polyclads, also indirect developers with an intermediate free-living larval stage and subsequent metamorphosis are found. A comparative study of developmental diversity may help understanding major questions in evolutionary biology, such as the evolution of cleavage patterns, gastrulation and axial specification, the evolution of larval types, and the diversification and specialization of organ systems. In this review, we present a thorough overview of the embryonic development of the different groups of free-living (turbellarian) platyhelminths, including the Catenulida, Macrostomorpha, Polycladida, Lecithoepitheliata, Proseriata, Bothrioplanida, Rhabdocoela, Fecampiida, Prolecithophora and Tricladida, and discuss their main features under a consensus phylogeny of the phylum.

Figure 1

Diversity of body plans and phylogeny of free-living platyhelminthes. Left side, consensus tree of various published phylogenetic reconstructions. The Catenulida is the sister group of the Rhabditophora [15-18], the Macrostomorpha the sister group to all other Rhabditophora [2,15,19]. The Polycladida is the sister group to the Neoophora [2], and the Lecithoepitheliata is sister group to all other Neoophora [2,20]. Subsequently, the Proseriata is the sister group of all other Neoophora except the Lecithoepitheliata [16,20,21], while the Neodermata is sister group to Rhabdocoela and Adiaphanida (Fecampiida, Prolecithophora, Tricladida) [15,16,20,21]. The Rhabdocoela is sister group to Adiaphanida [15-17,20,21]. Within the Adiaphanida, Fecampiida is sister group to Prolecithophora plus Tricladida [15-17,20,21]. According to Willems and coworkers, the Bothrioplanida is sister group to Adiaphanida plus Neodermata, although their overall tree topology is different than depicted here, where Bothrioplanida is sister group to the Eulecithophora [22]. 1 entolecithal eggs, 2 quartet spiral cleavage, 3 hull cells made from macromeres, 4 larvae, 5 ectolecithal eggs, 6 hull cells made from micromeres, 7 loss of spiral cleavage, 8 disperse cleavage (Blastomerenanarchie), 9 hull cells made of yolk cells (at least in some representatives), 10 embryonic pharynx and "yolk larvae". Right side, live images of adult representatives of the major taxa of free-living flatworms with their developing embryos. Stenostomum sthenum with two developing zooids, fresh water, about 1 mm long. Mid-stage embryo, about 150 μm in diameter. Macrostomum lignano, marine, about 1 mm long. 4-cell stage, about 150 μm in diameter. Prosthiostomum siphunculus, marine, about 1 cm long. Several embryos per cocoon, several cocoons per egg plate. Embryos about 130 μm in diameter. Geocentrophora sphyrocephala, fresh water, about 1 mm long. No embryonic stage provided. Monocelis fusca, marine, about 1.2 mm long. Egg capsule of an unidentified proseriate, about 150 μm in diameter. Rhynchomesostoma rostratum, fresh water, about 1.3 mm long. Egg capsule with late embryo (note eyes) of a summer egg, about 170 μm in diameter. Procerodes littoralis, marine, about 4 mm long. 2 cocoons, the one to the right opened. Cocoon about 1 mm in diameter. Lower left corner hatched juvenile. Anterior of adult specimens to the left.

Figure 2

Summary of the embryonic development of Macrostomorpha and Polycladida. (A-I), schematic representations of the early macrostomid (modified from [43]) and polyclad development (adapted from [45,46]). In macrostomids, early cleavage follows the typical quartet spiral cleavage pattern (A) up to the 8-cell stage, after which the four vegetal macromeres 2A-2D flatten (B) and form a yolk mantle that covers the embryo (C-D) that will be eventually replaced by the definitive epidermis. The rest of the blastomeres remain in the inner region and form an embryonic blastema from which the organs of the juvenile develop. Polyclads, on the contrary, exhibit a quite conserved quartet spiral mode of development (E-I), except that macromeres 4A-4D are smaller than the micromeres 4a-4d (H). Gastrulation occurs through epiboly of the animal micromeres over the vegetal macromeres (I). As a peculiarity of polyclad development, the macromeres 4A-4D (represented with a slashed line in H) and the micromeres 4a-4c degenerate, and thereby, the whole endoderm and a large part of the mesoderm is originated by the 4d micromere. In all schemes, an idealized animal-vegetal axis cross section of the embryo is represented (animal to the top, vegetal to the bottom), unless otherwise indicated. Yolk granules are colored in light blue, hull cells in orange and embryonic cells in gray. Drawings are not to scale. eb embryonic blastema, ec ectoderm, eym embryonic yolk mantle, ma macromere, mec mesoectoderm, men mesoendoderm, mi micromere.

Figure 3

Larval types and juveniles of Polycladida. (A) Müller's larvae of a cotylean (Prosthiostomum siphunculus) and (B) an acotylean species (Planocera multitentaculata), both hatching with eight lobes and three eyes (two cerebral eyes and one epidermal eye). (C) Goette's larva of the acotylean Imogine mediterranea, hatching with four lobes and a cerebral and an epidermal eye. (D-E) Kato's larva of the acotylean Planocera reticulata, hatching with eight lobes and 12 eyes and being dorsoventrally flattened. (D) Ventral side with four lobes around the mouth visible, (E) from dorsal. (F) Directly developing juvenile of the acotylean Pseudostylochus obscurus, hatching with no lobes and four eyes. (G) Directly developing juvenile of an undetermined acotylean, hatching with no lobes and 12 eyes. All scale bars are 50 μm. Photograph (C) is courtesy of Mehrez Gammoudi.

Figure 4

Summary of the embryonic development of Lecithoepitheliata and Proseriata. (A-H), schematic representations of the early development of lecithoepitheliates and proseriates (both modified from [29]). Lecithoepitheliates exhibit regular quartet spiral cleavage (A) and gastrulate by epiboly of the micromeres over the vegetal macromeres (B). During gastrulation, however, the micromeres 2a-2d and 3a-3d at the edge of the blastopore differentiate into hull cells, which engulf a portion of the yolk (in X. steinöcki, C) or the whole portion of maternally supplied vitellocytes (in G. applanata). The inner mass of blastomeres differentiates into an embryonic blastema that occupies the future ventral side of the embryo, and in X. steinböcki a second hull membrane is formed to incorporate the remaining yolk cells inside the eggshell (D). In proseriates, quartet spiral cleavage is only observed up to the 8-cell stage (E). After that, the embryo develops first into a coelogastrula (F) and later into a compact discoidal stereoblastula in which 6 peripheral blastomeres differentiate into a hull membrane that engulfs the yolk cells (G). As in lecithoepitheliates, the inner blastomeres form a discoidal embryonic blastema that occupies the future ventral side of the embryo (H). In all schemes, an idealized animal-vegetal axis (ventral-dorsal axis in D and H) cross section of the embryo is represented (animal/ventral to the top and vegetal/dorsal to the bottom). Yolk cells are colored in light blue, primary hull cells in orange, secondary hull cells in green and embryonic cells in gray. Drawings are not to scale. bl blastomere, bp blastopore, eb embryonic blastema, ec ectoderm, en endoderm, fhm first hull membrane, hm hull membrane, ma macromere, me mesoderm, mi micromere, hc hull cells, phc primary hull cells, pm primary mesoderm, shm second hull membrane, yc yolk cell.

Figure 5

Summary of the embryonic development of Bothrioplanida and Rhabdocoela. (A-H), schematic representations of the early development of bothrioplanids (modified from [26]) and rhabdocoels (modified from [67]). Bothrioplana lays eggs containing two oocytes and many yolk cells, which are fusing to a yolk syncytium before the egg is laid. The oocytes undergo two meiotic divisions and give rise to 8 "blastomeres" (gametes) (A), which further divide to build an embryonic blastema (B). Migrating blastema cells (C) provide hull cells enveloping the yolk syncytium and the blastema cells, which are accumulating in the brain primordium and the pharynx primordium (D). In rhabdocoels, the first cell division is equatorial, giving rise to an animal micromere and a vegetal macromere (E). Proliferation of these two initial cells forms a discoidal embryonic blastema, which is first placed in the middle of the egg (F) and later moves to one side (G), which will become the future ventral side of the embryo. The epidermis differentiates from this embryonic blastema, as do the other organs, and engulfs the mass of external yolk cells (H). In all schemes, an idealized animal-vegetal axis (ventral-dorsal axis in D, G and H) cross section of the embryo is represented (animal/ventral to the top, vegetal/dorsal to the bottom in bothrioplanids and vegetal/ventral to the bottom, animal/dorsal to the top in rhabdocoels). Yolk cells are colored in light blue, hull cells in orange and embryonic cells in gray. Drawings are not to scale. bl blastomere, "bl" "blastomeres" which are gametes, brp brain primordium, eb embryonic blastema, ep epidermis, hc hull cells, mb migrating blastomeres, pp pharynx primordium, yc yolk cell, ycn yolk cell nuclei in a yolk syncytium ys.

Figure 6

Summary of the embryonic development of Adiaphanida. (A-L), schematic representations of the early development of fecampiids (modified from [77]), prolecithophorans (modified from [79]) and triclads up to the incorporation of the external yolk cells by the embryo. In fecampiids, early cleavage seems not to be of the disperse type (A), as in the other adiaphanids. After cleavage, the embryo forms an open pouch (B) and incorporates inside this cavity part of the yolk cells (C). Subsequently, the embryo extends its walls to the periphery of the yolk mass, incorporating the remaining yolk and adopting a hemispherical shape (D). The yolk becomes restricted to the posterior part of the embryo, while the blastomeres in the opposite pole proliferate and form an embryonic blastema. In prolecithophorans, disperse cleavage is observed (E), although micromeres and macromeres are still recognizable. After a few cell divisions, blastomeres form an internal epidermal layer (F) that eventually covers the whole embryo and the external yolk cells after an inverse epibolic movement (G-H). The remaining blastomeres form an embryonic blastema on one side of the embryo, as observed in other neoophoran flatworms (H). In triclads, the formation of a yolk-derived syncytium where disperse cleavage takes place is observed in early embryos (I). Once a certain number of blastomeres is reached, some of them differentiate into two transitory organs (primary epidermis and embryonic pharynx (J), that will be used to ingest the maternally supplied yolk cells (K). After yolk ingestion, the remaining undifferentiated blastomeres proliferate and differentiate into the definitive organs (L), replacing the transitory ones. In all schemes, an idealized cross section of the embryo is represented. In (L), ventral to the bottom and anterior to the left. Yolk cells are colored in light blue, primary hull cells in orange and embryonic cells in gray. Drawings are not to scale. bl blastomere, eb embryonic blastema, ec epidermal cavity, epi epidermis, eph embryonic pharynx, epp embryonic pharynx primordium, es eggshell, pb polar body, pep primary epidermis, yc yolk cell, ys yolk syncytium.

Figure 7

The phylotypic stage in free-living flatworms. (A-F), schematic representations of embryos of the major groups of free-living flatworms at the moment of organ specification (stage 6 according to [67]). Regardless of their particular early steps of embryogenesis, free-living flatworm embryos exhibit the greatest similarity at this point of development, being also the stage at which the basic body plan is defined. In all groups, an anterior neuropile and a mid-posterior ventral pharynx start to differentiate, whereas the abundant yolk (either external or internal) adopts a dorsal position. In macrostomids and neoophoran groups, the definitive epidermis differentiates from the ventral side of the embryo to the most dorsal regions, superseding the yolk mantle or the hull membranes, respectively. These similarities led some authors to propose this time point as the phylotypic stage of Platyhelminthes [75], a concept that we extend in this work to other groups of free-living flatworms not previously considered. In all schemes, anterior is to the left and ventral to the bottom. Drawings are not to scale.