Persistent expression of BMP-4 in embryonic chick adrenal cortical cells and its role in chromaffin cell development
- Katrin Huber1Email author,
- Aylin Franke1Email author,
- Barbara Brühl1,
- Shlomi Krispin3,
- Uwe Ernsberger1,
- Andreas Schober1,
- Oliver Bohlen und von Halbach1,
- Hermann Rohrer2,
- Chaya Kalcheim3 and
- Klaus Unsicker1Email author
© Huber et al. 2008
Received: 16 February 2008
Accepted: 22 October 2008
Published: 22 October 2008
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.
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.
BMP-4 may serve to promote specific chromaffin traits, but is not sufficient to convert sympathetic neurons into a chromaffin phenotype.
bone morphogenetic protein
Chinese hamster ovary
The neural crest (NC) plays a paradigmatic role for studying the diversification of multipotential progenitor cells into distinct cell types. Sympathetic neurons and the endocrine chromaffin cells of the adrenal medulla and extra-adrenal locations are derived from the NC . Both cell types share many characteristics – for example, the synthesizing machinery for noradrenaline (see  for a review) – but are very distinct in other aspects. It is widely believed that chromaffin cells and sympathetic neurons develop from the NC via a common sympathaodrenal (SA) progenitor, which has the capacity to give rise to both sympathetic neurons and chromaffin cells. SA progenitors develop in the trunk region near the dorsal aorta [3–6]. In this location they acquire catecholaminergic neuronal features, and then are supposed to re-migrate to the sites of the secondary sympathetic ganglia and the adrenal gland. Chromaffin cell differentiation is believed to involve the inhibition of terminal neuronal differentiation , the downregulation of neurofilament (NF), lack of neurites, and the development of large 'chromaffin' dense-core vesicles [2, 8–11] However, the differential cues determining either a neuroendocrine or neuronal fate have not been identified as yet. Tissues surrounding NC cells and SA progenitor cells during their migration and at their final locations are considered to be important for the induction of a sympathetic neuronal or chromaffin cell phenotype.
Glucocorticoids secreted by the adrenal cortex have long been thought to be essential for chromaffin cell differentiation [11–14]; however, analysis of glucocorticoid receptor-deficient mice revealed that their adrenal chromaffin cells are largely normal . Other factors provided locally by the adrenal gland, such as transforming growth factor-β, have been shown to be involved in the regulation of chromaffin cell proliferation, but not in chromaffin cell phenotype determination .
Bone morphogenetic proteins (BMPs) comprise a family of growth factors that were first identified according to their osteogenic properties [17–19]. Subsequently, they were found to be expressed widely in vertebrate embryonic structures and shown to be involved in a variety of key embryonic processes such as dorsal-ventral axis specification, epithelio-mesenchymal interactions, and apoptosis . BMP-4 and BMP-7 play an important role in the specification of SA progenitors from the NC in the avian embryo [3, 4, 21] and are expressed in the wall of the dorsal aorta. Overexpression experiments of BMP4/7 and the use of noggin, an inhibitor of BMP-4/7, showed that BMPs are necessary and sufficient for the early induction of a neuronal and catecholaminergic phenotype in NC cells that aggregate in the vicinity of the dorsal aorta [3–5, 21].
It has recently been suggested that BMP-4 is required only transiently for an early step of sympathetic neuron differentiation but may block subsequent steps of terminal neuronal differentiation. This hypothesis was based on the observation that NC cells that were treated with BMP-4 form ganglion-like clusters and extend neurites only after withdrawal of BMP-4 . This suggested the possibility that high and maintained BMP expression may result in catecholaminergic cells without neuronal properties, that is, chromaffin cells. We now demonstrate that BMP-4 is expressed in cortical (interrenal) cells of the developing chick adrenal gland but is not detectable in sympathetic ganglia. We provide a detailed analysis of the temporal and spatial pattern of BMP-4 and BMP receptor (BMPR) expression in the embryonic chick adrenal gland. We show that noggin, which inhibits BMP activity, reduces numbers of catecholaminergic cells in explant cultures of the adrenal gland. However, our results from BMP-4 overexpression experiments at the sites of secondary sympathetic ganglia suggest that prolonged exposure of SA cells to BMP-4 promotes the expression of chromaffin traits but is not sufficient to alter the proportion of chromaffin-like cells in the ganglia.
Materials and methods
Fertilized White Leghorn eggs were incubated in a humidified egg chamber at 38°C until embryonic day (E)3, E4, E5, E6, E7 or E9. On the indicated day of incubation, whole embryos were harvested and the stage according to the criteria of Hamburger and Hamilton  was determined. Embryos were either fixed in 4% paraformaldehyde overnight or the adrenal anlagen were dissected for tissue cultures or RNA-isolation.
Chinese hamster ovary (CHO) cells producing Xenopus noggin and dhfr-CHO control cells were a kind gift from Richard Harland and Dale Frank. Cell lines were grown as described previously . Supernatant was collected after a 4-day culture period and was then concentrated 20-fold using a minicon concentrator (CS 15; Millipore, Schwalbach, Germany).
For explant cultures the adrenal anlagen, including the adjacent mesenchyme, were dissected from stage 23 using sharpened insect needles. To prepare collagen gels, 5 μl sodium bicarbonate (5%) was added to 95 μl of rat tail collagen (90%) in DMEM. The collagen solution was put into a 3.5 cm diameter petridish (Costar, Schiphol-Rijk, Netherlands) and adrenal explants were placed on top. After the gel had polymerized, 4 ml of DMEM medium (Invitrogen, Gaithersburg, MD, USA) supplemented with 10% foetal calf serum and antibiotics (penicillin, streptomycin, neomycin (PSN); Invitrogen) were added. The medium contained 1% supernatant of either noggin-producing CHO cells or control dhfr-CHO cells. Explant cultures were incubated in a 95% air/5% CO2 atmosphere at 37°C. Every two days 50% of the medium was changed. The explants were fixed after 1, 3 or 5 days in culture and processed for electron microscopy or cryoembedding followed by immunofluorescence staining or in situ hybridisation (see below). For 5-bromo-2'-deoxy-uridine (BrdU) labelling and detection, a BrdU-labelling and Detection Kit I (Roche; Mannheim, Germany) was used. The BrdU-labelling solution was prepared according to the manufacturer's instructions in culture medium with and without noggin and added after a culture period of 3 days 1 hour before fixation.
To prepare cryosections, paraformaldehyde-fixed tissues were rinsed three times with phosphate buffer and then placed in 30% sucrose in phosphate-buffered saline (PBS) for cryoprotection. Following overnight immersion in sucrose, the tissue was coated with Tissue TEK® O.C.T™ compound (Sakura Finetek Europe B.V, Zoeterwoude, Netherlands, frozen on dry-ice and stored at -70°C until further processing. The tissue was then cut into 12 μm serial sections, mounted on Superfrost™ slides and air-dried for 30 minutes before performing in situ hybridisation or immunfluorescence staining.
Non-radioactive in situ hybridisation on cryosections and preparation of digoxigenin-labelled probes for chick tyrosine hydroxylase (TH), chick achaete scute-homologue 1 (CASH-1)  chick Phox2B , chick neurofilament-M , chick BMP-4 , chick BMPRIA and IB , chick steroidogenic factor 1 (SF-1) and chick Sox10  were carried out using a modification of the protocol of D Henrique (IRFDBU, Oxford, UK) as previously described . Chick SF-1 (base-pairs 509–1,288) was cloned by reverse transcription (RT)-PCR using a pGEM-T vector system (Promega, Mannheim, Germany) following the manufacturer's instruction.
For TH immunfluorescence-staining, sections were pretreated with 10% normal rabbit serum in PBS and 0.1% Triton X-100, followed by overnight incubation with polyclonal sheep anti-tyrosine hydroxylase antibody (TH, 1:200; Chemicon International, Temecula, CA, USA) at 4°C. Specimens were rinsed in PBS and incubated with a Cy3™-conjugated rabbit anti-sheep antibody (1:200; Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature. Specimens were then rinsed in PBS, counterstained with 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI; 1:1,000) for 10 minutes, and mounted with Fluorescent Mounting Medium (Dako Hamburg, Germany).
For TH immunohistochemistry slides were pretreated with 3% hydrogen peroxide in PBS for 15 minutes. After incubation with primary antibody as described above, sections were incubated with a biotinylated rabbit anti-sheep antibody (1:200; Vector Laboratories Burlingame, CA, USA), rinsed with PBS and incubated for 1 h with avidin and biotinylated horseradish-peroxidase-macromolecular complex (Vector: Elite ABC reagent) according to the manufacturer's instructions. Sections were then rinsed with PBS and stained with 3-amino-9-ethylcarbazol (AEC; Sigma-Aldrich, Taufkirchen, Germany) according to the manufacturer's instructions. After rinsing with PBS, sections were mounted with Kaiser's glycerol gelatine (Merck, Darmstadt, Germany). HNK-1 (CD57) immunolabelling was performed as previously described .
RNA isolation and RT-PCR
RT-PCR was used to determine the expression of BMP-4 mRNA in adrenal anlagen explant cultures. Total RNA was isolated from tissues using Trizol (Life Technologies, Karlsruhe, Germany) according to the manufacturer's guidelines for extraction of RNA from small amounts of tissue. Before reverse transcription samples were digested with DNase (Roche) for 15 minutes at 37°C followed by inactivation at 70°C for 5 minutes. First-strand cDNA was synthesized in a final volume of 25 μl. Reaction mixtures consisted of 1 μg of total RNA and final concentrations of 1× first strand buffer (1× first-strand buffer (New England Biolabs, Frankurt, Germany): 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 5 mM MgCl2 (Biolabs), 10 mM dithiothreitol (DTT)) and 1 mM each of dNTPs (Biolabs), 50 ng/μl oligo-dT primer 18 (Biolabs), 1 U/μl RNase inhibitor (Roche), and 20 U/μl Moloney murine leukemia virus reverse transcriptase (Biolabs). Before adding buffer, dNTPs, and reverse transcriptase, the reaction mixture was heated to 75°C for 10 minutes. After adding the final components, incubation at 37°C for 2 h followed. Finally, the reaction mixture was heated for 10 minutes at 65°C. Negative controls were carried out by omitting the reverse transcriptase.
Following reverse transcription, PCR amplification of the cDNA was carried out using specific primers for chick BMP-4 (5'AGGAGCTTCCACCATGAAGA3' and 5'CGGCTAATCCTGACGTGTTT3'; 413 bp PCR product) and chick GAPDH (5'GTCAACGGATTTGGCCGTAT3' and 5'AATGCCAAAGTTGTCATGGATG3'; 489 bp PCR product). Reactions were performed in an Eppendorf Mastercycler Gradient thermocycler. (Eppendorf, Hamburg, Germany) Reagents were assembled in a final volume of 50 μl with 1 μl of first-strand cDNA, 1 μM forward primer, 1 μM reverse primer, 1× PCR buffer (10× PCR buffer: 200 mM Tris-HCl, pH 9.0, and 500 mM KCl (Promega, Mannheim, Germany), 2.5 mM MgCl2, and 0.1 mM each of dNTPs, Taq DNA polymerase (0.5 μl, 2.5 U; Promega) and RNase-free water to 50 μl. cDNAs were amplified for 30 cycles. One round of amplification consisted of 45 s at 94°C, 45 s at 57.3°C, and 1 minute at 72°C. PCR reactions (12.5 μl) were run on agarose gels (Life Technologies, Karlsruhe, Germany) in 1× TAE buffer (0.04 M Tris-acetate and 0.001 M EDTA), and reaction products were visualized after soaking gels in 0.5 μg/ml ethidium bromide solution in distilled water for 10 minutes, with a transilluminator (Intas, Göttingen, Germany). Pictures were taken by a computer-assisted gel documentation system (Intas).
Overexpression of BMP-4 at the site of developing secondary sympathetic ganglia
Infections of chick embryos with RCAS-BMP-4 viruses and control RCAS viruses were performed as described by Reissmann et al. . Embryos were implanted with infected fibroblasts at day 2 at the level of the wing bud, fixed at day 8, and staged as described above. Tissues were kryo-embedded, cut into 12 μm transverse serial sections and then processed for neurofilament-M in situ hybridisation followed by TH immunohistochemistry as described above. Numbers of TH-positive/NF-positive and TH-positive/NF-negative cells in sympathetic ganglia were determined in every fifth section. The analyzed region extended from the superior thoracic aperture 1.5 mm into the caudal direction. This region was infected by the RCAS virus in all experimental embryos as shown by in situ hybridisation for RCAS-RT.
For electron microscopy, tissue was fixed by immersion in a mixture of glutaraldehyde (1.5%) and paraformaldehyde (1.5%) in phosphate buffer at pH 7.3 for 48 h and rinsed several times with cacodylate buffer (0.1 M). Organs were then post-fixed in 1% OsO4/1.5% potassium hexacyanoferrate, rinsed in 0.1 M cacodylate buffer and 0.2 M sodium maleate buffer (pH 6.0) and block-stained with 1% uranyl acetate. Following dehydration through increasing concentrations of ethanol, the tissue was Epon-embedded. Ultrathin sections (50 nm) were examined with a Zeiss EM10.
For counts and measurements of 'chromaffin' granules in secondary sympathetic ganglia, serial ultrathin sections (50 nm) were photographed, digitalized, and stored on a personal computer. The subsequent analysis was performed by an experimenter blinded to the treatment and stage by coding the images. The coded images were analyzed using the software ImageTool 3.0 (University of Texas, Health Science Center, San Antonio, USA). The first image that was analyzed was randomly selected (one of the first three images) and starting with that image, every fourth image was analysed.
Two different parameters were analyzed: total numbers of granules within the image; and the mean surface area of large granules. The total numbers of granules were determined by using the 'count and tag' plug-in of ImageTool. Total numbers were determined in material derived from stages 31, 32 and 33 (with and without BMP-4 treatment). The data are presented as mean numbers of granules (± standard error of the mean). Using the same images, the mean surface areas of large granule profiles were calculated. Large granules were defined as granules with a profile area bigger than 0.01 μm2. Each granule meeting this criterion was measured using ImageTool. The data are presented as mean profile area (± standard error of the mean).
For statistical evaluation, a one-way ANOVA, followed by post-hoc test (Newman-Keuls Multiple Comparison test) was performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA).
BMP-4 is expressed in adrenal cortical cells during embryonic development
BMP-receptors are expressed in chromaffin cells of the developing adrenal gland
Numbers of TH-positive cells in adrenal explant cultures are reduced by noggin-treatment
Expression of BMP-4 in the adrenal cortical anlagen and concomitant expression of BMPR mRNA in adrenal chromaffin progenitor cells suggested a putative function of BMP-4 in chromaffin cell development. Noggin, a secreted inhibitor of BMP-4/7 , provides a useful tool for neutralizing BMP-4/7 in vivo or in vitro, and thereby interfering with BMP signalling. To examine the possible involvement of BMP signalling in chromaffin cell development, explants of the adrenal anlagen from S23 chick embryos were prepared and treated with noggin. Explants contained the cortical area and adjacent TH-positive and TH-negative SA progenitor cells, which had not yet colonized the cortex by the time of excision (Figure 2A–D).
We next studied the expression of markers specific for adrenal cortical and medullary cells in more detail using antisense and sense riboprobes for SF-1, BMP-4, CASH-1, Phox2B, TH, and NF. All these markers could be detected in the adrenal explants, reflecting a bona fide in vivo situation. In addition, cells with the typical ultrastructural features of chromaffin cells and sympathetic neurons could be identified by electron microscopy (not shown).
BMP-4 overexpression at the site of developing sympathetic ganglia promotes chromaffin cell differentiation, but is not sufficient to induce a sympathetic neuronal to chromaffin cell shift
Ultrastructure of sympathetic ganglia upon BMP-4 overexpression
The molecular bases of chromaffin progenitor specification are still enigmatic. A classic model had postulated a common progenitor cell for sympathetic neurons and neuroendocrine chromaffin cells (the SA progenitor), and a crucial role for glucocorticoids in blocking neuronal and promoting neuroendocrine differentiation [7, 11, 12, 40]. Analysis of the glucocorticoid receptor knockout [15, 41] failed to support this hypothesis: glucocorticoid receptor-deficient mice had normal numbers of adrenal chromaffin cells, which resembled their wild-type counterparts in virtually all structural and chemical aspects.
In our search for alternative cues, we report in this study that adrenal cortical cells (interrenal cells in the chick) express BMP-4 starting at the beginning of cortical cell assembly. At early stages, BMP-4 mRNA is detected in the wall of the dorsal aorta and in adjacent tissues, particularly those extending ventrally and laterally. These regions include the developing adrenal cortical cells expressing the orphan nuclear receptor SF-1. In addition, they engulf the area lateral to the aorta where NC-derived cells are found and adrenal chromaffin cells differentiate. With ongoing development, BMP-4 expression outside the wall of the aorta becomes restricted to adrenal cortical cells, which by then intermingle with the differentiating adrenal chromaffin cells to form the chick adrenal gland. BMP-4 is continuously expressed by cortical cells at least until E15. Thus, throughout this developmental period, differentiating chromaffin cells are surrounded by cells with high BMP-4 expression. This differs from the situation encountered by the cells destined to form secondary sympathetic ganglia. At their initial differentiation site, the primary sympathetic ganglia, the wall of the dorsal aorta is the major source of BMP-4. On the migration route from primary to secondary sympathetic ganglia, BMP-4 expression is hardly detectable. In secondary sympathetic ganglia, BMP expression is detectable by RT-PCR  (K. Tsarovina and H Rohrer, unpublished), yet barely detectable by in situ hybridisation (present study; UE unpublished observations). Taken together, BMP-4 expression levels differ dramatically between sites of adrenal gland and secondary sympathetic ganglion formation, provoking the question of whether continuously elevated BMP-4 levels constitute a molecular cue required for chromaffin cell differentiation and counteracting neuronal differentiation.
BMPs have been firmly established in their role in the development of autonomic sympathetic and parasympathetic neurons (see [6, 43, 44] for reviews). BMPs synthesized by cells in the wall of the dorsal aorta trigger the initial development of NC cells towards noradrenergic sympathetic neurons [3–5]. BMPs induce expression of the transcription factors MASH1, Phox2a/b, HAND2, and Gata2/3 [3, 45, 46]. Overexpression of BMP-4 [3, 22] and BMP depletion by noggin  demonstrates that BMPs are sufficient and necessary to induce the expression of the enzymes of noradrenalin biosynthesis, TH and dopamine β-hydroxylase (DBH), and the neuronal markers neurofilament-L, SCG10, neurexin I, and synaptotagmin I in NC-derived precursors. Thus, the available data suggest that BMPs are the decisive stimulus triggering a network of transcription factors necessary for the differentiation of NC cells into noradrenergic sympathetic neurons.
The observation that withdrawal of BMP-4 after a short period in NC cell cultures promotes the formation of ganglion-like aggregates of cells extending neurites and expressing TH, neurexin 1, and synaptotagmin I  supports the idea that short exposure to BMP suffices to induce noradrenergic and neuronal differentiation. To test whether prolonged BMP availability suppresses neuronal properties in vivo, the effect of virus-mediated overexpression of BMP-4 at sites of sympathetic ganglion formation was analyzed. The expression of the neuronal marker neurofilament-M mRNA in sympathetic neurons, which is barely detectable in chick chromaffin cells throughout embryonic development ( and this study), appears unaltered in ganglia at sites of BMP-4 overexpression (this study). The grafted BMP-4-secreting cells were clearly biologically effective, as judged by the massive local overproduction of cartilage, alterations in spinal cord patterning and structure of the dorsal aorta. There are several possible explanations for the failure of BMP-4 to ectopically increase numbers of chromaffin cells, beyond the possibility that high levels of BMP-4 may not specifically induce a chromaffin phenotype. Thus, BMP-4 may not have the capacity to convert committed sympathetic neuronal into chromaffin progenitors, at least not in the given cellular and molecular context. Or chromaffin cells might have been generated, but did not survive.
Even so, BMP-4 distinctly promoted differentiation of chromaffin properties in the sympathetic ganglia, as judged by the increase in the number and size of chromaffin granules. In sympathetic ganglia of chick embryos at E6-9, two cell populations with differently sized granules are present [47, 48]. A growing population of cells shows scarce dense core vesicles with approximate diameters of 100 nm while a transient population of cells, which is predominantly located in upper lumbar sympathetic ganglia  and was not found in the upper thoracic ganglia analysed in the present study, shows larger granules of up to 300 nm in diameter. It is not clear which of the cell populations is affected by BMP-4 overexpression, and it is currently not clear whether neurofilament-M expression differs between the two populations. One possibility is an effect of BMP-4 on granule size in sympathetic neuron precursors while neurofilament-M expression levels remain refractory. Alternatively, cells with larger granules that may show low neuronal marker expression similar to chromaffin cell precursors may respond with an increase in granule size to BMP treatment. The growing evidence of an early divergence between sympathetic neuron and adrenal chromaffin cell precursors may support the latter scenario.
Several lines of evidence argue for both similarities and differences in the signalling networks involved in the generation of sympathetic neurons and chromaffin cells (see  for a review). An initial study analyzing the SA cell lineage in mice lacking MASH1 reported that the neuronal progeny of the SA lineage was largely eliminated, whereas adrenal chromaffin cells were hardly affected . Our re-analysis of the MASH1 knockout revealed that MASH1 was required for orchestrating the normal differentiation program of a majority, but not all, chromaffin cells . Similar to the MASH1 knockout, our analysis of Phox2B-/- mice provided evidence that chromaffin cells are apparently distinctly different from sympathetic neurons in their requirement for Phox2B . Recently, it has been shown that the zinc-finger factor Insm1 (IA1) is expressed early during SA development and that a null mutation of Insm1 affects the development of chromaffin cells and sympathetic neurons differentially . Thus, these analyses of MASH1, Phox2B and Insm1 deficient mice suggest that the SA progenies populating sympathetic ganglia and adrenal glands are not identical with regard to their requirements for these transcription factors. The distinct requirements may reflect distinct origins and distinct characters of SA progenitors, and/or differences in the cellular/molecular environments of sympathetic ganglia and the adrenal gland, respectively. Similar to mice, in the chick embryo differences between sympathetic neuron and adrenal chromaffin precursors have been noted from the earliest stages of differentiation . The presence of neuron-like cells with high neurofilament-L expression levels in adrenal tissue throughout development  and the presence of chromaffin-like cells with low neurofilament-M expression in sympathetic ganglia (this study) suggest an early diversification of lineages that cannot be overcome by differences in the environment.
What, then, is the function of BMP-4 in the adrenal gland? Our experiments applying noggin for 3 or 5 days to adrenal explants isolated at S23 strongly suggest that adrenal BMP-4 augments numbers of TH-positive cells, and does so by inducing TH in TH-negative SA progenitors rather than by stimulating proliferation monitored by BrdU incorporation. The notion that adrenal BMP-4 induces TH in SA progenitors that are still TH-negative at the time of their migration into the adrenal anlagen is also supported by our observation that a substantial number of SA progenitors, which are Sox10-, MASH1- and/or Phox2B-positive, but TH-negative, can be found in the vicinity and inside the chick (S23) and E12.5 mouse adrenal anlage [38, 45]. Thus, adrenal BMP-4 would serve a similar role as BMP-4 secreted from the dorsal aorta, that is, to induce NC cells to become SA progenitor cells. It is also conceivable that adrenal cortical BMP-4 may act in an autocrine fashion on BMPR-bearing cortical cells in functions that remain to be elucidated.
Our study has revealed adrenal cortical cells as a site of BMP-4 synthesis. Adrenal BMP-4 probably serves to induce SA-specific transcription factors and TH in cells that colonize the adrenal anlage in a very immature state.
We thank Nicole Karch and Jutta Fey for excellent technical assistance. We are grateful to Richard Sparla for providing the RT-PCR data. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 488, TP A6).
- Le Douarin NM, Kalcheim C: The Neural Crest. 2nd edition. Cambridge: Cambridge University Press; 1999.
- Unsicker K: The chromaffin cell: paradigm in cell, developmental and growth factor biology. J Anat 1993, 183:207–221.PubMed
- Reissmann E, Ernsberger U, Francis-West PH, Rueger D, Brickell PM, Rohrer H: Involvement of bone morphogenetic protein-4 and bone morphogenetic protein-7 in the differentiation of the adrenergic phenotype in developing sympathetic neurons. Development 1996, 122:2079–2088.PubMed
- Schneider C, Wicht H, Enderich J, Wegner M, Rohrer H: Bone morphogenetic proteins are required in vivo for the generation of sympathetic neurons. Neuron 1999, 24:861–870.View ArticlePubMed
- Shah NM, Groves AK, Anderson DJ: Alternative neural crest cell fates are instructively promoted by TGF beta superfamily members. Cell 1996, 85:331–343.View ArticlePubMed
- Goridis C, Rohrer H: Specification of catecholaminergic and serotonergic neurons. Nat Rev Neurosci 2002, 7:531–541.View Article
- Michelsohn AM, Anderson DJ: Changes in competence determine the timing of two sequential glucocorticoid effects on sympathoadrenal progenitors. Neuron 1992, 8:589–604.View ArticlePubMed
- Coupland RE: The Natural History of the Chromaffin Cells. London and Colchester: Spottiswoode, Ballanntyne and Co. Ltd; 1995.
- Coupland RE, Tomlinson A: The development and maturation of adrenal medullary chromaffin cells of the rat in vivo: a descriptive and quantitative study. Int J Dev Neurosci 1989, 7:419–438.View ArticlePubMed
- Langley K, Grant NJ: Molecular markers of sympathoadrenal cells. Cell Tissue Res 1999, 298:185–206.View ArticlePubMed
- Unsicker K, Krisch B, Otten J, Thoenen H: Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: Impairment by glucocorticoids. Proc Natl Acad Sci USA 1978, 75:3498–3502.View ArticlePubMed
- Anderson DJ, Axel R: A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. Cell 1986, 47:1079–1090.View ArticlePubMed
- Doupe AJ, Landis SC, Patterson PH: Environmental influences in the development of neural crest derivatives: glucocorticoids, growth factors and chromaffin cell plasticity. J Neurosci 1985, 5:2119–2142.PubMed
- Seidl K, Unsicker K: The determination of the adrenal medullary cell fate during embryogenesis. Dev Biol 1989, 136:481–490.View ArticlePubMed
- Finotto S, Krieglstein K, Schober A, Deimling F, Lindner K, Brühl B, Beier K, Metz J, Garcia-Arraras JE, Roig-Lopez JL, Monaghan P, Schmid W, Cole TJ, Kellendonk C, Tronche F, Schütz G, Unsicker K: Analysis of mice carrying targeted mutations of the glucocorticoid receptor gene argues against an essential role of glucocorticoid signalling for generating adrenal chromaffin cells. Development 1999, 126:2935–2944.PubMed
- Combs SE, Krieglstein K, Unsicker K: Reduction of endogenous TGF-beta increases proliferation of developing adrenal chromaffin cells in vivo. J Neurosci Res 2000, 59:379–383.View ArticlePubMed
- Urist MR: Bone: formation by autoinduction. Science 1965, 150:893–899.View ArticlePubMed
- Wang EA, Rosen V, D'Alessandro JS, Bauduy M, Cordes P, Harada T, Israel DI, Hewick RM, Kerns KM, LaPan P, Luxenberg DP, McQuaid D, Moutsatsos IK, Nove J, Wozney JM: Recombinant human bone morphogenetic protein induces bone formation. Proc Natl Acad Sci USA 1990, 87:2220–2224.View ArticlePubMed
- Kingsley DM, Bland AE, Grubber JM, Marker PC, Russell LB, Copeland NG, Jenkins NA: The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGF beta superfamily. Cell 1992, 71:399–410.View ArticlePubMed
- Liu A, Niswander LA: Bone morphogenetic protein signalling and vertebrate nervous system development. Nat Rev Neurosci 2005, 6:945–954.View ArticlePubMed
- Varley JE, McPherson CE, Zou H, Niswander L, Maxwell GD: Expression of a constitutively active type I BMP receptor using a retroviral vector promotes the development of adrenergic cells in neural crest cultures. Dev Biol 1998,196(1):107–118.View ArticlePubMed
- Patzke H, Reissmann E, Stanke M, Bixby JL, Ernsberger U: BMP growth factors and Phox2 transcription factors can induce synaptotagmin I and neurexin I during sympathetic neuron development. Mech Dev 2001, 108:149–159.View ArticlePubMed
- Hamburger V, Hamilton HL: A series of normal stages in the development of the chick embryo. J Exp Zool 1951, 88:49–92.
- Lamb TM, Knecht AK, Smith WC, Stachel SE, Economides AN, Stahl N, Yancopolous GD, Harland RM: Neural induction by the secreted polypeptide noggin. Science 1993, 262:713–718.View ArticlePubMed
- Ernsberger U, Patzke H, Tissier-Seta JP, Reh T, Goridis C, Rohrer H: The expression of tyrosine hydroxylase and the transcription factors cPhox-2 and Cash-1: evidence for distinct inductive steps in the differentiation of chick sympathetic precursor cells. Mech Dev 1995, 52:125–136.View ArticlePubMed
- Stanke M, Junghans D, Geissen M, Goridis C, Ernsberger U, Rohrer H: The Phox2 homeodomain proteins are sufficient to promote the development of sympathetic neurons. Development 1999, 126:4087–4094.PubMed
- Zopf D, Hermans-Borgmeyer I, Gundelfinger ED, Betz H: Identification of gene products expressed in the developing chick visual system: characterization of a middle-molecular-weight neurofilament cDNA. Genes Dev 1987, 1:699–708.View ArticlePubMed
- Francis PH, Richardson MK, Brickell PM, Tickle C: Bone morphogenetic proteins and a signalling pathway that controls patterning in the developing chick limb. Development 1994, 120:209–218.PubMed
- Ernsberger U, Patzke H, Rohrer H: The developmental expression of choline acetyltransferase (ChAT) and the neuropeptide VIP in chick sympathetic neurons: evidence for different regulatory events in cholinergic differentiation. Mech Dev 1997, 68:115–126.View ArticlePubMed
- Shoval I, Ludwig A, Kalcheim C: Antagonistic roles of full-length N cadherin and its soluble BMP cleavage product in neural crest delamination. Development 2007, 134:491–501.View ArticlePubMed
- Luo X, Ikeda Y, Parker KL: A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 1994, 77:481–490.View ArticlePubMed
- Guillemot F, Lo LC, Johnson JE, Auerbach A, Anderson DJ, Joyner AL: Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons. Cell 1993, 75:463–476.View ArticlePubMed
- Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF: The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 1999, 399:366–370.View ArticlePubMed
- Huber K: The sympathoadrenal cell lineage: specification, diversification, and new perspectives. Dev Biol 2006, 298:335–343.View ArticlePubMed
- Unsicker K, Huber K, Schütz G, Kalcheim C: The chromaffin cell and its development. Neurochem Res 2005, 30:921–925.View ArticlePubMed
- Massagué J, Chen YG: Controlling TGF-beta signalling. Genes Dev 2000, 14:627–644.PubMed
- Zimmerman LB, De Jesús-Escobar JM, Harland RM: The Spemann organizer signal Noggin binds and inactivates bone morphogenetic protein 4. Cell 1996, 86:599–606.View ArticlePubMed
- Ernsberger U, Esposito L, Partimo S, Huber K, Franke A, Bixby JL, Kalcheim C, Unsicker K: Expression of neuronal markers suggests heterogeneity of chick sympathoadrenal cells prior to invasion of the adrenal anlagen. Cell Tissue Res 2005, 319:1–13.View ArticlePubMed
- Gut P, Huber K, Lohr J, Bruhl B, Oberle S, Treier M, Ernsberger U, Kalcheim C, Unsicker K: Lack of an adrenal cortex in Sf1 mutant mice is compatible with the generation and differentiation of chromaffin cells. Development 2005, 132:4611–4619.View ArticlePubMed
- Kande ER, Schartz JH, Jessel TM: Principles of Neural Science. 4th edition. New York: McGraw-Hill; 2000.
- Cole TJ, Blendy JA, Monaghan AP, Krieglstein K, Schmid W, Aguzzi A, Fantuzzi G, Hummler E, Unsicker K, Schütz G: Targeted disruption of the glucocorticoid receptor gene blocks adrenergic chromaffin cell development and severely retards lung maturation. Genes Dev 1995, 13:1608–1621.View Article
- Lein PJ, Beck HN, Chandrasekaran V, Gallagher PJ, Chen HL, Lin Y, Guo X, Kaplan PL, Tiedge H, Higgins D: Glia induce dendritic growth in cultured sympathetic neurons by modulating the balance between bone morphogenetic proteins (BMPs) and BMP antagonists. J Neurosci 2002, 22:10377–10387.PubMed
- Ernsberger U: Evidence for an evolutionary conserved role of bone morphogenetic protein growth factors and phox2 transcription factors during noradrenergic differentiation of sympathetic neurons. Induction of a putative synexpression group of neurotransmitter-synthesizing enzymes. Eur J Biochem 2000, 267:6976–6981.View ArticlePubMed
- Howard MJ: Mechanisms and perspectives on differentiation of autonomic neurons. Dev Biol 2005, 277:271–286.View ArticlePubMed
- Howard MJ, Stanke M, Schneider C, Wu X, Rohrer H: The transcription factor dHAND is a downstream effector of BMPs in sympathetic neuron specification. Development 2000, 127:4073–4081.PubMed
- Tsarovina K, Pattyn A, Stubbusch J, Müller F, Wees J, Schneider C, Brunet JF, Rohrer H: Essential role of Gata transcription factors in sympathetic neuron development. Development 2004, 131:4775–4786.View ArticlePubMed
- Luckenbill-Edds L, van Horn C: Development of chick paravertebral sympathetic ganglia. I. Fine structure and correlative histofluorescence of catecholaminergic cells. J Comp Neurol 1980, 191:65–76.View ArticlePubMed
- Ross S, Fischer A, Unsicker K: Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure, and quantitative determinations of catecholamines and somatostatin. Cell Tissue Res 1990, 260:147–159.View ArticlePubMed
- Huber K, Bruhl B, Guillemot F, Olson EN, Ernsberger U, Unsicker K: Development of chromaffin cells depends on MASH1 function. Development 2002, 129:4729–4738.PubMed
- Huber K, Karch N, Ernsberger U, Goridis C, Unsicker : The role of Phox2B in chromaffin cell development. Dev Biol 2005, 279:501–508.View ArticlePubMed
- Wildner H, Gierl MS, Strehle M, Pla P, Birchmeier C: Insm1 (IA-1) is a crucial component of the transcriptional network that controls differentiation of the sympatho-adrenal lineage. Development 2008, 135:473–481.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.