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Fig. 1. Developmental progression of A. rubens from fertilized egg to bipinnaria larva. (A) Unfertilized immature oocyte with the germinal vesicle. (B) Fertilized oocyte without germinal vesicle. Note the presence of fertilization membrane. (C) Fertilized oocyte with polar bodies. (D) Two-cell stage embryo following the first cleavage. (E) Four-cell stage embryo. Cleavages are roughly equal, giving rise to blastomeres of roughly equal size within the fertilization membrane. (F) Blastula-stage embryo, consisting of approximately 250 cells. Embryos are round at this stage, due to constraints imposed by the fertilization membrane. (G) Blastula-stage embryo following the loss of the fertilization membrane. (H) Swimming blastula-stage embryo. (I) Blastula-stage embryo. Elongation has taken place along the anteroposterior axis, and thickening and flattening of the vegetal plate is evident along the vegetal end of the embryo. (J) Early gastrula-stage embryo. Archenteron has invaginated and has thinner walls at the blind end than in the rest of the tube. (K) Mid-gastrula stage embryo. Archenteron has thickened relative to early gastrula-stage embryos seen in (J). Additionally, many mesenchymal cells have separated from the tip of the archenteron. (L) The lumen of archenteron has expanded and two coelomic pouches have formed in the developing gut. (M) Immature bipinnaria stage larva. The gut has differentiated into the esophagus, stomach, and intestine, and coelomic pouches are well-developed. Numerous mesenchymal cells are present. (N) Lateral view of bipinnaria larva. A well-developed, tripartite gut is present, as well as the hydropore canal, which connects one of the coelomic pouches to the exterior of the animal. (O) Bipinnaria larvae with tripartite gut and two elongate coelomic pouches. Green algae are present in the stomach of the animal, showing that it has started feeding. GV, germinal vesicle; PB, polar bodies; FM, fertilization membrane; VP, vegetal plate; A, archenteron; MeCs, mesenchymal cells; CP, coelomic pouch; St, stomach; Oe, esophagus, HG, hind gut. Scale bars are 75 µm.
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Fig. 2. Musculature of bipinnaria larva of A. rubens. (A) Maximal projection confocal image showing the oral view of myosin heavy chain (MHC, false colored in magenta) immunoreactivity and cell nuclei visualized with DAPI (gray) in a 2-week-old larva. Strong MHC immunostaining can be seen in three populations of muscle cells: fibrous longitudinal muscles encircling the esophagus, and two clusters of aboral muscles associated with the dorsal surface. (B) Lateral view of maximum projection confocal image showing a high-magnification view of MHC-immunoreactive aboral muscles located on the dorsal surface of the larva, as well as the esophageal muscles. (C) Oral view of maximum projection confocal image showing a high-magnification view of MHC-immunoreactive muscles surrounding the esophagus. (D) Bright-field (with DIC) image showing the esophageal muscles surrounding the stomach, which exhibit MHC-immunoreactivity as shown in (C). (E) High-magnification image showing MHC-immunoreactivity in aboral muscle cells. These cells are arranged into two clusters on the dorsal surface of the animal and are thicker at one end with a tapering morphology at the other end. (F) Bright-field (with DIC) image of aboral muscle cells and their proximity to mucous cells embedded in the ectoderm of the larva. OeMs, esophageal muscles; AMs, aboral muscles; St, stomach, Sts, stomach sphincter; MC, mucous cell; Hg, hind gut.
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Fig. 3. Architecture and morphology of the nervous system in A. rubens embryos and larvae. (A) Maximum projection in oral view showing synaptotagmin-immunoreactivity revealed by 1E11 antibodies (green) in the A. rubens bipinnaria larva. Strong immunoreactivity is present in the ciliary bands around the mouth and along the gut. In addition to these major areas of labeling, synaptotagmin-immunoreactive projections are also present in other regions of the larva. (B) Synaptotagmin-immunoreactivity in a maximum projection ventral view of a bipinnaria larva. Strong staining is present in the pre-oral ciliary band and the oral hood, as well as in the lateral ganglia and post-oral ciliary band. Innervation can be seen lining the esophagus as well as around the mouth. (C) High-magnification maximum projection image of synaptotagmin-immunoreactive neurons in the oral hood with orally directed projections. Additionally, labeling of clustered neurons of the oral ganglia can be seen on either side of the mouth. (D) High-magnification maximum projection image of synaptotagmin-immunoreactive neurons of the oral hood shown in (C), which can be interpreted as sensory cells with downward projecting processes. (E) High-magnification maximum projection image of synaptotagmin-immunoreactive neuronal cell bodies of the oral ganglia proximal to the mouth and with immunostained processes distributed along the length of the esophagus. (F) Gastrula stage maximum projection embryo stained with serotonin antibodies (magenta) and with cell nuclei labeled using DAPI (blue). Immunoreactivity is strongest in numerous ectodermal cells, interpreted to be neuronal precursors. Immunostaining in the archenteron is interpreted as background staining. (G) Maximum projection ventral view of an immature bipinnaria showing serotonin immunoreactive cells and processes along the ventral surface. These cells are interpreted to be serotonergic neurons and are arranged into 2 clusters in the lateral ganglia. Immunostaining in the stomach is interpreted as background staining. (F) High-magnification maximum projection image of serotonin immunoreactive cells in the lateral ganglia, showing stained cell bodies and neuronal processes. Abbreviations: Pre, pre-oral ciliary band; OH, oral hood; Post, post-oral ciliary band; LG, lateral ganglia; Oe, esophagus; OG, oral ganglia; SC, sensory cells.
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Fig. 4. Neuronal subpopulations expressing ELAV or neuropeptides (AN-peptide, SALMFamide-S2) in A. rubens larvae. (A) Maximum projection confocal image of the oral view of the A. rubens bipinnaria showing ELAV-immunoreactivity (orange). Immunostaining is strongest in distinct subpopulations of cells within the pre-oral and post-oral ciliary bands. (B) High-magnification confocal image in a maximum projection of ELAV-immunoreactive cells in the pre-oral ciliary band and oral hood. Note that neuronal processes are not labeled by the ELAV-antibodies and immunoreactivity is present in some, but not all, ciliary band neurons. (C) High-magnification light microscope image of the oral hood and pre-oral ciliary band showing AN-peptide immunoreactive neuronal cells and processes (red). (D) High-magnification maximum projection light microscope image of the post-oral ciliary band showing AN-peptide immunoreactive neuronal cells and processes. (E) High-magnification maximum projection light microscope image of the post-oral ciliary band showing AN-peptide immunoreactive neuronal cells and processes. (F) Maximum projection confocal image of SALMFamide-S2-immunoreactive processes (pink) in the anterior loop of the post-oral ciliary band and in projections extending onto the oral surface in a larva that has also been labeled for acetylated tubulin (green) and with a nuclear marker (DAPI; blue). (G) High-magnification image showing SALMFamide-S2-immunoreactive cells and their processes in the post-oral ciliary band. Abbreviations: Pre; pre-oral ciliary band; Post, post-oral ciliary band; St, stomach; Hg, hindgut; OH, oral hood; PCs, pyramidal cells.
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Fig. 5. Neuronal subpopulations expressing SoxB2 or the neuropeptide ArPPLN1b and cells undergoing proliferation in A. rubens larvae. (A) Light microscope image showing immunostaining of a sub-population of cells expressing the transcription factor SoxB2 (green) in the mouth region of a bipinnaria larva. (B) Antibodies to the neuropeptide ArPPLN1b (red) reveal immunostained cells and processes around the mouth of a bipinnaria larva in a maximum projection confocal image. (C) As in bipinnaria larvae, in immature bipinnaria, ArPPLN1b -immunoreactivity is strongest in a subpopulation of cells around the mouth. (D) Cell proliferation in a bipinnaria larva labeled using the marker EdU (magenta) together with immunostaining for synaptotagmin using 1E11 antibodies (green) and DAPI (blue). EdU-positive cells are located throughout the larva but are most strongly concentrated in the ciliary bands. Cell proliferation is most evident along the edges of the pre-oral lobe and the edges of the mouth. Maximum projection of the confocal image. (E) Enlargement of D showing the oral hood (arrow). (F) Projection of a single confocal slide of a portion of the ciliary band showing an EdU-positive nucleus adjacent to the 1E11 staining. Abbreviations: Oe, esophagus; St, stomach; OH, oral hood; Post, post-oral ciliary band.
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Fig. 6. Diagrammatic representation of neuronal subpopulations in A. rubens bipinnaria larva. (Left) Oral view showing subpopulations of neurons expressing ELAV, SoxB2, and the neuropeptides AN-peptide and ArPPLN1b. The mouth appears to be a center of neuronal complexity, with at least two distinct subpopulations of neurons present. (Right) Aboral view showing subpopulations of serotonergic neurons and neurons expressing neuropeptides (SALMFamide-S2 and AN peptide).
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