First published online June 7, 2004
Journal of Experimental Biology 207, 2409-2416 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01045
Macrophage involvement for successful degeneration of apoptotic organs in the colonial urochordate Botryllus schlosseri
Ayelet Voskoboynik1,2,
Baruch Rinkevich2,*,
,
Anna Weiss1,
Elizabeth Moiseeva2 and
Abraham Z. Reznick1,*
1 Department of Anatomy and Cell Biology, The Bruce Rappaport Faculty of
Medicine, Technion – Israel Institute of Technology, Haifa,
Israel
2 National Institute of Oceanography, Oceanographic and Limnological
Research, Tel-Shikmona, PO Box 8030, Haifa 31080, Israel
View larger version (148K):
[in a new window]
|
Fig. 1. Control B. schlosseri ramet (A,C,E) and a 6 mg BHT
l-1-treated ramet (B,D,F). (A) Control ramet in stage D. The zooid
resorption has already started and fully developed buds ready for the takeover
stage are visible; (B) BHT-treated ramet after 5 days under BHT
administration, reaching the first day of blastogenic stage D; (C) control
ramet 1 day later, already at blastogenic stage A. The old generation of
zooids was completely resorbed. (D) The corresponding BHT-treated ramet, 1 day
later (6th day of BHT exposure). The old gene ration of zooids in the center
still exists. The new generation of zooids dispersed in the tunic matrix
without the typical patterning of systems. (E) The control ramet 1 day later,
at normal blastogenic development, already at stage B; (F) the BHT-treated
ramet after 7 days of BHT exposure. The old generation of zooids was not
resorbed, while the new generation of zooids developed abnormal round shapes
of zooids with open, inhalant siphons and accumulated deep pigmentation. The
tunic looks opaque. z, zooid; z (b), new generation zooid; b, bud; am,
ampulla; t, tunic. Scale bars, 1 mm.
|
|
View larger version (134K):
[in a new window]
|
Fig. 2. Azan Heidenhains stain of sections from stage D control zooids (A,C,E),
compared to 6 mg BHT l-1-treated zooids (B,D,F) after 3 days
arrested at stage D. (A) A control ramet with two resorbed zooids at stage D.
(B) BHT-treated zooid after 3 days arrested at stage D. Internal organs, such
as endostyle, branchial sac and siphon, are intact. (C) Enlargement of the
deteriorating endostyle area of a control zooid. (D) Enlargement of the
BHT-treated zooid endostyle area with unspoiled structures including
morphologically normal epithelial cells. (E) Stage D 22 control, the
deteriorated zooid branchial sac stigmata area. Leftover cells and macrophages
carrying debris are seen. (F) Third day arrested stage D zooid, branchial sac
stigmata area. The organ is intact. z, zooid; bs, branchial sac; en,
endostyle; s, siphon; st, stigmata; mac, macrophage. Scale bars, 5 µm.
|
|
View larger version (98K):
[in a new window]
|
Fig. 3. Histological sections of control blastogenic stage D primary and secondary
buds (A,C,E,G) as compared to 6 mg BHT l-1-treated primary and
secondary buds after 10 days exposure, on third day of arrested stage D
(B,D,F,H). Azan Heidenhains stain. (A) Control ramet, primary bud at
blastogenic stage D. Only a few cells can be seen in the bud blood vessels.
(B) BHT-treated primary bud after 3 days arrested at blastogenic stage D. The
bud periphery blood vessels are thick and packed with enormous numbers of
cells. (C) Enlargement of the endostyle area in the primary control bud; only
few blood cells can be seen near the endostyle epithelium. (D) The endostyle
of the treated primary bud, surrounded by an unusual mass of cells. (E) A few
cells circulating in the peripheral blood vessel of the primary control bud
and the lacunas around its digestive system. (F) Large numbers of blood cells
circulating in the treated primary bud peripheral blood vessel and its
digestive system. (G) Secondary bud control ramet has large empty spaces
(subdivisions of the future organs) lined by epithelium of thin blood vessel.
(H) BHT-treated secondary bud has dilated blood lacuna filled with m any
cells. b, bud; bs, branchial sac; bv, blood vessel; ds, digestive system; en,
endostyle; s siphon; sb, secondary bud.
|
|
View larger version (132K):
[in a new window]
|
Fig. 4. Histological section of a control ramet at blastogenic stage D and its
corresponding BHT-treated ramet, 3 days arrested at stage D. (A) A `packed'
macrophage (arrow) is seen with ingested inclusions between the control zooid
23 stigmata; (B) BHT-treated ramet, empty macrophages (arrowheads) are located
between zooid stigmata; (C) packed macrophages (arrowheads) in the control
ramet bud; (D) BHT-treated bud with an empty macrophage (arrowhead); (E) a
packed macrophage (arrow) in control zooid blood vessel; (F) BHT-treated zooid
blood vessel with empty macrophages (arrowheads). Azan Heidenhains stain. bv,
blood vessel; mac, macrophage; pig, pigment cell; sg, stigmata. Scale bars, 1
µm.
|
|
View larger version (118K):
[in a new window]
|
Fig. 5. DNA fragmentation detection staining (fragEL) of control ramet (A,B) and
BHT-treated ramet, 3-day blastogenic stage D arrested (C,D). A and C reveal
DNA fragmentation staining. B and D are, respectively, their negative control
for background staining. bs, branchial sac; ds, digestive system; en,
endostyle; s, siphon. Scale bars, 10 µm.
|
|
View larger version (24K):
[in a new window]
|
Fig. 6. Effect of BHT on lipid oxidation levels, (A) at different stages of the
blastogenic cycle of control B. schlosseri colonies; (B) in BHT (6 mg
l-1)-treated, ethanol-treated and control ramets at blastogenic
stage D. Lipid oxidation was determined as MDA using the HPLC based
Thiobarbituric acid test (see Materials and methods for details). The MDA
levels were standardized to 10 µg protein. n, ramets number. Data are mean
± S.D. N values are given above each bar.
|
|
© The Company of Biologists Ltd 2004
