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Figure 1. Scanning electron micrographs showing different early embryological stages of Saccoglossus kowalevskii . (A) The 8 hour post fertilization (h pf) blastula shows a prominent depression at the vegetal pole (vp). Inset shows blastomeres at higher magnification (high mag). Partial constrictions demonstrate ongoing cleavage (arrowhead). (B) The flattened gatrula (24 h pf) shows the invagination of the archenteron at the blastoporus region (bp). Inset: a single cilium (ci) is developed on each cell‘s surface. (C) Around 30 h pf gastrulation is completed. Embryos display an elongated shape. The multiciliated telotroch (tt) is developed about 50 μm from the blastopore. (D) The cells of the blastula stage are of cuboid shape, and covered apically with microvilli (mv). The cells are interconnected by occluding cell junctions (ocj). (E) The cells of the gastrula are highly prismatic enclosing the blastocoel (bc). (F) The blastocoel is reduced by the endodermal germ layer almost completely. Endodermal cells (edc) are columnar, bordering the lumen of the archenteron (ar) with their apical cell surface. Long cilia protrude into the lumen. Ectodermal cells (ecc) exhibit a columnar shape, with sometimes very narrow elongated parts, conveying a pseudostratified appearance. ap animal pole, pf perianal field.
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Figure 2. 3D reconstruction of the embryo of Saccoglossus kowalevskii at 36 h pf. Rows from left to right: dorsal, ventral, left, and right view. Columns from top to bottom: The merge row (A-D) shows the embryo with all reconstructed structures. Ectoderm row (E-H) shows external shape of embryo, telotroch not shown. Endoderm row (I-L) reveals the transparent endodermal tissue (light green) and the position of the endodermal lumen (yellowish green). Mesoderm row (M-P) shows the position of the anterior protocoel (blue) and the primordal meso- (pink) and metacoelic (red) tissue. Download interactive 3D-PDF as Additional file
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Figure 3. Histology and fine structure of the embryo ofSaccoglossus kowalevskiiat 36 h pf.A - C, Electron micrographs. D Semithin sagittal sections. E-F Scanning electron micrographs. (A) The lumen of the archenteron (ar) is still continuous with the anterior primordal protocoel (ppc). (B) The apical cell processes of the primordal protocoelic cells (pcc) are goblet-shaped and their basal portion is resting on ecm (arrowheads) separating the protocoel from the ectoderm (ec). (C) The epithelial endodermal cells (edc) are filled with several vesicles (vs) apically. Inset: Each cilium is connected to the cytoplasm by an anchoring complex. (D) Sagittal sections of two specimens. At the onset of mesoderm formation, the endoderm (ed) shows two shallow constrictions (arrowheads) mirroring the embryo’s future tripartite body organization. The lower embryo is slightly older indicated by the completely separated protocoel (pc). The primordal mesoderm (pmd) starts to establish laterally, at the middle and posterior regions of the endoderm. The extracellular matrix (ecm) is indicated by the dotted line. (E-F) Sagittally dissected embryo showing the internal organization of the posterior end (note the position of the telotroch (tt)). Ectoderm and endoderm have close contact to each other. A third layer of cells, the primordal mesoderm, reaches between ecto- and endoderm. ac accessory centriole, bb basal body, ecc ectodermal cell, ci cilium, cr ciliary rootlet, mi mitochondrion, mv microvilli, nn nerve net, yo yolk, za zonula adherens.
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Figure 4. 3D reconstruction of the embryo of Saccoglossus kowalevskii at early kink stage (~ 56 h pf). Rows from left to right: dorsal, ventral, left and right view. Columns from up to down: The merge row (A-D) shows the embryo with all reconstructed structures. Epidermis row (E-H) shows the external shape of embryo. The telotroch is not shown. Endoderm row (I-L) reveals the transparent endodermal tissue (light green) and the straight course of the endodermal lumen (yellowish green). Lateral evaginations (double arrowheads) in the collar region illustrate the connections to the pockets that constitute the mesocoela. Mesoderm row (M-P) shows the position of the anterior protocoel (blue) and the paired meso- (pink) and metacoelic (red) compartments. The anlage of the pericardium (purple) emerges in the posteriormost region of the proboscis (pr) within the ectoderm. Download interactive 3D-PDF as Additional file
3. co collar, mo mouth opening, stA anlage of the stomochord, tr trunk.
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Figure 5. Histology and fine structure of the early kink stage of Saccoglossus kowalevskii. (A) Semithin sagittal section showing the inner organization of the embryo. The lateral evagination of the endodermal wall constitutes the rudiment of the earlier connection to the mesocoela. Fine structure of the tubular connection is shown in C. (B) The longitudinal epitheliomuscle cells (lec) of the protocoel (pc) are rich in myofilaments (myo) and mitochondria (mi). Neighbouring cells are interconnected by zonulae adherentes (za). (C) The cells comprising the former connection to the mesocoela are monociliated and resemble those of the endodermal lining. Inset: Each cilium (ci) is anchored to the cytoplasm by a short horizontal rootlet and a second vertical rootlet. (D) The endodermal cells (edc) are columnar and monociliated. They are covered by numerous bulbus microvilli (mv) and several yolk granules (yo) are present within the cytoplasm. (E) Cross section of the mesocoelic mesoderm composed of two layers. The proximal mesothelium (pms) is made up of flattened cells, while the distal mesothelium (dms) contains more cuboid and voluminous cells. Note the course of the ecm (arrowheads). (F) The proximal flattened cells contain basal myofilaments in a circular arrangement. There is a virtual lumen present between both cell layers as indicated by interspersed cilia (see S2D for high mag). (G) The distal cells contain basal myofilaments in a longitudinal arrangement. (H) SEM of a dissected embryo showing the same area as seen in E. The mesodermal is composed of a distal and proximal layer of cells. ac accessory centriole, cr ciliary rootlet, ec ecdoderm, ecc ectodermal cell, ed endoderm, lu lumen, ms mesocoel, msc mesodermal cell, mt metacoel, nu nucleus, vs vesicles.
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Figure 6. 3D-reconstruction of the embryo of Saccoglossus kowalevskii at early kink stage (~ 96 h pf). Rows from left to right: dorsal, ventral, left and right view. Columns from up to down: The merge row (A-D) shows the embryo with all reconstructed structures. Note the asymmetric development of the anlagen of the gill pores (double arrowheads) in C and D and O and P. Epidermis row (E-H) shows the external shape of embryo. The telotroch is not shown. Endoderm row (I-L) reveals the transparent endodermal tissue (light green) revealing the subdivision into an anterior pharynx and posterior intestine region. The anlagen of the gill pores (gpA) can easily be discerned. The anlage of the stomochord (stA) is protruding shortly into the protocoel. Mesoderm row (M-P) shows the position of the anterior protocoel (blue) and the paired meso- (pink) and metacoelic (red) compartments. The pericardium (purple) is bulb-shaped and protruding into the protocoelic cavity. Download interactive 3D-PDF as Additional file
5. co collar, mo mouth opening, stA anlage of the stomochord, tr trunk.
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Figure 7. Histology and fine structure of the late kink stage in Saccoglossus kowalevskii (~ 96 h pf). (A) sagittal section showing the internal organization. The anlagen of the 1st gill pores (gpA) are visible. The meso- (ms) and metacoelic (mt) cavities enlarge to form central lumina. (B) The pericardium (pd) is surrounded by a basement membrane (arrowheads) and furthermore bulb-shaped by protruding into the protocoelic cavity (pc). (C) The protocoelic cells lining the pericardium are differentiated into podocytes (po) resting on prominent blood sinus (bs) nested underneath the basement membrane (bm). (D) The elliptic anlage of the gill pore is constituted by monociliated cells though, the cytoplasm of individual cells contain several centrioles (ct) indicating ciliogenesis. (E) The majority of the protocoelic cells are differentiated into myoepithelial cells. Inner longitudinal muscle cells alternate with circular muscle cells as indicated by the arrangement of myofilaments (myo). (F) SEM of a dissected embryo revealing the goblet-shape of the apical cell processes of the protocoelic cells. (G) The meso- and metacoelic lining cells constitute a sqamous epithelium holding a single cilium (ci) at the cell surface. dms distal mesodermal cell, ed endoderm, ep epidermis, gpc gill pore cell, mi mitochondrion, nn nerve net, nu nucleus, pdc pericardial cell, pe pedicels, pms proximal mesodermal cell, pph primordal pharynx, stA anlage of the stomochord, yo yolk, za zonula adherens.
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Figure 8. 3D-reconstruction of the embryo of Saccoglossus kowalevskii at 1 gill slit stage (~ 132 h pf). Rows from left to right: dorsal, ventral, left and right view. Columns from up to down: The merge row (A-D) shows the embryo with all reconstructed structures. The first gill pore (gp) is opened on the left side whereas the right side is still closed. Epidermis row (E-H) shows the external shape of embryo. The telotroch is not shown. The collar cord (cc) is invaginated by a process similar to chordate neurulation. Endoderm row (I-L) shows the transparent endodermal tissue (light green) revealing the right-curved course of the oesophagus (oe). Only the left gill pore is opened. The still short stomochord (st) is protruding into the protocoel. At the posterior end of the digestive tract a hindgut region (hg) can be distinguished. Mesoderm row (M-P) shows the position of the anterior protocoel (blue) and the paired meso- (pink) and metacoelic (red) compartments. The pericardium (purple) is protruding into the protocoelic cavity. Download interactive 3D-PDF as Additional file
6. i intestine, mo mouth opening, ph pharynx.
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Figure 9. Histology and fine structure of the 1 gill slit stage of Saccoglossus kowalevskii (~ 132 h pf). (A) Slightly oblique sagittal section displaying the opening of the mouth (mo) and the left gill pore (gp). (B) Pharyngeal lining cells. Inset: The slender microvilli (mv) produce a glycokalyx (gx) covering the cells. (C) Duct of the gill pore. Cilia (ci) and microvilli are projecting into the lumen of the duct. (D) High mag of the apical cell surfaces of the cells lining the intestinal region. The entire surface area is enlarged to form microvilli. E The cells lining the duct of the gill pores are interconnected by zonulae adherentes (za). The apical cytoplasm contains numerous centrioles (ct). Inset: The duct lining cells are developing into multicilated cells. (F) The proximal (pmt) and distal (dmt) cells of the metacoel constitute flattened epithelial cells. (G) Epithelial cells constituting the mesocoelic lining. (H) Cross section of the mesocoelic lining cells (msc). Blood vessels (bv) are situated between the basement membranes (arrowheads, bm). Inset: The mesocoelic cells contain myofilaments (myo) within the very basal portions of the cytoplasm. P ac accessory centriole, cr ciliary rootlet, ep epidermis, i intestine, lu lumen, mi mitochondrion, ms mesocoel, mt metacoel, nn nerve net, nu nucleus, pc protocoel, ph pharynx, yo yolk.
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Figure 10. 3D-reconstruction of the juvenile of Saccoglossus kowalevskii at 2 gill slit stage (~ 432 h pf). Rows from left to right: dorsal, ventral, left and right view. Columns from up to down: The merge row (A-D) shows the juvenile with all reconstructed structures. Note the characteristic post-anal tail (pat). Epidermis row (E-H) shows the external shape of juvenile, condensed nervous structures and the skeletal elements. There is only one skeletal rod (skr) developed on the right side yet, whereas two are already present on the left side. Endoderm row (I-L): The stomochord (st) is projecting into the protocoel and supported ventrally by the proboscis skeleton (sk). The oesophagus (oe) is positioned asymmetrically on the right body half of the juvenile. The anus (an) is opened at the posterior end of the hindgut region (hg). Mesoderm row (M-P) shows the position of the anterior protocoel (blue) and the paired meso- (pink) and metacoelic (red) compartments. The glomerulus (gl) is visible covering the anterior tip of the stomochord. Download interactive 3D-PDF as Additional file
8. cc collar cord, dns dorsal nerve cord, gp gill pore, i intestine, pd pericardium, ph pharynx, ps proboscis stem, vnc ventral nerve cord.
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Figure 11. Detail images of the juvenile of Saccoglossus kowalevskii at 2 gill slit stage (~ 432 h pf). (A-E) 3D-reconstruction, (F-H) histological sections. (A,B) Detail images showing the left and right sides of the collar and anterior trunk region. Note the asymmetric development of the gill pores (gp). (C) The pericardium (pd) is overlying the heart sinus (hs), which is in turn connected to the glomerulus (gl) by a pair of lateral vessels (lv). Note, the dorsal vessel which supplies the heart sinus from posterior is not shown. (D) View from posterior, the right meso- and metacoel are hidden. The dorsal nerve cord (dnc) terminates just before the anus (an). The metacoel is extended into the post-anal tail (pat). (E) Dorsal view focussed on the pharyngeal region (ph). The skeletal rods (skr) develop asymmetrically and ar of unequal number. The proboscis pore (pp) opens dorsally on the left side of the base of the proboscis. (F) Cross section of the trunk region. The ventrolateral metacoelic cells constitute the substantial longitudinal muscles (lm) of the trunk. (G) Cross section of the collar region. The collar cord (cc) runs within the dorsal mesentery. (H) Longitudinal section showing the position of the proboscis pore, stomochord, pericardium and heart sinus. The majority of the protocoelic cavity (pc) is filled with longitudinal muscles. Anterior is to the left. ep epidermis, hg hindgut, mc mouth cavity, ms mesocoel, mt metacoel, oe oesophagus, ps proboscis stem, sk proboscis skeleton, st stomochord, vnc ventral nerve cord.
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Figure 12. Histology and fine structure of the juvenile ofSaccoglossus kowalevskiiat the 2 gill slit stage (~ 432 h pf). (A) Histological section, (B-H) Transmission electron micrographs. (A) Sagittal section showing internal structures. The proboscis stalk is flanked by elongations of the mesocoelic cavity, which are filled with longitudinal muscle strands (lm) completely. (B) Cells lining the roof of the mouth cavity. Glandular mucus cells (glc) are interspersed. (C) Vacuolated cells lining the ventrolateral regions of the anterior pharynx. (D) The stomochord is oval in cross sections and its vacuolated cells surround a central lumen (lu). (E) Horizontal section of the gill pore (gp). The proximal duct of the gill pore is extremely high ciliated. Inset: high mag of the cilia (ci) and their associated basal structures. (F) The intestinal lining is composed of columnar cells. (G,H) special intestinal cells containing zymogen-like granules (zg) or distended amount of rough endoplasmatic reticulum (dER). cr ciliary rootlet, epc epidermal cell, hg hindgut, mo mouth opening, mi mitochondrion, ms mesocoel, mt metacoel, muc mucus cell, mv microvilli, ne neurites, nu nucleus, pc protocoel, ph pharynx, skr skeletal rod, va vacuole, vs vesicles, za zonula adherens.
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Figure 13. Fine structure of the juvenile of Saccoglossus kowalevskii at 2 gill slit stage (~ 432 hpf). (A) Cells lining the proximal duct of the proboscis pore. These cells have developed circular myofilaments (myo). (B) Longitudinal section showing the musculature of the protocoel. Outer circular muscles cells alternate with inner longitudinal muscle cells. (C) Podocytes (po) resting on the basement membrane (bm) covering the glomerular sinus (gls). Pedicels (pe) form fenestrations between them. (D) SEM showing the highly flattened metacoelic cells resting on the intestinal region. The area containing the nucleus is protruding into the coelomic cavity. (E) Cross section of the pericardial cells (pdc) covering the heart sinus (hs). Basal myofilaments are orientated in longitudinal direction. (F) Cells lining the distal duct of the proboscis pore. These cells are flattened and highly microvillar (mv). (G) Cross section of the mesocoelic lining cells (msc). Myofilaments of the somatic lining are longitudinally arranged. Inset: in contrast, myofilaments of the visceral lining cells are orientated in circular direction. (H) The somatic lining of the metacoel is hyperdeveloped to form the substantial longitudinal strands of the ventral musculature within the trunk region. Inset: numerous adhesion plaques (adj) anchor the myofilaments to the extracellular matrix (ecm). ac accessory centriole, ci cilium, cr ciliary rootlet, cmyo circular myofilaments, lmyo longitudinal myofilaments, ms mesocoel, mt metacoel, ne neurites, nu nucleus, phc pharyngeal cell, za zonula adherens.
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Figure 14. Coelom formation in Enteropneusta. (A – E) Schematic drawings of different types of coelom formation compiled and newly colored from text books
[3,49,92]. For a more detailed explanation see text and Table
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