Tseng AS et al. (2007), Apoptosis is required during early stages of ta...

XB-ART-34997

Dev Biol 2007 Jan 01;3011:62-9. doi: 10.1016/j.ydbio.2006.10.048.

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Apoptosis is required during early stages of tail regeneration in Xenopus laevis.

Tseng AS , Adams DS , Qiu D , Koustubhan P , Levin M .

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The Xenopus tadpole is able to regenerate its tail, including skin, muscle, notochord, spinal cord and neurons and blood vessels. This process requires rapid tissue growth and morphogenesis. Here we show that a focus of apoptotic cells appears in the regeneration bud within 12 h of amputation. Surprisingly, when caspase-3 activity is specifically inhibited, regeneration is abolished. This is true of tails both before and after the refractory period. Programmed cell death is only required during the first 24 h after amputation, as later inhibition has no effect on regeneration. Inhibition of caspase-dependent apoptosis results in a failure to induce proliferation in the growth zone, a mispatterning of axons in the regenerate, and the appearance of ectopic otoliths in the neural tube, in the context of otherwise normal continued development of the larva. Larvae amputated during the refractory stage exhibit a much broader domain of caspase-3-positive cells, suggesting a window for the amount of apoptosis that is compatible with normal regeneration. These data reveal novel roles for apoptosis in development and indicate that a degree of apoptosis is an early and obligate component of normal tail regeneration, suggesting the possibility of the existence of endogenous inhibitory cells that must be destroyed by programmed cell death for regeneration to occur.

???displayArticle.pubmedLink??? 17150209
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Species referenced: Xenopus laevis
Genes referenced: casp3.2 hpse itih3
???displayArticle.antibodies??? Casp3 Ab5 H3f3a Ab11 Tuba4b Ab5

Phenotypes: Xla Wt + M50054 (A, B, C) [+]

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	Fig. 1. Endogenous pattern of apoptosis in Xenopus regeneration. Legend: Larvae were processed for immunohistochemistry with an antibody to activated caspase-3, a marker of apoptosis. Positive signals are deep purple. Hpa labels on each panel indicate stage in hours-post-amputation. Larvae in panels B–E were amputated at st. 40; the animal in panel A was not amputated, and that in panel G was amputated during the refractory period (st. 46). (A) Intact larvae at st. 45 possessed small numbers of randomly distributed apoptotic cells; tails at all stages both before and after this period exhibited the same pattern. Red arrowhead indicates one set of positive cells. (B) Six hours post-amputation, no specific signal was detected near the wound swelling. (C) At 12 hpa, a discrete focus of caspase-3-positive cells is located in the nascent regeneration bud (red arrow). Inset: sectioning reveals an epithelial component to the staining that is not obvious in a whole mount, shown also in superficial (C′) and deep (Cʺ) sections that reveal epithelial and mesenchymal caspase-3-positive cells. (D) At 24 hpa, the apoptotic region has expanded (red arrows). (E) When amputation was performed at st. 48 (after the refractory period), tails at 24 hpa likewise exhibit a degree of apoptosis in the bud. (F) At 48 hpa, apoptosis can be detected in cells along the axis (red arrows). (G) In larvae amputated during the refractory period (st. 46), significantly more apoptosis can be detected at 24 hpa-the refractory swelling is almost entirely apoptotic in contrast to the small apoptotic region in regenerating tails, and foci of apoptotic cells can be seen extending ventrally along the amputation edge border (red arrows). Dorsal is up in panel A; dorsal is to the right in panels B–G.
	Fig. 2. Apoptosis is required for regeneration during the first 24 h. Legend: (A) In contrast to control embryos that regenerate a tail within 7 days, larvae treated with a specific apoptosis inhibitor between stages 40 and 46 completely failed to regenerate. (B) Close-up of the amputation site from an inhibited tail, 7 days after amputation. The graphs in panels C and C′ represent averages of replicate experiments performed 3 times each. (C) The effect was concentration dependent: compared to control embryos (regeneration index 281 ± 10), those treated with 12 μM or 35 μM of the inhibitor exhibited regeneration indexes that were 38% and 12% of the controls, respectively. (C′) The same concentration-dependent effect was observed for NS3694, a different apoptosis blocker, significantly reducing the ability of the animals to regenerate: compared to control embryos (regeneration index 294 ± 6), those treated with 12 μM or 35 μM of the inhibitor exhibited regeneration indexes that were 38% and 8% of the controls. (D) This effect was highly time dependent: treatment that lasted only over the first 24 h post-amputation-inhibited regeneration to almost the same degree as inhibition lasting over 7 days, while inhibition that began at 24 h and continued throughout 7 days had no significant effect on regeneration. RI for three replicates combined is noted in the axis labels. Dorsal is to the top in panel A, and to the left in panel B.
	Fig. 3. Apoptosis inhibition results in ectopic otoliths. Legend: At st. 46, control larvae possess two otoliths that are visible lateral to the neural tube from the dorsal view (A, green brackets, close-up inset). Larvae exposed to apoptosis inhibitor between stages 40 and 46 develop two ectopic otoliths located medial–dorsal and adjacent to the endogenous otoliths (B). (C) Sectioned larva stained for mineralized tissues (red arrows) revealing the ectopic mineralized tissues to be located outside and medial to the otocyst, possibly within brain tissue; normal otoliths occur ventral to the position of the ectopic otoliths and are thus not present in this planar section. (C′) Transverse section showing ectopic otolith (red arrow) next to the neural tube (the two brown circles are melanocytes). (D) Staining for mineralized tissue in whole mount also reveals ectopic otoliths (red arrows) in the hindbrain. Green bracket indicates endogenous otoliths. Red arrowheads indicate ectopic otoliths. Anterior is to the right in panel C, and to the left in panels A, B and D.
	Fig. 4. Apoptosis inhibition results in inhibition of cell proliferation and neuronal mispatterning near the amputation site. Legend: H3P-positive cells are easily distinguishable from pigment cells by their color (blue vs. brown) and morphology (dendritic melanocytes vs. small round nuclear H3P stain). (A) Control larvae at 48 hpa contain many proliferating cells in the growth region (identified by staining for phosphorylated Histone 3B, a marker of G2/M transition; four such cells are indicated by red arrowheads; average was 65). (B) In contrast, larvae treated with apoptosis inhibitor have many fewer cells at the G2/M transition in their growth region (average of 32, SD = 9.3, p < 0.01 compared to controls). When placed side by side with control animals (C), irradiated animals are significantly shorter and narrower (C′); this effect of global reduction of cell proliferation was not observed in drug-treated embryos (compare embryo trunk widths in Fig. 2A), showing that a direct and general inhibition on cell proliferation was not associated with exposure to apoptosis inhibitors. At 72 hpa, staining for axons reveals the early presence of neurons throughout the regeneration bud. In contrast to control regeneration where axons run parallel to the tail's main axis (D), apoptosis-inhibited larvae's axons do not extend all the way to the end of the regeneration bud and are present in tangles, often curling perpendicular to the main axis (D′). Dorsal is to the left in panels A and B.

References [+] :

Abdel-Karim, Mitotic activity in the blastema and stump tissues of regenerating hind limbs of Xenopus laevis larvae after amputation at ankle level. An autoradiographic study. 1990, Pubmed, Xenbase