Hensey C and Gautier J (1998), Programmed cell death during Xenopus developmen...

XB-ART-14012

Dev Biol 1998 Nov 01;2031:36-48. doi: 10.1006/dbio.1998.9028.

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Programmed cell death during Xenopus development: a spatio-temporal analysis.

Hensey C , Gautier J .

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Programmed cell death (PCD) is an integral part of many developmental processes. In vertebrates little is yet known on the patterns of PCD and its role during the early phases of development, when embryonic tissue layers migrate and pattern formation takes place. We describe the spatio-temporal patterns of cell death during early Xenopus development, from fertilization to the tadpole stage (stage 35/36). Cell death was analyzed by a whole-mount in situ DNA end-labeling technique (the TUNEL protocol), as well as by serial sections of paraffin-embedded TUNEL-stained embryos. The first cell death was detected during gastrulation, and as development progressed followed highly dynamic and reproducible patterns, strongly suggesting it is an important component of development at these stages. The detection of PCD during neural induction, neural plate patterning, and later during the development of the nervous system highlights the role of PCD throughout neurogenesis. Additionally, high levels of cell death were detected in the developing tail and sensory organs. This is the first detailed description of PCD throughout early development of a vertebrate, and provides the basis for further studies on its role in the patterning and morphogenesis of the embryo.

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Species referenced: Xenopus laevis
Genes referenced: dnah5 dnai1 pcbd1

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	FIG. 1. Whole-mount TUNEL staining reveals dying cells in gastrulation-stage embryos. (A) Stage 10.5, vegetal view, no TUNEL staining was detected in this embryo. (B) Stage 10.5, lateral view, a small patch of TUNEL staining is evident in the equatorial region of the embryo. (C) Stage 10.5, vegetal view, hundreds of TUNEL-positive nuclei were evident in the circumblastoporal region. (D) Stage 10.5, lateral view, TUNEL-stained nuclei covered the surface of the embryo except for the blastopore. (E) Stage 11.5, vegetal view, a patch of TUNEL staining in the equatorial region extends to the edge of the blastopore. (F) Stage 11.5, lateral view, two bilaterally symmetrical patches of TUNEL staining border the blastopore. (G) Stage 11.5, animal view, TUNEL staining was detected in the animal pole and extended vegetally to the edge of the blastopore. (H) Stage 11.5, lateral view, a partially exogastrulating embryo showing an accumulation of TUNEL-positive nuclei in the blastopore (arrowhead). (I) Stage 12.5, animal view (left), lateral view (right). A dorsal median stripe of TUNEL-stained nuclei extended from the blastopore. (J) Stage 12.5, animal view (left), lateral view (right). TUNEL-stained nuclei were detected across the dorsal side of the embryo. Throughout gastrulation TUNEL staining was never detected in the blastopore, indicated by the dotted red line. The embryos shown are representative of the TUNEL staining observed following analysis of 237 embryos of which 67% were stained. Embryos with more than 5 TUNEL-stained nuclei were considered TUNEL positive.
	FIG. 2. Programmed cell death as detected by whole-mount TUNEL staining at the neural plate stage. (A) Stage 13, dorsal (top) and lateral (bottom) view. The dorsal side of the embryo is TUNEL stained with a band of staining broadening toward the anterior end of the embryo, delineating the neural plate. (B) Stage 13, dorsal (top) and lateral (bottom) view. Two broad bands of staining extend laterally from the blastopore slit. (C) Stage 13, dorsal (top) and lateral (bottom) view. A broad medial stripe of staining was detected on the dorsal side. (D) Stage 14, lateral view, anterior is left, no TUNEL staining was detected. (E) Stage 15, lateral view, anterior is left. Intense TUNEL staining was detected in the anteriormost portion of the embryo. Light staining was evident posteriorly. (F) Stage 14, dorsal (top) and lateral (bottom) view, anterior is left. A semicircular band of staining in the anteriormost portion of the embryo, corresponding to the anterolateral edge of the neural plate. (G) Stage 15, end on anterior view (top), lateral view (bottom), anterior is left. TUNEL staining formed a horseshoe-shaped band of staining in a more anteroventral position than stage 14. The embryos shown are representative of the staining observed following staining of 58 stage 13 embryos of which 67% were TUNEL positive, and 37 stage 14/15 embryos of which 52% were TUNEL positive. Embryos with more than 5 TUNEL-stained nuclei were considered TUNEL positive.
	FIG. 3. Programmed cell death as detected by whole-mount TUNEL staining at the neural fold stage. A, B, and C, stage 17. Dorsal, lateral, and end-on anterior views are shown for each embryo (left to right). (A) TUNEL staining was detected in the anteriormost portion of the embryo with a light scattering of staining along the length of the neural fold. (B) Strong TUNEL staining in the anteriormost portion of the embryo, corresponding to the optic placodes (arrowhead). Staining extends from these placodes along the neural fold. (C) Strongest staining was in the anterior embryo with patches of TUNEL staining corresponding to the optic (arrowhead), and olfactory (arrow) placodes, and developing brain. Dying cells were found in defined stripes on each side of the dorsal midline corresponding to the primary sensory neurons. A scattering of dying cells was detected throughout the neural fold. Lines 1 and 2 mark the approximate location of transverse sections shown in Figs. 7B and 7C, respectively. The cell death patterns are representative of the staining observed following staining of 33 embryos of which 64% were TUNEL positive. Embryos with more than 5 TUNEL-stained nuclei were considered TUNEL positive.
	FIG. 4. Programmed cell death as detected by whole-mount TUNEL staining at the late neural stage. Each panel shows a lateral (top), and a dorsal (bottom) view. (A) Stage 18, TUNEL staining in the anteriormost portion of the developing brain. A clear asymmetry is present. (B) Stage 18, TUNEL staining in the posterior of the embryo, along the neural fold. (C) Stage 18, bilaterally symmetrical staining in the anterior developing brain, and faint staining in the posterior of the embryo. (D) Stage 23, bilaterally symmetrical diffuse staining in the developing head possibly corresponding to migrating neural crest cells. (E) Stage 23, diffuse staining in the developing head, showing a distinct rightâleft asymmetry. (F) Stage 25, diffuse TUNEL staining throughout the head and tailbud. The staining shown is representative of the programmed cell death seen following staining of 89 embryos of which 98% were TUNEL positive. Embryos with more than 5 TUNEL-stained nuclei were considered TUNEL positive.
	FIG. 5. Programmed cell death as detected by whole-mount TUNEL staining during stages 26â28. Each panel shows a lateral view (left), and a dorsal view (right). (A) Stage 26, the embryo looks younger in the lateral view due to curvature. TUNEL staining is evident in the ventral forebrain and midbrain in addition to the eye vesicle. Staining was also evident in the posterior half of the spinal cord (arrowheads), and in the tailbud. Line indicates the approximate location of the transverse section shown in figure 7D. (B) Stage 26, staining in the midbrain, posterior spinal cord and tailbud. (C) Stages 27â28, staining in the midbrain, hindbrain, and eye vesicles. (D) Stages 27â28, bilaterally symmetrical staining in the midbrain. The patterns of cell death shown are representative of the cell death detected following staining of 36 embryos, all of which were TUNEL positive. Embryos with more than 5 TUNEL-stained nuclei were considered TUNEL positive.
	FIG. 6. PCD as detected by whole-mount TUNEL staining during stages 29â35. Each panel shows a lateral (top), and dorsal view (bottom). (A) Bilaterally symmetrical TUNEL staining is detected in the fore- and midbrain; lighter staining is detected in the eye vesicle. Line indicates the approximate location of a sagital section shown in Fig. 7F. (B) TUNEL staining is detected in the brain, eye vesicles, the posterior half of the spinal cord, and the elongating tail. (C) Dying cells were detected in the brain and developing eye. (D) TUNEL staining in the rhombencephalon, the outer ring of the eye vesicle the branchial arches (arrowheads), and the otic vesicle. Staining was also evident along the length of the spinal cord and in the tail tip. The staining is representative of the patterns seen following staining of 92 embryos of which 99% were TUNEL stained. Embryos with more than 5 TUNEL-stained nuclei were considered TUNEL positive.
	FIG. 7. Sections of TUNEL-stained embryos from stages 13 to 35. (A) Stage 13, arrowhead indicates TUNEL staining in the presumptive neuroectoderm. (B) Stage 17, transverse section (1) indicated in Fig. 3C, shows TUNEL staining in circular patches which correspond to the developing brain and sensory placodes. (C) Stage 17, a transverse section (2) indicated in Fig. 3C, shows TUNEL staining in the region of the neural crest. (D) Stage 26, a transverse section indicated in Fig. 5A, TUNEL staining was detected in both sides of the spinal cord giving rise to two bilaterally symmetrical dorsal stripes, Fig. 5A (arrowheads). (E) Stage 31/32, a transverse section through the head as indicated in Fig. 6B shows TUNEL staining in the brain and optic stalk. Most intense staining is evident in the ventral diencephlon. (F) Stage 29/30, a sagital section of the embryo indicated in Fig. 6A, shows TUNEL staining in the mesencephalon, diencephalon, and telencephalon regions of the brain.