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Figure 1. Subdivisions of the holothurian ENS. (A) Mesothelial plexus labeled with monoclonal antibody RN1 showing an extensive fiber plexus and fiber bundles (arrowhead) while (B) other markers (antiâ€PH3) only labeled discrete fiber populations. (C) Confocal image showing RN1 label fiber (green) distribution in relation to the muscle layer (labeled with Cy3â€labeled phalloidin, red). (D) Connective tissue plexus as labeled by RN1. Arrow points to a neuronâ€type cell. (E) Neuroendocrine cell labeled with RN1 in the luminal epithelium. (F) RN1 labeling of fibers in the mesothelial layer of the mesentery. (G) Diagram showing the intestinal tissue layers. CT, connective tissue layer; Le, luminal epithelial layer. Nuclei are stained with DAPI (red) in (D), (E), (F). Bar (A, B) 10 μm; (C, E, F) 20 μm.
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Figure 2. Degenerating fibers during early formation of the intestinal rudiment. (A) Fibers, labeled here with RN1 (green), are absent from the regenerating intestinal primordium at the distal tip of the mesentery (asterisk) in a 3 dpe specimen. (B) In the 5 dpe intestinal rudiment (asterisk), that has largely increased in size, there are few RN1â€labeled fibers (arrowheads) and these are disorganized and in the process of degenerating. DAPIâ€stained nuclei are shown in red. Bar 50 μm.
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Figure 3. Temporal profile of the appearance of neuronal fibers in the intestinal mesothelium. Labeling with αâ€GFS provides an overview of the pattern of innervation of the regenerating intestine mesothelium. (A) The mesothelium of 10 dpe is devoid of fibers. (B) Few fibers are initially observed in the mesothelium of 14 dpe specimens. The density and organization of fibers increase as regeneration progresses by 21 dpe (C). (D) At 28 dpe fiber innervation closely resembles that of (E) the normal nonâ€eviscerated intestine. Arrowheads signal some of the fiber varicosities in the mesothelium. Me, mesothelial layer; CT, connective tissue layer. Bar 10 μm.
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Figure 4. Differential innervation pattern between the distal and proximal areas of the regenerating rudiment. In 7 dpe regenerating intestinal rudiment nerve fibers immunoreactive to αâ€PH3 (His) are not present in (A) proximal or (B) distal mesothelium, but can be found in (C) the adjacent mesentery. At 14 dpe, PH3â€immunoreactive fibers are found in (D) the proximal but not in (E) the distal mesothelium of the intestinal rudiment. (F) PH3â€immunoreactive fibers are particularly abundant in the connecting region of the mesentery and the intestinal rudiment. A few PH3â€immunoreactive fibers are observed in (G) the distal 21 dpe rudiment mesothelium (arrow) and by 28 dpe fibers are found in both (H) proximal and (I) distal mesothelium. Differential innervation by Pax6â€immunoreactive fibers in (J) the proximal mesothelium of 14 dpe intestinal rudiments but not in (K) the distal portion. Notice the abundant fiber immunoreactivity (arrow) at the mesentery intestinal junction. Antiâ€galaninâ€immunoreactive fibers can be found in the mesothelium of both (L) proximal and (M) distal mesothelium of 14 dpe intestinal rudiments. Bar 50 μm.
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Figure 5. Quantification of fiber regeneration in different regions of the intestinal rudiment. (A) Relative density of RN1â€labeled nerve fibers in the mesothelium of the intestinal rudiment during the various stages of regeneration and in the nonâ€eviscerated (normal) specimens. The relative amount of fibers practically disappears during the early stages and then regenerates to levels similar to normal by 35 dpe. Each point represents the mean ± SD of at least three animals. (B) Proximal/distal fiber density ratios for various fiber populations. Fibers were labeled with antibodies against GFS, galanin, βâ€tubulin, PH3 (histone) or with monoclonal antibody RN1. See Materials and methods for quantification procedure. Each point represents the mean ± SE of at least three animals.
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Figure 6. Regeneration of perpendicular fibers in the connective tissue of the intestinal rudiment. These varicose fibers (arrow) that cross from the mesothelium into the connective tissue are first observed in the proximal area of the rudiment by 14 dpe (A) using the RN1 antibody, while they are absent in the distal area of the rudiment at the same stage (B). Arrowheads signal some of the fiber varicosities in the mesothelium. Me, mesothelial layer; CT, connective tissue layer. Bar 20 μm.
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Figure 7. Regeneration of fiber bundles. Antiâ€Î²â€tubulin immunoreactivity shows the late appearance of fiber bundles during intestinal regeneration. The bundles are not present in (A) 14 dpe intestines and can be seen to begin to organize in (B) 21 dpe intestinal regenerates. The asterisk (*) shows nerve fibers at different focus planes that might appear to be nerve bundles. (C) At 28 dpe the bundles are organized similarly to those in (D) the normal uneviscerated intestines. (E) Quantification of bundles using immunoreactivity to βâ€tubulin shows a sharp rise between 21 and 28 dpe that appears to take place faster in the medial section of the rudiment. Bar 10 μm. Each point represents the mean ± SD of at least three animals.
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Figure 8. Regeneration of cells labeled with neuronal markers. Cells labeled with neuronal markers are not observed in the normal mesentery; some cells labeled with RN1 (green) appear during the early stages of regeneration in the mesentery mesothelium at (A) 3 and (B) 5 dpe (arrows). (C), (D) RN1â€labeled cells are also found within the connective tissue of the growing rudiment and (C) some show long neuriteâ€like extensions (arrow) while others (D) are small round cells (arrowheads) weakly labeled by the antibody. (E) Immunoreactivity to calbindin (green) shows the initial appearance of cells in the mesothelium of a 10 dpe specimen. DAPI nuclei stain (red). Bar 20 μm.
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Figure 9. Regeneration of neuroendocrine cells. (A) Initial appearance of calbindinâ€immunoreactive cells in a 10 dpe luminal epithelium is observed soon after lumen formation. At 14 dpe (B) calbindinâ€immunoreactive cells acquire neuroendocrine morphology and by 28 dpe (C) the number of cells has increased significantly (the figure shows two adjacent luminal epithelia). GFSâ€immunoreactive cells (D) also appear during the second week of regeneration showing their extended fiber connections. (E) Pax6â€immunoreactive neuroendocrine cells appear at 21 dpe. Bar 20 μm.
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Figure 10. Temporal stages of enteric nervous system regeneration. (A) Extrinsic innervation occurs via the mesentery. Nervous fibers remain within the mesentery even when most of the nervous innervation to the forming intestinal rudiment is degraded and lost. As regeneration continues, fibers originating in neurons present in the mesentery or other tissues such as the radial nerve (not shown) reâ€enter the proximal regions of the new intestinal rudiment and eventually arrive at the distal regions covering the complete intestinal rudiment. The extrinsic innervation preferentially involves the muscle in the mesothelial layers. (B) Intrinsic innervation. Neuronâ€like cells appear during the second week of regeneration in the mesothelium, connective tissue and luminal epithelial layers. Fibers originating from these neurons are also found within these tissues, their density increasing as the new intestine forms and acquires the characteristics of the normal mature intestine. Formation of nervous bundles is shown in both extrinsic and intrinsic innervation panels, since their formation most probably involves both components.
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Figure 11. Quantification of nerve fibers in the intestinal regenerating rudiment. The mesothelia of normal or regenerating intestine were divided into areas proximal, medial, and distal relative to the attachment to the mesentery (proximal right [PR], proximal left [PL], medial right [MR], medial left [ML], distal right [DR], distal left [DL]). The microscope ruler scale (containing five sections) was placed over the region to be measured. Fiber presence was quantified by the number of sections in which fibers were found. Thus the range of measurements was from 0 to 5. A similar strategy was used to quantify fibers in the connective tissue, except that the regenerate was divided into two by an imaginary (dashed) line through the middle. The total number of fibers was counted in each half.
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