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First published online February 15, 2008
Journal of Experimental Biology 211, 731-740 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.011148
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Physiological characterization of stolon regression in a colonial hydroid

Kimberly S. Cherry Vogt, Gabrielle C. Geddes, Lori S. Bross and Neil W. Blackstone*

Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115, USA


Figure 1
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  		Fig. 1. Images of genetically identical colonies of P. carnea growing on 18 mm diameter glass coverslips. Polyps are bright and circular; stolons are darker and web-like. (A) An untreated control colony exhibits a more runner-like morphology. (B) A colony treated with 100 µmol l–1 vitamin C exhibits a more sheet-like morphology (see Blackstone et al., 2005Go).

 

Figure 2
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  		Fig. 2. Bright-field and fluorescent micrographs of stolon tips from colonies of P. carnea. (A,B) A healthy stolon tip (A) from a control colony exhibits relatively little peroxide-related fluorescence (B). (C,D) A regressing stolon tip (C) from a control colony exhibits an elevated level of peroxide-related fluorescence (D). (E,F) A regressing stolon tip (E) after 60 min treatment with 5 mmol l–1 H2O2 (F) exhibits an elevated level of peroxide-related fluorescence. (G,H) A regressing stolon tip (G) after 60 min treatment with 0.5 mmol l–1 of SNAP (H) exhibits an elevated level of peroxide-related fluorescence. Scale bar, 25 µm.

 

Figure 3
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  		Fig. 3. Bright-field and fluorescent micrographs of stolon tips from colonies of P. carnea. (A,B) A healthy stolon tip (A) from a control colony exhibits relatively little NO-related fluorescence (B). (C,D) A regressing stolon tip (C) after 60 min treatment with 5 mmol l–1 H2O2 (D) exhibits an elevated level of NO-related fluorescence. (E,F) A regressing stolon tip (E) after 60 min treatment with 0.5 mmol l–1 SNAP (F) exhibits an elevated level of NO-related fluorescence. Scale bar, 25 µm.

 

Figure 4
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  		Fig. 4. Treatment with H2O2 or SNAP leads to an increase in ROS- and RNS-related emissions. There is an increase in (A) ROS emissions from peroxide-treated stolon tips, (B) ROS emissions from SNAP-treated stolon tips, (C) RNS emissions from peroxide-treated stolon tips, and (D) RNS emissions from SNAP-treated stolon tips. Each graph shows mean ± s.e.m. luminance (grayscale from 0 to 4095) for three stolon tips per replicate colony (filled bars, control; unfilled bars, treatment).

 

Figure 5
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  		Fig. 5. Bright-field and fluorescent micrographs of stolon tips from colonies of P. carnea. (A–C) A healthy stolon tip (A) from a control colony has numerous cells as visualized by propidium iodide (PI) staining (B), but only a few of these cells exhibit DNA fragmentation as visualized by TUNEL staining (C). (D–F) A regressing stolon tip that was treated for 60 min with 5 mmol l–1 H2O2 (D) also has numerous cells as visualized by PI staining (E), and the majority of these cells exhibit DNA fragmentation as visualized by TUNEL staining (F). Note that the tip of the stolon has regressed in D, E and F, thus, there is no fluorescence in this area in E and F. Scale bar, 25 µm.

 

Figure 6
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  		Fig. 6. Treatment with H2O2 leads to an increase in DNA fragmentation in the cells of stolon tips. (A) There is an increase in DNA fragmentation in cells of peroxide-treated stolon tips. Mean ± s.e.m. luminance (grayscale from 0 to 4095) of FITC-labeled dUTP for three stolon tips per replicate colony (filled bars, control; unfilled bars, 5 mmol l–1 H2O2). (B) There is no significant difference between treatments in cells labeled with PI in stolon tips. Mean ± s.e.m. luminance (grayscale from 0 to 4095) of PI-labeled cells for three stolon tips per replicate colony (filled bars, control; unfilled bars, 5 mmol l–1 H2O2).

 

Figure 7
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  		Fig. 7. TEM micrographs of a healthy stolon. (A) Epitheliomuscular cells (EMCs) in control stolons are found just inside of the perisarc and show the characteristic columnar shape. (B) The ectoderm is relatively wide (as indicated by the space between the perisarc and mesoglea) and nuclei can typically be seen suspended in a large vacuole. (C) The mitochondria of a healthy EMCs are usually about 0.5–0.75 µm. (D) Healthy EMCs near the stolon tip can be quite large as shown here. (E) The tip of a non-regressed stolon has a smooth and rounded appearance. P, perisarc; E, ectoderm; MG, mesoglea; N, nucleus; M, mitochondrion; V, vacuole. Scale bars, 1 µm.

 

Figure 8
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  		Fig. 8. TEM micrographs of a regressed stolon treated with 5 mmol l–1 H2O2. (A–C) The epitheliomuscular cells (EMCs) in a regressed stolon exhibit vacuolation of the cytoplasm (A). Treated stolons are sometimes contracted (B; as indicated by the space between the perisarc and the ectoderm). Cells of a regressed stolon have relatively normal appearing mitochondria (C) and compact nuclei with flocculent condensation of chromatin (A,B). (D,E) Cells in the tip of the regressed stolon are shrunken (D) compared to their control counterparts (e.g. Fig. 7D). Once a stolon regresses, the cell membranes in the tip form numerous small vesicles (E). P, perisarc; E, ectoderm; MG, mesoglea; N, nucleus; M, mitochondrion; V, vacuole, Ve, vesicle. Scale bars, 1 µm.

 

Figure 9
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  		Fig. 9. Following treatment with H2O2, gastrovascular flow within stolon tips virtually ceases. Oscillations in lumen width (µm) over time (seconds) for stolon tips of a control colony (filled circles) and a colony treated with 5 mmol l–1 H2O2 (unfilled circles). The treated colony shows much smaller amplitudes and similar periods of oscillations; total stolon width was the same for both colonies.

 




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