Development of chromaffin cells depends on MASH1 function
Katrin Huber1,
Barbara Brühl1,
François Guillemot2,
Eric N. Olson3,
Uwe Ernsberger1 and
Klaus Unsicker1,*
1 Neuroanatomy, Interdisciplinary Center for Neurosciences (IZN), University of Heidelberg, INF 307, D-69120 Heidelberg, Germany
2 IGBMC,CNRS/INSERM/Université Louis Pasteur, BP 163, 67404 Illkirch Cédex, Cu de Strasbourg, France
3 Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75239-9148, USA
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Fig. 1. Expression of MASH1 mRNA in a majority of SA progenitors within the developing adrenal gland. Cross-sections through the adrenal gland of E14.5 (A,B) and E16.5 (C,D) wild-type mice. Digoxigenin-labeled antisense RNA-probes for MASH1 mRNA (A,C) and TH mRNA (B,D) were employed. Sense controls did not result in any staining (not shown). sg, sympathetic suprarenal ganglion, where MASH1 mRNA expression at E16.5 is hardly detectable. Scale bars: 100 µm.
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Fig. 2. Lack of increase in the number of TH-immunoreactive cells in the adrenal gland of Mash1–/– mice. (A-F) Photomicrographs showing cross-sections through the adrenal glands of wild-type (A-C) and Mash1–/– mice (D-F) at E13.5 (A,D), E16.5 (B,E) and P0 (C,F) stained with an antibody against TH. (G) Counts of TH-immunoreactive cells in serial sections of the adrenal glands of Mash1–/– mice and wild-type littermates. Data are presented as mean±s.e.m. At least six adrenal glands of three different animals for each group have been analyzed. Scale bar: 100 µm.
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Fig. 3. Phox2b and TH immunoreactivities in adrenal glands of E14.5 Mash1–/– (B) and Mash1+/+ (A) mice. Double immunofluorescence-staining using antibodies against TH (red cytoplasmic stain) and Phox2b (green nuclear stain). (A) In adrenal glands of wild-type mice virtually all Phox2b-immunoreactive cells are also positive for TH. (B) By contrast, adrenal glands of Mash1–/– mice display many Phox2b-positive cells that are TH-negative (arrowheads). (C) Quantification of numbers of Phox2b and TH positive cells in the adrenal glands of wild-type and Mash1–/– mice. Data are presented as mean±s.e.m. At least six adrenal glands of three different animals were analyzed per group. Scale bar: 50 µm.
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Fig. 4. Apoptosis within groups of TH-positive (red) adrenal medullary cells of Mash1–/– (B,D) and Mash1+/+ (A,C) mice monitored by TUNEL staining (green). There were no TUNEL-positive nuclei in wild-type adrenal glands at E14.5 (A), and only few at E16.5 (C, arrowhead). MASH1 deficient adrenal glands displayed only very few apoptotic nuclei at E14.5 (B, arrowhead), but there were numerous TUNEL-positive nuclei at E16.5 (D, arrowheads). Scale bar: 50 µm.
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Fig. 5. In situ hybridization for Phox2a (A,C) and Hand2 (E,G) on sections of the adrenal glands of E13.5 Mash1+/+ (A,E) and Mash1–/– (C,G) mice. TH in situ hybridization has been carried out in adjacent sections (B,D,F,H). The expression of Phox2a and Hand2 appears unaltered in Mash1–/– mice. Note that in Mash1–/– the cells expressing Phox2a and Hand2 (C,G) outnumber the TH-expressing cells (D,H). Scale bars: 100 µm.
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Fig. 6. Electronmicrographs of adrenal medullae of wild-type (A,C) and Mash1–/– mice (B,D) at E14.5 (A,B) and E16.5 (C,D). Most adrenal medullary cells in Mash1–/– mice lack chromaffin granules and ultrastructurally resemble immature neuroblasts. Note the similarity of adrenal medullary cells of E16.5 Mash1–/– to cells in the sympathetic suprarenal ganglion of E16.5 wild-type mice (E). Scale bars: 2 µm. (F) Semi-quantitative analysis of percentages of adrenal medullary cells that lack chromaffin granules, cells with predominantly small granules (core diameter <50 nm) and cells with typical large chromaffin granules (core diameter  100 nm) in Mash1–/– mice and wild-type littermates at E16.5. At least 100 cells per group were analyzed in random sections through different levels of the adrenal gland.
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Fig. 7. Expression of neurofilament 68 mRNA is strongly enhanced in Mash1–/– adrenal glands (B) at E14.5 when compared with wild type (A). Arrowheads indicate adrenal neurofilament-expressing cells. Arrows indicate the following sympathetic ganglia: sg, sympathetic paravertabral ganglion; srg, suprarenal ganglion; pag, pre-aortic ganglion. Scale bar: 100 µm.
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Fig. 8. Expression of Ret mRNA as revealed by in situ hybridization is strongly enhanced in the adrenal medulla of Mash1 knockout mice at E16.5. Photomicrographs show cross-sections through the adrenals of wild-type (A,B) and Mash1–/– mice (C,D). Arrows indicate sympathetic ganglia that are positive for Ret (A) and TH (D). Appropriate sense controls have been performed (not shown). am, adrenal medulla. Scale bar: 100 µm.
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Fig. 9. PNMT is present in few surviving adrenal medullary cells of Mash1 knockout mice at P0. Cross-section through the adrenal glands of newborn wild-type (A) and Mash1–/– (B) mice (B). Double immunofluorescence staining for TH (red) and PNMT (green). Cells that are positive for TH and PNMT appear yellow. Scale bar: 100 µm.
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Fig. 10. (A,C) PNMT immunofluorescence staining on sections of the adrenal gland of newborn Mash1–/– mice (B,D). In situ hybridization for neurofilament 68 (B) and Ret (D) have been carried out on adjacent sections. Note that there is no overlap of PNMT immunoreactive cells (A,C, arrowheads) and neurofilament 68 (B, arrowheads) or Ret (D, arrowheads) expressing cells. Scale bar: 100 µm.
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© The Company of Biologists Ltd 2002
