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Activity regulates programmed cell death of zebrafish Rohon-Beard neurons

¹ Department of Physiology and Biophysics, University of Colorado Health Sciences Center, Denver, CO 80262, USA
² Program in Neurosciences, University of Colorado Health Sciences Center, Denver, CO 80262, USA

View larger version (134K): [in a new window]   Fig. 1. Development regulates the distribution of anti-acetylated tubulin immunoreactivity in the peripheral processes of Rohon-Beard neurons. The distribution of aat in the major peripheral processes was examined using whole-mount immunocytochemistry (see Materials and Methods). The peripheral processes of RB cells were viewed in the focal plane of the skin of embryos that had been mounted laterally. In all views, anterior and dorsal are towards the left and upwards, respectively. (A) At 27 hpf, aat immunoreactivity displayed a uniform, continuous distribution (arrows) within the major peripheral processes of RB neurons. Within the spinal cord, aat immunoreactivity was present in emerging spinal tracts and RB somata. At this time, the embryo has just developed the ability to respond rapidly to tactile stimulation. (B) At 36 hpf, the distribution of aat immunoreactivity within peripheral processes remained continuous and uniform. In the spinal cord, aat immunoreactivity was more intense within spinal tracts when compared with spinal tracts of 27 hpf embryos. (C) By 40 hpf, aat immunoreactivity within peripheral processes began to display an altered distribution consisting of varicosities (asterisks). However, continuous, uniform labeling (arrows) was still evident in some processes. Within the spinal cord, aat immunoreactivity remained intense. (D) At 48 hpf, aat immunoreactivity was now only present as varicosities in the peripheral processes of RB cells. Within the spinal cord, aat immunoreactivity was no longer present. Scale bar: 20 µm.

View larger version (92K): [in a new window]   Fig. 2. Anti-acetylated tubulin immunoreactivity is distributed within varicosities of intact Rohon-Beard peripheral processes. (A) In a 60 hpf wild-type embryo, zn-12 immunoreactivity reveals the peripheral processes of RB cells are intact and form a mesh of fine fibers. (B) By contrast, aat immunoreactivity appears as discrete varicosities within a subset of the peripheral processes, referred to here as the major peripheral processes. (C) In double-antibody experiments, the peripheral processes of a 60 hpf embryo were co-labeled for aat (FITC, yellow) and zn-12 (Rhodamine, red) immunoreactivities. The former recognizes a mature form of tubulin, whereas the latter binds to an extracellular sugar epitope on the plasma membrane. Even though the peripheral processes appear intact at the level of the plasma membrane (red, arrows), aat immunoreactivity is found in only discrete subregions (yellow) of the peripheral processes, indicating that redistribution of aat immunoreactivity occurs before process fragmentation. Scale bars: 10 µm.

View larger version (110K): [in a new window]   Fig. 3. Anti-acetylated tubulin immunoreactivity within Rohon-Beard peripheral processes differs between touch-insensitive mao mutant and sibling embryos at late but not early times. The peripheral processes of sibling control (A) and mao mutant (C) embryos displayed similar patterns of aat immunoreactivity (arrows) at the initial stages of touch sensitivity (compare with Fig. 1A,B). However, at 48 hpf, the pattern of aat immunoreactivity of mao mutants (D) differed from that of sibling controls (B) and resembles that observed at earlier stages in either sibling, mutant or wild-type embryos (Fig. 3A,C, 1A,B). In all examples, images were taken from skin regions at caudal levels of the spinal cord. Scale bar: 20 µm.

View larger version (84K): [in a new window]   Fig. 4. The mao mutation diminishes the normal loss of Rohon-Beard cell bodies. (A) Dorsal views of two segments from 36 (top) and 72 (bottom) hpf mao mutant and sibling embryos that had been processed for HuA immunocytochemistry (see Materials and Methods). At 36 hpf, mutant and sibling embryos have similar numbers of RB somata. However, at 72 hpf, RB somata have been eliminated in the sibling but retained in the mutant embryo. (B) RB cell counts for mao mutant and sibling embryos at 36 (left) and 72 (right) hpf. Each column represents the mean±s.e.m. for data collected from eight to ten embryos. At 72 hpf, there are significantly more RB somata in mutants versus siblings (P<0.0001). (C) In lateral views of 72 hpf sibling and mutant embryos (top and bottom, respectively), dorsal root ganglia (asterisks) and any remaining RB cells (arrows) can be seen. In siblings, only the dorsal root ganglia are present. By contrast, mutants possess both dorsal root ganglia and RB cells. Scale bars: 20 µm in A; 40 µm in C.

View larger version (109K): [in a new window]   Fig. 5. Direct visualization of Rohon-Beard cells in mao mutant and sibling embryos. RB cells in mao sibling (A) and mutant (B) 72 hpf embryos were directly visualized with Hoffman optics. RB cells (arrows) are easily recognized on the basis of their dorsal position and relatively large size within the spinal cord (Grunwald et al., 1988). More RB cells are present in mao mutants than sibling embryos. Scale bar: 20 µm.

View larger version (36K): [in a new window]   Fig. 6. The mao mutation diminishes an early sign of programmed cell death – DNA fragmentation. (A) Mao mutant (bottom) and sibling (top) 36 hpf embryos were co-processed for HuA immunocytochemistry (left) and TUNEL (middle; see Materials and Methods). In the merged images (right), fewer TUNEL-positive RB cells are present in the mao mutant versus the sibling embryo. Scale bar:10 µm. (B) Cell counts of TUNEL-positive RB neurons indicate that there are significantly fewer TUNEL-positive RB neurons in 36 hpf mao mutants compared with siblings (P<0.05; n=4 and n=3, mutants and siblings, respectively).

View larger version (90K): [in a new window]   Fig. 7. Pharmacological block of neural activity phenocopies the effect of the mao mutation on Rohon-Beard peripheral processes. (A) Wild-type embryos were treated with 0.006% tricaine between 24-72 hpf. Untreated (top) and tricaine-treated (bottom) embryos develop normally without obvious gross morphological differences. Scale bar: 200 µm. (B) aat immunoreactivity in a 60 hpf wild-type (top) is present in varicosities, as found in mao sibling embryos of similar age (Fig. 3B; data not shown). By contrast, tricaine-treated embryos (bottom) do not develop aat-positive varicosities. Thus, tricaine-treatment phenocopies the effect of the mao mutation on RB peripheral processes (Fig. 3D). Scale bar: 20 µm.

View larger version (47K): [in a new window]   Fig. 8. Tricaine treatment phenocopies the effect of the mao mutation on Rohon-Beard cell death. (A) In lateral views of control (top) and tricaine-treated (bottom) embryos, HuA immunocytochemistry reveals dorsal root ganglia (asterisks). In addition, RB cells (arrows) are revealed in the tricaine-treated but not the control embryo. Scale bar: 40 µm. (B) RB cell counts indicate that there is a significant difference in the number of RB neurons present in 72 hpf tricaine-treated and control embryos (P< 0.000001; tricaine treated, n=9; untreated, n=10). Tricaine-treatment suppresses the normal loss of RB cells, as does the mao mutation.

View larger version (16K): [in a new window]   Fig. 9. Genetic and pharmacological silencing of Na+ channels reduce RB cell death to the same extent. Numbers of RB somata are plotted as a function of time for wild-type (white squares), tricaine-treated (black squares), mao mutant (black circles), and mao sibling (white circles) embryos. At 36 and 54 hpf, the number of RB somata were similar for mao mutants and siblings. At 60 hpf, differences are first noted between the number of RB somata in mao sibling versus mutant embryos (*, P<0.05-0.0001). A similar 10% difference is noted between the number of RB somata in wild-type and tricaine-treated embryos (#, P<0.05-0.000001). At subsequent stages, the differences between the number of RB somata between wild-type and mao sibling embryos versus tricaine-treated and mao mutant embryos increases. For example, at 72 hpf, there are approx. three times as many RB somata in mao mutant or tricaine-treated embryos versus mao sibling or wild-type embryos. At 78 hpf, there are approx. ten times as many RB somata in mao mutant and tricaine treated embryos versus either wild-type or sibling embryos. At all stages, the effects of the mao genetic lesion and pharmacological suppression on RB cell death are similar, and the numbers of RB somata in tricaine-treated and mao mutant embryos are similar. The number of embryos examined for each data point range between 5 and 10.