Malikova MA et al. (2007), Apoptosis regulates notochord development in Xe...

XB-ART-36590

Dev Biol 2007 Nov 15;3112:434-48. doi: 10.1016/j.ydbio.2007.08.047.

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Apoptosis regulates notochord development in Xenopus.

Malikova MA , Van Stry M , Symes K .

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The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed.

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Species referenced: Xenopus laevis
Genes referenced: pcdh1

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	Fig. 1. Apoptosis occurs in the notochord but not somitic mesoderm. Cell death was assessed in intact embryos at different stages of development by TUNEL assay, in which nicked DNA is visualized by terminal-UTP-nicked-end labeling. Longitudinal sections indicating anterior notochord (center) and somites (top and bottom of frames) at stages 21 (A) and 25 (B) are shown. Note the presence of apoptotic cells in the notochord (arrows) but not in the somites. Anterior is to the right in both frames. (C) Graph indicating the number of apoptotic cells in the notochord at stages 17–25. Error bars indicate 95% confidence intervals. A minimum of 8 embryos in 3 independent experiments were used for this analysis. Scale bar in panel B is 100 μm.
	Fig. 2. Apoptosis in the notochord proceeds in an anterior to posterior direction. Embryos were fixed at stages 19–25, TUNEL stained, embedded and sectioned sagittally. (A) The anteroposterior distribution of TUNEL-positive cells in the notochord was assessed in each sagittal section at different developmental stages and notochord positions (A = anterior; M = middle; P = posterior). Error bars indicate 95% confidence intervals. (B) TUNEL-stained, sagittal sections of notochord at stages 22 (top panels) and 25 (bottom panels). Anterior, middle and posterior regions of the notochord are shown. At stage 25 the anterior and middle regions are fully vacuolated. Note the A to P change in the distribution of apoptotic nuclei with developmental stage. A minimum of 8 embryos in 3 independent experiments were used for this analysis. Scale bar in panel B is 100 μm.
	Fig. 3. Apoptosis regulates the extension of axial mesoderm explants. Embryos were injected with either Bcl-2 or GFP mRNA into the dorsoanterior marginal zone at the 4-cell stage. Open-faced Keller explants were dissected at stage 10.25 and cultured until sibling embryos reached stage 25, when they were fixed and analyzed. (A) The length-to-width ratio (LWR) of axial mesoderm explants was assessed by calculating the ratio of the longest aspect of each explant (length) to its width at the constriction point where the mesoderm extends from neural ectoderm. (B) The LWR of axial mesoderm explants derived from embryos microinjected with control (GFP, n = 31) or Bcl-2 (n = 34) mRNA was calculated. Note that explants in which apoptosis was prevented by Bcl-2 injection have a greater LWR than control explants. (C) Open-faced Keller explants comprise the prospective ectoderm (), neural ectoderm () and mesoderm (). Following convergent extension, differentiated notochord is flanked by somites (represented enlarged in the indicated box). (D) The length of the notochord was also measured in control (GFP mRNA-injected (n = 14), DNase I treated – a positive control for TUNEL assay – (n = 10) and no TUNEL enzyme – a negative control for TUNEL assay – (n = 8)) and Bcl-2 mRNA-injected (n = 22) explants. Note that the notochord in Bcl-2-injected explants elongated to a greater extent than in control explants. (E) Cell death was assessed by TUNEL assay (middle panels). The notochord is visualized by immunohistochemistry using the notochord antibody Tor70 (lower panels). DNase I treated explants are shown as a positive control for the TUNEL assay. Background autofluorescence is visible in all explants. (F) Higher magnification images of area within white boxes in GFP control and Bcl-2 explants shown in panel E. Note that apoptosis of mesoderm cells in mock-injected control explants (TUNEL-positive fluorescein-labeled cells) is blocked in Bcl-2-injected explants. Error bars in panels B and D indicate 95% confidence intervals. Scale bars in panels E and F 500 μm and 200 μm, respectively.
	Fig. 4. Inhibition of apoptosis in vivo causes severe arching of the dorsal axis. Embryos were injected into the prospective notochord region with (A–C) control GFP mRNA alone or with (D–F) Bcl-2 mRNA at the 4-cell stage. At stage 30, the embryos were fixed and processed for immunohistochemistry using (B, E) GFP and (C, F) Tor70 antibodies. Note that the embryos that received Bcl-2 mRNA develop with an arched dorsal axis. A minimum of 14 embryos in 3 independent experiments were used for this analysis.
	Fig. 5. Inhibition of apoptosis does not increase notochord extension in vivo but causes the notochord to kink severely. Embryos were injected into the prospective notochord region with (A, B, D, F) GFP mRNA (Con) alone or with (A, C, E, G) Bcl-2 mRNA at the 4-cell stage. At stages (A) 19, (A–C) 23, (A, D, E) 30 and (A, F, G) 35 the embryos were fixed and processed for immunohistochemistry using Tor70 and notochord length was measured in wholemount (A). Note that in the embryos that received Bcl-2 mRNA the notochord is kinked (C, E, G; arrowheads in panels C, G) compared to controls (B, D, F). Arrows point to the boundary between kinked and non-kinked notochord (C, E). Error bars indicate 95% confidence intervals. In panel A differences that are statistically significant (p < 0.05) in the unpaired t test are indicated by *. For stages 23–35 a minimum of 12 embryos in 3 independent experiments were used for this analysis; for stage 19 a minimum of 6 embryos were used. Anterior is to the right. Scale bar in panel G is 100 μm.
	Fig. 6. Inhibition of apoptosis severely disrupts notochord structure. Embryos were injected into the prospective notochord region with (A, B, E, F, I, J) GFP mRNA or (C, D, G, H, K, L) Bcl-2 mRNA at the 4-cell stage. At stages 23 (A–D), 30 (E–H) and 35 (I–L) the embryos were fixed, processed for immunohistochemistry using Tor70, TUNEL stained and then embedded and sagittally sectioned. Note that inhibition of apoptosis by Bcl-2 disrupts the structure of the notochord such that by stage 30, the notochord structure is severely compromised, with extensive kinking and a lack of vacuolization. Arrows in panels A, E and I indicate the anteroposterior limit of vacuolization. Dotted lines indicate the position of the notochord in panels A, C, E, G, I and K. Anterior is to the left in all frames. Scale bar in panel L is 100 μm.
	Fig. 7. Anteroposterior limit of notochord vacuolization changes with embryonic stage. Sagittal histological sections of GFP-injected embryos at stage 23, 30 and 35 were examined for the presence of vacuoles in the notochord, which begins in the anterior at stage 23. The anterior–posterior limit of the vacuoles is indicated as a percentage of total notochord length. A minimum of 5 embryos were analyzed at each stage. Error bars indicated 95% confidence interval.
	Fig. 8. Notochord structure is disrupted in an anterior to posterior progression with developmental stage when apoptosis is inhibited. Embryos were injected into the prospective notochord region with GFP mRNA (hatched bars) or Bcl-2 mRNA (solid bars) at the 4-cell stage. At stages 19, 23 (), 30 () and 35 (▪), the embryos were fixed, processed for immunohistochemistry using Tor70 and GFP antibodies, TUNEL stained and then embedded and sagittally sectioned. Notochord disruption was assessed in 4 different sectors that encompassed the percent of the notochord length from the anterior, 0–25%, 26–50%, 51–75% and 76–100%. The following values were used to score the severity of the phenotype, grade 0 for normal, grade 1 for a mild phenotype including a rippled notochord with contiguous borders, grade 2 for highly kinked notochord but with contiguous borders, and grade 3 for severe phenotypes including breakages in the notochord borders and a loss of structure. Note that, at stage 23, the notochord is disrupted only in the most anterior regions but at later stages, this disruption extends posteriorly such that by stage 35 the entire notochord is affected. Error bars indicate 95% confidence intervals. A minimum of 4 embryos in 3 independent experiments were used for this analysis.
	Fig. 9. Inhibition of apoptosis severely disrupts notochord structure. Embryos were injected into the prospective notochord region with (A, D, G) GFP mRNA or (B, C, E, F, H, I) Bcl-2 mRNA at the 4-cell stage. Two examples of Bcl-2-injected embryos are shown. At stages 23 (A–C), 30 (D–F) and 35 (G–I) the embryos were fixed, processed for immunohistochemistry using (A′–I′) Tor70 and (A″–I″) anti-GFP antibodies, (A–I) TUNEL stained and then embedded and sagittally sectioned. Note that inhibition of apoptosis caused the notochord to kink and develop with broken edges and with malformed or missing vacuoles. Arrows in panels D, D″, G and G″ point to vacuoles in wild type notochords. Compare to arrows in panels E″ and F″ that show misshapen vacuoles in Bcl-2-injected embryos. Arrowheads in panels B″, C″ and F′ indicate brakes in the notochord sheath. Dotted lines in panels C, F and I indicate the position of the notochord boundaries and correspond to frames panels C′, C″, F′, F″, I′ and I″. All frames taken at approximately the notochord anteroposterior midpoint. Anterior is to the left or down in all frames. Scale bar in panel I″ is 20 μm.
	Fig. 10. The integrity of the sheath is compromised when apoptosis is inhibited in the notochord. Embryos were injected into the prospective notochord region with (A–C) GFP mRNA or (D–F) Bcl-2 mRNA at the 4-cell stage. At stage 35 the embryos were fixed, processed for immunohistochemistry using (A, D) anti-laminin and (B, E) Tor70 antibodies before being embedded and sagittally sectioned. Confocal microscopy (Axiovert LSM 200 M with 5 Pascal version 4 software; Zeiss) was used to analyze co-localization of the two markers (C, F) that outline the notochord, which overlapped 95–97%. Note that inhibition of apoptosis caused the notochord sheath to break. Anterior is to the left and dorsal is up in all frames. Scale bar in panel F is 40 μm.
	Fig. 11. The temporal expression of notochord markers is similar in control and Bcl-2-injected embryos. Embryos were injected into the prospective notochord region with (A, C, E, G) GFP mRNA or (B, D, F, H) Bcl-2 mRNA at the 4-cell stage. At stages 23 (A, B, E, F), 35 (C, D) and 37 (G, H) the embryos were fixed and processed for in situ hybridization using probes for AXPC (A–D) and procollagen IIA (E–H). The embryos were then photographed in wholemount (E, F) or embedded and transversely sectioned (A–D, G, H). Note that inhibition of apoptosis did not alter the pattern of expression of either marker. Notochord (n), pronephros (p). Dotted lines in panels C and D outline the notochord. Arrows in panels E and F point to the notochord. Anterior is to the left in panels E and F. Scale bar in panel D is 100 μm and applies to panels A–D, in panel F is 500 μm and applies to panels E and F and in panel H is 100 μm and applies to panels G and H.
	Fig. 12. Neural tube and somite extension mirrors that of the notochord. At stage 35, embryos that had been injected with GFP (A–D, I–L) or Bcl–2 (E–H, M–P) mRNA were subject to immunohistochemistry for notochord (A, C, E, G, I, K, M, O), skeletal muscle (B, D, F, H) or neural tube (J, L, N, P). Arrowheads in panels L and P indicate the neural tube. Anterior is to the left. Scale bar in panel N is 500 μm and applies to top 8 panels, and in panel P is 100 μm and applies to lower 8 panels.
	Fig. 13. Somites are disorganized when notochord apoptosis is inhibited. At stage 35, embryos that had been injected with GFP (A) or Bcl-2 (B) mRNA were subject to immunohistochemistry for skeletal muscle. Note severe disruption of somite structure in panel B. Anterior is to the left. Scale bar is 100 μm.

References [+] :

Adams, The mechanics of notochord elongation, straightening and stiffening in the embryo of Xenopus laevis. 1990, Pubmed, Xenbase