Børve A and Hejnol A (2014), Development and juvenile anatomy of the nemerto...

ECB-ART-43511

Front Zool 2014 May 09;11:50. doi: 10.1186/1742-9994-11-50.

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Development and juvenile anatomy of the nemertodermatid Meara stichopi (Bock) Westblad 1949 (Acoelomorpha).

Børve A , Hejnol A .

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INTRODUCTION: Nemertodermatida is the sister group of the Acoela, which together form the Acoelomorpha, a taxon that comprises bilaterally symmetric, small aquatic worms. While there are several descriptions of the embryology of acoel species, descriptions of nemertodermatid development are scarce. To be able to reconstruct the ground pattern of the Acoelomorpha it is crucial to gain more information about the development of several nemertodermatid species. Here we describe the development of the nemertodermatid Meara stichopi using light and fluorescent microscopic methods. RESULTS: We have collected Meara stichopi during several seasons and reconstruct the complex annual reproductive cycle dependent on the sea cucumber Parastichopus tremulus. Using common fluorescent markers for musculature (BODIPY FL-phallacidin) and neurons (antibodies against FMRFamide, serotonin, tyrosinated-tubulin) and live imaging techniques, we followed embryogenesis which takes approximately 9-10 weeks. The cleavage pattern is stereotypic up to the 16-cell stage. Ring- and longitudinal musculature start to develop during week 6, followed by the formation of the basiepidermal nervous system. The juvenile is hatching without mouth opening and has a basiepidermal nerve net with two dorsal neurite bundles and an anterior condensation. CONCLUSIONS: The development of Meara stichopi differs from the development of Acoela in that it is less stereotypic and does not follow the typical acoel duet cleavage program. During late development Meara stichopi does not show a temporal anterior to posterior gradient during muscle and nervous system formation.

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Genes referenced: fmo1 LOC100887844 LOC100888622 LOC115919910 LOC115923516 LOC115925415 LOC578198 LOC590297 pole tubgcp2

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	Figure 1. Collection of Meara stichopi. A) Three individuals of Meara stichopi from a collection in June. Individuals are not gravid and the size range is between 1–2 mm. B) Sea cucumber Parastichopus tremulus, the host of M. stichopi (photo courtesy of Mattias Ormestad, kahikai.org, anterior to the right). C) The “Schander sled”, after dredging in 250 m depth in the Lysefjorden. Red P. tremulus sea cucumbers visible in the mesh. D) Opened foregut of P. tremulus with adult M. stichopi (arrows). Gut content visible on top.
	Figure 2. Egg deposition of gravid M. stichopi and model of annual life cycle. A) Gravid adult of Meara stichopi collected in September. The characteristic double statocyst (dst) at the anterior end is indicated. B) Close-up of oocytes in different stages of the adult (black arrows). Note that smaller oocytes are located more distally than the large oocytes. Anterior to the left, the gut is labeled with the dotted line. C) Five eggs (white arrows) deposited in a glass bowl by an adult M. stichopi oriented with the anterior end to the embryos. D) Two eggs in jelly deposited on the bottom of the glass bowl. Small dots surrounding the eggs are motile spermatozoa (arrowheads). E) Model for annual cycle of M. stichopi. According to the model, fertilized eggs exit the sea cucumber through the gut and develop between 9–12 weeks in the sediment. After hatching, the juveniles initially do not have a mouth opening and survive from the nutrients of the yolk. We presume the juveniles are ingested by the sea cucumbers, where they are able to adhere to the foregut of the sea cucumber, and live as commensals. The juveniles grow to adults over the next months and start to become gravid in August-October. Fertilization occurs in the foregut of the sea cucumber and eggs are deposited, probably exiting the gut of the sea cucumber through the anus. The adults disintegrate after egg deposition and are digested by the sea cucumber. The approximate variation of development covers a period of three months, which also includes the time window when gravid adults are observed to deposit eggs.
	Figure 3. Timing of the cell divisions of an embryo of M. stichopi up to the 50-cell stage. Cell divisions of a single embryo recorded with time-lapse microscopy. The lineage of the vegetal blastomeres indicated with dark blue and dark red branches, the animal blastomeres in light blue and orange branches. The duration of the cell cycle increases during the course of development from 24 hours to 3 days. A-E) Fertilized egg and cleavage stages imaged with Nomarski optics. A) Fertilized egg with egg shell, B) 2-cell stage. C) 4-cell stage, D) 8-cell stage E) 16-cell stage, A-E, same embryo. F) different embryo in a 48-cell stage. Scale bar: 30 μm.
	Figure 4. Early cleavage pattern of Meara stichopi embryos. Nuclear labeling with Propidium Iodide (magenta), cell cortices and spindle with BODIPY FL-Phallacidin (green) Left row Maximum Intensity Projections, right row optical sections. A) 2-cell stage (3 days after fertilization). One of the polar bodies (pb) is visible at the animal pole. A’) shows an optical section through the same embryo. Propidium iodide is labeling the chromatin in the nucleus (nc) as well as the centrosomes (ct). Both blastomeres are equal in size. B) After 4.5 days, the 4-cell stage has large blastomeres at the vegetal pole, and two smaller daughter blastomeres at the animal pole. B’) shows a section of the embryo in B). The spindles are arranged for the future direction of cell division. C) After 5.5 days the 8-cell stage is composed out of four larger cells at the vegetal pole with four blastomeres at the animal pole. C’ shows an optical section of the embryo of C), with spindles arranged to the future plane of division. D) 16-cell stage reached 7 days after fertilization. The size differences between the blastomeres are less prominent and the arrangement is variable. D’) BODIPY FL-phallacidin labeled cell borders as well as the centrosomes, while the chromatin is labeled by propidium iodide. E) 24-cell stage after 8.5 days. E’) shows a median section of the embryo shown in E). The blastocoel is bordered with the phallacidin labeled cell cortex of the outer blastomeres. F) 64-cell stage 10.5 days after fertilization. F’ shows the cells that have been internalized (blastomeres labeled with arrowhead) during the transition from the 24 to the 64 cell stage. Sister blastomeres are connected by white bars, animal pole is indicated with an asterisk. Scale bar: 30 μm.
	Figure 5. Later development of M. stichopi embryos including muscle formation. Nuclear labeling with Propidium Iodide (magenta), muscle fibers with BODIPY FL-Phallacidin (green) and anti-tyrosinated tubulin (yellow). Left row Maximum Intensity Projections, right row optical sections. A) Embryo two weeks after fertilization with ~180 cells labeled with BODIPY FL-phallacidin. A’) shows the inner cell mass (encircled by dotted line) in an optical section of the embryo shown in A). B) Embryo with ~500 cells three weeks after fertilization. B’) shows the nuclei close to the cell membrane of each cell. C) 4-week old embryo composed out of approximately 700 cells. C’) Optical section of C), with actin filaments visible that indicate the beginning of the formation of muscle fibers (arrows). D) Dorsal view on 5–6 week old embryo composed out of ~800 cells. The actin fibers of the myocytes are visible in all areas of the embryo. D’) Optical section of D) with subepidermal signal of BODIPY FL-phallacidin visible in multiple areas of the embryo. E) The labeling of tyrosinated-tubulin in 6–7 week old embryo shows the cilia in the epidermis of the embryo (yellow), dorsal view. The phallacidin labeling of the musculature has become more prominent but is still irregular. E’) Optical cross section through another embryo in the same age as E) The propidium iodide labeled nuclei and the musculature, dorsal view. F) The 7–8 week old embryo shows regularly arranged muscle fibers corresponding to the future pattern of the ring-musculature. F’) most nuclei are located at the apical pole of the epidermal cells (white arrows). Other nuclei are located also at the base of the epidermis (red arrows), likely the nuclei of the neural precursors of the basiepidermal nerve net. Scale bar 30 μm in all images except F’ 10 μm.
	Figure 6. Meara stichopi hatchlings, general morphology and serotonergic cells. Optical stacks of different juveniles labeled with antibodies and BODIPY FL-Phallacidin. Anterior is indicated with an asterisk. A) Dorsal view of hatchling labeled with anti-tyrosinated tubulin antibody (magenta) and BODIPY-phallacidin (green). The basiepidermal nerve net is located just above the ring and longitudinal musculature of the juvenile. Two bilateral neurite bundles (dnb) are extending from anterior to the posterior along the body with a more anterior concentration of axon tracks. A prominent cross nerve (crn) is visible more posterior. The musculature is forming a spindle-shaped sheath around the body and is composed out of ring musculature and longitudinal muscles. B) Ventral view of hatchling of Meara stichopi labeled with anti-tyrosinated tubulin antibody (magenta), BODIPY FL-phallacidin (green) and anti-serotonin antibody (yellow). The location of the future mouth is indicated (fmo), but the mouth is not formed yet. The anti-serotonin antibody is labeling cells that are located in the epidermis on the ventral side of the animal. The shape of these cells is indicating a sensory function and a higher concentration of these cells is found anterior. Similar sensory cells are also found on the dorsal side of the hatchling (not shown). The inlet shows a close up of an optical section of the hatchling. The epidermal serotonergic sensory cells (ssc) are directly connected to the muscular system and possess extensions to the outer epidermis. Scale bar 15 μm
	Figure 7. Morphology hatchlings of Meara stichopi: FMRFamide signal. Different optical sections through a hatchling of Meara stichopi labeled with anti-tyrosinated tubulin (magenta) and anti-FMRFamide (cyan) antibodies, anterior to the left. A) Dorsal section shows neurite bundles (dnb). A basiepidermal ‘commissural’ neurite bundle (cnb) is connecting the two bilateral longitudinal bundles. The longitudinal neurites extend to the posterior end, where the two strands are connected. The dorsal crossing nerves are visible (crn). B) More ventral optical section of the confocal stack. The basiepidermal nerve net (bepnn) is visible and FMRFamide-signal is detected internally around the double statocyst. C) Ventral optical section of the same hatchling as in A) and B). Subepidermal cells that are labeled with the anti-FMRFamide antibody are visible (seamidc). The nature of these cells remains unclear. Scale bar 20 μm.
	Figure 8. Musculature of hatchlings of Meara stichopi. Musculature of two different stages of Meara stichopi juveniles, anterior to the left. A) Ventral view on a juvenile that hatched in the laboratory. The muscle sheath is surrounding the whole body and no mouth opening is formed yet. B) A ventral view on a larger and older juvenile collected from the gut of the sea cucumber with the mouth opening (mo) present. C) Optical cross-section through the animal shown in B). Internal muscle strands (im) extend from the dorsal to the ventral side. D) Longitudinal optical section through juvenile shown in B). The dorso-ventral internal muscle is arranged along the anterior-posterior axis in a serial fashion. Scale bar 10 μm.
	Figure 9. Schematic drawings of the comparisons of early acoelomorph embryos up to the 16-cell stage. Comparison between the development up to the 16-cell stage between the nemertodermatids M. stichopi A) 2-cell B) 4-cell, C) 8-cell D) 16-cell stage and previously described N. westbladi (E-F, same arrangement of the stages as for M. stichopi) and acoel embryos (I-L, same arrangement of the stages as for M. stichopi) (see discussion in the text). The 16-cell stage of N. westbladi is shaded and labeled with a question mark because it has not been documented in [22] with photographs and own observations could not confirm this blastomere arrangement. Bars connect sister blastomeres in all stages.

References [+] :

Baguñà, The dawn of bilaterian animals: the case of acoelomorph flatworms. 2004, Pubmed