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Fig. 1. Time course of primary mouth development. (A) Frontal view of stages 26–27, black arrow indicates the presumptive primary mouth, scale bar = 260 μm. (B) Optical sagittal section at stages 26–27, white arrow indicates presumptive primary mouth and white arrowhead indicates the evaginated endoderm. Anterior is to the right, scale bar = 200 μm. (C) Histological sagittal section at stages 26–27, bracketed regions show juxtaposed endoderm and ectoderm in the presumptive primary mouth. Anterior is to the right, scale bar = 25 μm. (D) Frontal view of stages 34–35, black arrow indicates invaginating presumptive primary mouth, scale bar = 260 μm. (E) Optical sagittal section of stages 34–35, white arrow indicates presumptive primary mouth. Note that, while the anterior most portion of the embryo in this figure is mid-sagittal, the more posterior of the embryo is not. This is due to curling of the embryo during processing. Anterior is to the right, scale bar = 200 μm. (F) Histological sagittal section of stages 34–35, bracketed regions show juxtaposed endoderm and ectoderm in presumptive primary mouth. Anterior is to the right, scale bar = 25 μm. (G) Frontal view of stages 38–39, black arrow indicates the deeply invaginated presumptive primary mouth, scale bar = 270 μm. (H) Optical sagittal of stages 38–39, white arrow indicates buccopharyngeal membrane. Anterior is to the right, scale bar = 200 μm. (I) Histological sagittal section of stages 38–39, bracketed regions show juxtaposed endoderm and ectoderm in presumptive primary mouth. Cell is shown spanning the width of the forming buccopharyngeal membrane (black arrowhead). (J) Frontal view of stages 40–41, black arrow indicates the primary mouth, scale bar = 270 μm. (K) Optical sagittal section of stages 40–41, white arrow indicates the primary mouth, scale bar = 200 μm. Anterior is to the right, scale bar = 25 μm. (L) Histological sagittal of stages 40–41, showing the primary mouth. Anterior is to the right, scale bar = 25 μm. Abbreviations: end, endoderm; ect, ectoderm; cg, cement gland.
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Fig. 2. Laminin and fibronectin immunohistochemistry during primary mouth formation. Immunoreactivity is shown in green and the counterstains in red (nuclear stain propidium iodide and beta-actin). The presumptive primary mouth region is bracketed and anterior is to the right in all figures. (A) Sagittal section of a stage 22 embryo showing laminin-immunoreactivity between endoderm and ectoderm, scale bar = 60 μm. (B) Sagittal section of a stage 22 embryo showing fibronectin-immunoreactivity between the endoderm and ectoderm, scale bar = 60 μm. (C) Sagittal section of a stage 24 embryo showing discontinuous laminin-immunoreactivity (white arrow) in the presumptive primary mouth, scale bar = 60 μm. (D) Sagittal section of a stage 24 embryo showing discontinuous fibronectin-immunoreactivity (white arrow) in the presumptive primary mouth, scale bar = 60 μm. (E) Sagittal section of a stage 30 embryo showing the absence of laminin-immunoreactivity in the presumptive primary mouth, scale bar = 60 μm. (F) Sagittal section of a stage 30 embryo showing the absence of fibronectin-immunoreactivity in the presumptive primary mouth, scale bar = 60 μm. (G) Sagittal section of a stage 39 embryo showing the absence of laminin-immunoreactivity in the presumptive primary mouth, scale bar = 60 μm. (H) Sagittal section of a stage 39 embryo showing the absence of fibronectin-immunoreactivity in the presumptive primary mouth, scale bar = 60 μm. Abbreviations: cg, cement gland.
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Fig. 3. The role of cell death in primary mouth formation. (A) Frontal view of embryo showing TUNEL labeling in the presumptive primary mouth (arrow) at stages 34–35, scale bar = 300 μm. (B) Histological sagittal section of TUNEL labeling in the presumptive primary mouth at stages 34–35. Arrow indicates TUNEL labeled cell in the inner ectoderm. Anterior is to the right, scale bar = 36 μm. (C) Embryo at stage 40 following treatment with z-vad-fmk (250 μm). Shows that the primary mouth does not form (arrow), scale bar = 330 μm. (D) Untreated control embryo at stage 40 with normal primary mouth (arrow), compare with panel C, scale bar = 330 μm. (E) Optical sagittal section of stage 40 embryo treated with z-vad-fmk as in panel C. Arrow indicates the unperforated primary mouth. Anterior is to the right, scale bar = 310 μm. (F) Untreated control embryo at stage 40 with normal primary mouth (arrow), compare with panel E. Anterior is to the right, scale bar = 310 μm. (G) Histological sagittal section of stage 40 embryo treated with z-vad-fmk as in panel C. Tissue remains thick where an opening should occur (white arrow heads), presumptive primary mouth region is indicated by the black arrow. Anterior is to the right, scale bar = 15 μm. (H) Histological sagittal section of an untreated control embryo at stage 40 with a normal primary mouth (black arrow), compare with G. Anterior is to the right, scale bar = 15 μm. (I) Dissected eye from embryo at stage 40 treated with z-vad-fmk, showing fewer TUNEL positive cells, scale bar = 200 μm. (J) Dissected eye from untreated control embryo at stage 40, showing TUNEL labeling (compare with I), scale bar = 200 μm. (K) Graph showing average number of cells from one eye from each of 5 embryos treated with z-vad-fmk (ZVF) and untreated control (Cont.). Difference is significant based on the Mann–Whitney Rank Sum Test for non-normalized data (P = 0.008). Abbreviations: cg, cement gland.
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Fig. 4. Fate mapping the oral and surrounding regions. All figures show fluorescent DiI in red superimposed upon corresponding light micrograph. The left column show representative embryos at time 0 (stage 24) and the right column show representative embryos approximately 24 h after labeling (stage 35). (A) Frontal view of stage 24 embryo, just after DiI labeling in a medial position just dorsal to the cement gland in the region thought to be the presumptive primary mouth (arrow), scale bar = 200 μm. (B) Frontal view of a stage 35 embryo, 24 h after DiI labeling as in panel A. Shows labeling in the invaginating presumptive primary mouth (arrow), scale bar = 275 μm. (C) Frontal view of stage 24 embryo, just after DiI labeling in positions just lateral to the hatching gland (hg), the presumptive primary mouth is indicated (arrow), scale bar = 200 μm. (D) Frontal view of stage 35 embryo, 24 h after DiI labeling as in panel C. Shows no DiI in the stomodeum (arrow), scale bar = 275 μm. (E) Frontal view of stage 24 embryo just after DiI labeling in a dorsal region between the forming eyes, the presumptive primary mouth is indicated (arrow), scale bar = 200 μm. (F) Frontal view of stage 35 embryo, 24 h after DiI labeling as in E. Shows no DiI in the stomodeum (arrow), scale bar = 275 μm. (G) Ventral view of stage 24 embryo, an incision was made ventral to the cement gland and tissue pulled forward to expose and DiI label the anterior endoderm, scale bar = 200 μm. (H) Lateral view of stage 35 embryo, 24 h after DiI labeling as in G. Shows DiI labeled endoderm in the tissue underlying the stomodeum (arrow), scale bar = 275 μm.
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Fig. 5. (A) Summary of extirpation experiments. The first column of the table indicates the name of the tissue extirpated. The second column shows representative embryos just after extirpation at time zero (stage 24). The third column shows representative embryos 48 h after the operation (stages 40–41). (a) Frontal view of stage 24 embryo just after “Epidermis†(inner ectoderm and outer epidermal layers) in the presumptive primary mouth was extirpated, scale bar = 270 μm. (b) Frontal view of a stage 40 embryo, 48 h after extirpation as in panel A. Shows a normal primary mouth (arrow), scale bar = 350 μm. (c) Frontal view of stage 24 embryo, just after the Cement Gland was extirpated, scale bar = 270 μm. (d) Frontal view of a stage 40 embryo, 48 h after extirpation as in panel C. Shows a normal primary mouth (arrow), scale bar = 350 μm. (e) Lateral view of an embryo just after “Endoderm†(tissue underlying the ectoderm) in the presumptive primary mouth was extirpated, scale bar = 270 μm. (f) Frontal view of a stage 40 embryo, 48 h after extirpated as in e, g or i showing one of the possible abnormal phenotypes, an un-perforated primary mouth and large invagination (arrow), scale bar = 350 μm. (g) Frontal view of stage 24 embryo just after “ Neural†(tissue dorsal to the oral region) was extirpated, scale bar = 270 μm. (h) Frontal view of a stage 40 embryo, 48 h after extirpated as in e, g or i showing one of the possible abnormal phenotypes, an un-perforated primary mouth and medium size invagination (arrow), scale bar = 350 μm. (i) Frontal view of stage 24 embryo just after tissue lateral to the oral region was extirpated, scale bar = 270 μm. (j) Frontal view of a stage 40 embryo, 48 h after extirpated as in e, g or i showing one of the possible abnormal phenotypes, an un-perforated primary mouth and very small barely detectable invagination (arrow), scale bar = 350 μm. (B) Quantitative RT-PCR results performed on extirpated tissues. Numbers were scaled to a maximum value of 100% for each gene. (a) The epidermal marker cytokeratin xk81, is expressed at 42.9% in “Epidermisâ€Â + “Cement glandâ€, 0.68% in “Endodermâ€, 42.2% in “Neuralâ€, and 100% in “Lateral tissuesâ€. (b) The mesodermal marker, m-actin, is expressed at 14.8% in “Epidermisâ€Â + “Cement glandâ€, 28.3% in “Endodermâ€, 14.9% in “Neuralâ€, and 100% in “Lateralâ€. (c) The endodermal marker, edd is expressed at 7.7% in “Epidermisâ€Â + “Cement glandâ€, 100% in “Endodermâ€, 1.57% in “Neuralâ€, and 1.71% in “Lateral†tissues. (d) The neural marker, n-tubulin, is expressed at 27.7% in “Epidermisâ€Â + “Cement glandâ€, 20.0% in “Endodermâ€, 100% in “Neuralâ€, and 80.7% in “Lateral†tissues. (e) The neural crest marker, slug is expressed at 35.0% in “Epidermisâ€Â + “Cement glandâ€, 16.2% in “Endodermâ€, 37.61% in “Neuralâ€, and 100% in “Lateral†tissues. Abbreviations: Ep, Epidermis; Cg, Cement gland; En, Endoderm; N, Neural; L, Lateral.
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Fig. 6. Summary of transplant experiments. The first column shows schematic diagrams of the operations. The second (right) column shows representative embryos 48 h after operation. (A) The ectoderm from the presumptive primary mouth region of a Texas Red-Dextran labeled embryo was removed from a donor embryo and transplanted to the presumptive primary mouth of a recipient un-labeled embryo at the same stage. (B) Frontal view of a stage 40 embryo that was operated on as in A, resulting in formation of a normal primary mouth (arrow), scale bar = 250 μm. (C) The lateral flank ectoderm was removed from a donor embryo, labeled with Texas Red-Dextran, and transplanted to the presumptive primary mouth of a un-labeled recipient embryo at the same stage. (D) Frontal view of a stage 40 embryo that was operated on as in C, resulting in a normal primary mouth (arrow), scale bar = 250 μm.
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Fig. 7. (A) Summary of presumptive primary mouth endoderm and ectoderm transplants. (a) Schematic diagram of the operation. The ectoderm and endoderm from the presumptive primary mouth region and the cement gland were removed from a donor embryo, labeled with GFP, and transplanted to region next to the endogenous presumptive primary mouth of an un-labeled recipient embryo at the same stage. (b) Lateral view of a stage 40 embryo that was operated on as in a, resulting in an ectopic primary mouth (arrow), next to the endogenous primary mouth (arrowhead), scale bar = 250 μm. (c) Schematic diagram of the operation. The presumptive primary mouth ectoderm and endoderm and cement gland removed from a donor embryo, labeled with GFP, and transplanted to a region posterior to the branchial arches of an un-labeled recipient embryo at the same stage. (d) Lateral view of a stage 40 embryo that was operated on as in c, resulting in no obvious ectopic primary mouth, scale bar = 250 μm. (B) Summary of explant experiments. (a) Schematic diagram showing the ectoderm and underlying endoderm, including the cement gland that was removed and cultured. (b) A representative explant as dissected in a did not form any opening in the tissue, scale bar = 185 μm. (c) Schematic diagram showing the presumptive ectoderm, underlying endoderm, lateral and neural regions, including the cement gland that was removed and cultured. (d) A representative explant as dissected in c formed an opening through the tissue (arrow), scale bar = 185 μm.
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Fig. 8. Schematic diagram summarizing the morphological changes during primary mouth development. On the left is a sagittal section of an early tailbud embryo showing the different germ layers. The box indicates the presumptive primary mouth which is magnified in the subsequent pictures. Each picture represents the presumptive primary mouth at progressively later stages. The first morphological change at stage 22 is the dissolution of the basement membrane (BM) in the presumptive primary mouth region. The next anatomical change is the formation of a depression or stomodeum, which begins roughly at stage 30 and is more apparent by stage 32. Approximately 7–10 h after the stomodeum starts to form, at stages 34–35, we observed a burst of apoptosis. This was followed by thinning of the region and by stage 39 some cells could be seen to intercalate and form 1–2 cell layers, called the buccopharyngeal membrane. Finally, by stage 40, a perforation or hole can be seen in the buccopharyngeal membrane that over several hours widens to form an opening to the foregut, the primary mouth.
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