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Persistent Expression of BMP-4 in Embryonic Chick Adrenal Cortical Cells and Its Role in Chromaffin Cell Development

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Persistent Expression of BMP-4 in Embryonic Chick Adrenal Cortical Cells and Its Role in Chromaffin Cell Development

Katrin Huber et al. Neural Dev.

Abstract

Background: Adrenal chromaffin cells and sympathetic neurons both originate from the neural crest, yet signals that trigger chromaffin development remain elusive. Bone morphogenetic proteins (BMPs) emanating from the dorsal aorta are important signals for the induction of a sympathoadrenal catecholaminergic cell fate.

Results: We report here that BMP-4 is also expressed by adrenal cortical cells throughout chick embryonic development, suggesting a putative role in chromaffin cell development. Moreover, bone morphogenetic protein receptor IA is expressed by both cortical and chromaffin cells. Inhibiting BMP-4 with noggin prevents the increase in the number of tyrosine hydroxylase positive cells in adrenal explants without affecting cell proliferation. Hence, adrenal BMP-4 is likely to induce tyrosine hydroxylase in sympathoadrenal progenitors. To investigate whether persistent BMP-4 exposure is able to induce chromaffin traits in sympathetic ganglia, we locally grafted BMP-4 overexpressing cells next to sympathetic ganglia. Embryonic day 8 chick sympathetic ganglia, in addition to principal neurons, contain about 25% chromaffin-like cells. Ectopic BMP-4 did not increase this proportion, yet numbers and sizes of 'chromaffin' granules were significantly increased.

Conclusion: BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.

Figures

Figure 1
Figure 1
Transverse sections through a stage 21 (E3.5) chick embryo at the level of the developing adrenal gland. (A,B) Steroidogenic factor-1 (SF-1) mRNA (A), a marker for adrenal cortical tissue, is expressed at a location corresponding to the site of bone morphogenetic protein-4 (BMP-4) mRNA expression (B). (C-F) A cluster of cells that express the autonomic markers chick achaete-scute homologue (CASH-1, C) and Phox2B (D), but lack tyrosine hydroxylase (TH) (E) and neurofilament (NF) (F), are located in close proximity to the adrenal cortical anlage. Note that cells at a dorsolateral position of the dorsal aorta, corresponding to developing secondary sympathetic ganglia, express TH and NF. ac, adrenal cortical anlagen; da, dorsal aorta; sg, sympathetic ganglion. Bar: 100 μm.
Figure 2
Figure 2
Expression of bone morphogenetic protein-4 (BMP-4) in adrenal cortical cells at developmental stages S23 (B), S26 (F), S30 (J), and S35 (N). Adjacent sections were labelled for steroidogenic factor-1 (SF-1) (A,E,I,M), Phox2B (C,G,K,O) and tyrosine hydroxylase (TH) mRNA (D,H,L,P). The positions of the adrenal cortical anlagen (ac) or the adrenal gland (ag) are marked. (A-D) At S23, the TH/Phox2B-positive chromaffin progenitors are located outside, but closely attached to the adrenal cortical anlage. (E-H) At S26 they have started to invade the adrenal cortical anlage. (I-L) At S30 both cortical and TH-positive chromaffin cells are completely intermingled. da, dorsal aorta; sg, sympathetic ganglion. Bar: 100 μm.
Figure 3
Figure 3
Double in situ hybrization for bone morphogenetic protein-4 (BMP-4; blue) and tyrosine hydroxylase (TH; red) on a section of the adrenal gland of a developmental stage S35 (embryonic day 9) chick embryo. Note that BMP-4 is expressed in the adrenal cortical (interrenal) cells surrounding the TH-positive adrenal chromaffin cells. Bar: 100 μm.
Figure 4
Figure 4
In situ hybridisations for bone morphogenetic protein receptor (BMPR)1A (A,B,D,E,F,H) and BMPR1B (I,J,L) in developing chick adrenal glands at embryonic day (E)7 (A-D,I-L) and E9 (E-H). Neural crest-derived (chromaffin) cells have been labelled with HNK-1 (CD57, G,H merge) or tyrosine hydroxylase (TH) (C,D merge; K,L merge). Note that BMPR1A is expressed in both adrenal chromaffin and interrenal (cortical) cells (A-H) while BMPR1B is expressed only by interrenal cells (I-L). ag, adrenal gland; da, dorsal aorta; no, notochord. Bar: 175 μm.
Figure 5
Figure 5
RT-PCRs for bone morphogenetic protein-4 (BMP-4; upper panel) of adrenal explant cultures of a developmental stage S23 chick embryo after 4 days in vitro (lane a). Levels of BMP-4 mRNA are comparable to those in vivo at S23 (lane b) and S32 (lane c). RT-PCRs of GAPDH (lower panel) were run in parallel.
Figure 6
Figure 6
Quantification of tyrosine hydroxylase (TH)-immunoreactive cells in adrenal explant cultures after 0, 1, 3, and 5 days in vitro with or without noggin-treatment. Numbers of TH-positive cells in explants treated with noggin failed to increase, in contrast to those in control cultures. Counts of TH-immunoreactive cells were performed in every sixth section of the adrenal explant cultures. Data are presented as means ± standard error of the mean. At least seven explants for each group were analyzed.
Figure 7
Figure 7
(A) Numbers of bromo-deoxyuridine (BrdU)-positive cells expressed as a percentage of tyrosine hydroxylase (TH)-positive cell numbers in adrenal explant cultures after 3 days in vitro. Note that there is no significant difference between noggin-treated and control cultures. Data are presented as means ± standard error of the mean. Four explants were analysed per group and every fourth section was counted. (B) Ratio of Sox10-positive/TH-positive cells in adrenal explant cultures after 3 days in vitro. Data are presented as means ± standard error of the mean. Five explants were analyzed per group. Sox10 mRNA-positive cells were counted in every eighth section and the adjacent sections were analyzed for TH mRNA-positive cells.
Figure 8
Figure 8
In situ hybridisation for neurofilament (blue) combined with tyrosine hydroxylase (TH)-immunocytochemistry (red) on sections of sympathetic ganglia at the upper thoracic level (A,B) and of the adrenal gland (C) of embryonic day (E)8 chick embryos: (A,C) control embryo; (B) embryo that was transplanted with bone morphogenetic protein-4 (BMP-4)-expressing fibroblasts at E2. Note that the majority of cells in the sympathetic ganglia of both control and BMP-4-overexpressing embryos is neurofilament-positive, while in the adrenal gland most cells are TH-positive, but neurofilament-negative. (D) The percentage of TH-positive neurofilament-negative cells was not altered in BMP-4-overexpressing embryos. Bars: 100 μm.
Figure 9
Figure 9
(A-E) Electron micrographs of sympathetic ganglia at the upper thoracic level (A-D) and of the adrenal gland (E) of embryonic day (E)8 chick embryos: (A,C,E) control embryo; (B,D) embryo that was transplanted with bone morphogenetic protein-4 (BMP-4)-expressing fibroblasts at E2. Note that two major types of sympathoadrenal cells exist in sympathetic ganglia of both control and BMP-4 overexpressing embryos: cells without or with small granules in the cell body (A,B), and cells with large granules (C,D). Bar: 2 μm.
Figure 10
Figure 10
Numbers and area profiles of 'chromaffin' granules of cells in sympathetic ganglia. (A) The number of granules in the sympathetic ganglia was significantly enhanced in the group of the bone morphogenetic protein-4 (BMP-4)-treated embryos at stage 33. The data are presented as mean numbers of granules (± standard error of the mean). (B) BMP-4 treatment increased the mean profile area of 'chromaffin' granules in the sympathetic ganglia of stage 32 and 33 embryos. The data are presented as mean profile area (± standard error of the mean).*p ≤ 0.05; **p ≤ 0.01.

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