Sympathetic neurons and chromaffin cells share a common progenitor in the neural crest in vivo
© Shtukmaster et al.; licensee BioMed Central Ltd. 2013
Received: 17 December 2012
Accepted: 17 May 2013
Published: 18 June 2013
The neural crest (NC) is a transient embryonic structure unique to vertebrates, which generates peripheral sensory and autonomic neurons, glia, neuroendocrine chromaffin and thyroid C-cells, melanocytes, and mesenchymal derivatives such as parts of the skull, heart, and meninges. The sympathoadrenal (SA) cell lineage is one major sub-lineage of the NC that gives rise to sympathetic neurons, chromaffin cells, and the intermediate small intensely fluorescent (SIF) cells. A key question is when during NC ontogeny do multipotent progenitors segregate into the different NC-derived lineages. Recent evidence suggested that sympathetic, sensory, and melanocyte progenitors delaminate from the thoracic neural tube (NT) in successive, largely non-overlapping waves and that at least certain NC progenitors are already fate-restricted within the NT. Whether sympathetic neurons and chromaffin cells, suggested by cell culture studies to share a common progenitor, are also fate segregated in ovo prior to emigration, is not known.
We have conducted single cell electroporations of a GFP-encoding plasmid into the dorsal midline of E2 chick NTs at the adrenomedullary level of the NC. Analysis of their derivatives, performed at E6, revealed that in most cases, labelled progeny was detected in both sympathetic ganglia and adrenal glands, where cells co-expressed characteristic marker combinations.
Our results show that sympathetic neurons and adrenal chromaffin cells share a common progenitor in the NT. Together with previous findings we suggest that phenotypic diversification of these sublineages is likely to occur after delamination from the NT and prior to target encounter.
KeywordsChicken embryo Chromaffin cells Neural crest Single cell electroporation Sympathetic neurons Sympathoadrenal progenitors
Green fluorescent protein
The neural crest (NC) is a transient structure in developing vertebrate embryos that arises at the interface between the epidermal and neural ectoderm. Upon neural tube (NT) formation, NC cells delaminate from its dorsal aspect and undergo an epithelial-to-mesenchymal transition followed by migration to their target locations. NC cells give rise to many different cell types, including neurons and glia of the autonomic and sensory nervous systems, neuroendocrine chromaffin cells in the adrenal medulla and other locations, thyroid C cells, melanocytes, and selected populations of mesenchymal cells . The sympathoadrenal (SA) cell lineage is a major sub-lineage of the NC that gives rise to sympathetic neurons, intra- and extra-adrenal chromaffin cells, and the small intensely fluorescent (SIF) cells, which are intermediate between sympathetic neurons and chromaffin cells [2–5]. SA derivatives share several features, including the ability to synthesize, store, and release catecholamines, but also exhibit traits specific for each cell type, such as axons, dendrites, and neurofilaments, which are only found in sympathetic neurons but not chromaffin cells.
After delaminating from the NT, the earliest emigrating NC cells migrate ventrally to the area of the dorsal aorta [6–8]. Here, they acquire catecholaminergic features, instructed by bone morphogenetic proteins 2/4/7, which are secreted by cells of the wall of the dorsal aorta [9, 10]. Furthermore, a series of interacting transcription factors including MASH-1, Phox2a, Phox2b, dHand, Gata2/3, and Insm1, were found to be essential for the generation of the SA catecholaminergic phenotype [11–19]; for reviews see [2–6, 20, 21].
Cell culture studies using isolated SA progenitor cells from embryonic and early postnatal sympathetic ganglia and adrenal gland have suggested that glucocorticoids play a crucial role in the diversification of SA cells into sympathetic neurons and chromaffin cells [22–24]; for reviews see [2, 5, 6, 25], by suppressing a neuronal differentiation programme in SA cells destined to become chromaffin cells. However, analysis of mice deficient for the glucocorticoid receptor  or lacking an adrenal cortex  revealed that chromaffin cell differentiation was largely unimpaired suggesting that cues unrelated to glucocorticoid receptor signalling and the adrenal cortex triggered the chromaffin phenotype. This notion was corroborated and extended by studies showing that chick SA progenitors are already heterogeneous in terms of neurofilament-M (NF-M) expression prior to populating the adrenal gland and sympathetic ganglia  with presumptive neuronal cells co-expressing tyrosine hydroxylase (TH) and NF-M, and presumptive neuroendocrine cells being TH-positive and NF-M-negative. This indicated that specification of the respective phenotypes occurred either during migration or even prior to migration at the level of the NT.
A recent study by Krispin et al. [7, 29] showed that in the trunk NT of avian embryos, precursors for sympathetic ganglia, Schwann cells, sensory dorsal root ganglionic neurons, and melanocytes leave the NT in successive, largely non-overlapping waves, some being already distinct while still residing in the NT. Although adrenal chromaffin cells were not addressed in this study, the results raised the possibility that sympathetic neurons and chromaffin cells might also already be distinct at the level of the NT. We therefore conducted single cell electroporations (EPs) of green fluorescent protein (GFP)-DNA into pre-migratory NC cells at the level of somites 18–24 (“adrenomedullary level”), where both chromaffin cells and sympathetic neurons arise . The timing and location of EPs was directed to label the earliest progenitors that undergo delamination, previously shown to generate the sympathetic lineage [7, 29], and the progeny of labelled cells was then analysed at E6 following homing to their target areas. Our results indicate that in more than 80% of the cases the progeny of a single labelled cell ends up in both sympathetic ganglia and adrenal gland, being mostly NF-M-negative in the adrenal gland and NF-M-positive in sympathetic ganglia. This suggests that chromaffin cells and sympathetic neurons share a common progenitor in the NT and that diversification of the phenotypes occurs during migration or assembly in primary sympathetic ganglia at the dorsal aorta.
Results and discussion
Transfection of GFP-encoding DNA into single dorsal NT progenitors
Distribution of the progeny of single NC progenitors following target organ colonization
Number of GFP+ cells per clone in the various derivatives
Altogether, our data suggest that a single NC progenitor residing in the NT before delamination gives rise to both chromaffin cells and sympathetic neurons. Our findings therefore strongly support the notion that sympathetic neurons and chromaffin cells still share a common progenitor in the dorsal NT prior to delamination.
Consistent with this result, additional NC-derived sublineages are likely to be segregated only after emigration. For example, neurons and glia of sensory ganglia become segregated within the DRG themselves by Notch-dependent lateral inhibition . A similar mechanism might account for segregation of SA progenitors as members of the Delta/Notch family are expressed in subsets of ventrally migrating SA cells .
Although growing evidence suggests that fate restrictions in the NC may occur prior to cell emigration [7, 29, 36–38], this notion should be systematically examined for specific derivatives by single cell lineage analysis in vivo at the appropriate time and axial location. In our study, this was done by performing single cell EP into the dorsal NT at the time and axial levels from which adrenal chromaffin and sympathetic neuronal progenitors delaminate. Single cell EP is an established method that allows the labelling of an individual cell in the midline of the NT before delamination. Although in ovo single cell EP is a technically demanding technique, with a success rate in our experiments of only 12%, we preferred EP over all other available methods, since it lacks the potential physical damage to the embryo produced by biolistic gene gun, potential toxic effects of viruses , and does not harm the morphology of the tissue . Notably, in our experiment, a clear GFP signal was already visible 3.5 h after EP, likely before most labelled cells underwent mitosis. Consistently, in 71% of cases, only one cell in the dorsal NT was detected, while 27.5% of cases revealed two cells. In the latter case, either two cells were originally electroporated, or a single cell was labelled shortly before undergoing cell division.
Taken together, our results suggest that sympathetic neuronal and chromaffin NC precursors are not pre-specified at the NT level favouring the option that specification may occur after delamination. Since neuronal and chromaffin progenitors can already be distinguished in their locations prior to populating the final target sites, sympathetic ganglia and adrenal gland, respectively, specification must occur during migration from the NT to the dorsal aorta or early in the peri-aortic area. Underlying mechanisms need to be investigated.
Fertilized white Leghorn chicken (Gallus gallus) eggs were from commercial sources (Haas, France). Embryos were staged according to Hamburger and Hamilton .
In ovo manipulations
An expression construct encoding an enhanced version of GFP, the pCAGGS-AFP (5 μg/μL) [42, 43] was microinjected into the NT at the level of somites 18–24 of 18–24 somite-stage embryos (HH13). Care was taken to label cells opposite recently segmented epithelial somites as the dorsal midline area of the NT at this stage harbours progenitors that mostly generate sympathetic cells [7, 29]. For focal EP of dorsal NT cells, a tungsten L-shape negative electrode was placed underneath the blastoderm. The positively charged electrode with the DNA-filled micropipette was inserted into the lumen of the NT. DNA at a concentration of 5 μg/μL was injected into the lumen and the micropipette was lifted up, until the tip of the micropipette touched the NT (Figure 1A, [7, 29]). Immediately after injection four electrical pulses of current at a voltage of 60 V for 15 ms were applied at 100 ms intervals using an ECM® 830 electroporator (BTX Harvard Apparatus). Single cell labelling was achieved when the resistance of the glass micropipette was 1.8–2.2 MΩ (measured after the pulse) using a Multimeter (Alcron). Embryos were re-incubated for 3.5 h to monitor initial expression of GFP by inspection under a stereomicroscope with fluorescent attachment (Zeiss). Eggs were then re-incubated for an additional 20–24 h after EP to confirm that the cell had delaminated from the NT. Successfully electroporated embryos were then re-incubated until E6.
Embryo processing and sectioning
Embryos were fixed in 4% formaldehyde overnight, washed twice in PBS, subsequently dehydrated in ascending concentrations of ethanol and acetone, and embedded in paraffin (Leica Biosystems). Paraffin blocks were cut into 10 μm thin sections on a microtome (Leica), and sections were mounted on Superfrost slides (Langenbrinck). Paraffin sections were deparaffinized and rehydrated before further processing.
Immunohistochemistry and in situ hybridization
Deparaffinized sections were immunolabelled with antibodies for GFP and TH combined with in situ hybridization for NF-M. A non-radioactive in situ hybridization for chick-specific neurofilament-M (NF-M) was performed . The riboprobes were diluted 1:10 in Tris/EDTA-buffer. Detection was performed with an anti-DIG antibody coupled to alkaline phosphatase and 4-nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP; Roche). Then, sections were processed for double-immunostaining with antibodies against TH and GFP. Before applying the primary antibody, antigen retrieval was performed to uncover the epitope of the TH antigen. Then, sections were incubated in blocking solution (0.5% Triton-100, 2% BSA, 1 x PBS) for 1 h. Next, they were incubated over night at 4°C with the primary antibodies to GFP (dilution 1:200; Molecular Probes-Invitrogen), and TH (dilution 1:400; Chemicon – Milipore). Secondary antibodies were: Cy3TM-568 goat anti-mouse (IgG) for detection of TH (dilution 1:400; Invitrogen). For visualization of GFP secondary Biotin-SP-conjugated Donkey anti-rabbit (IgG) antibody (dilution 1:100; Jackson ImmunoResearch) was applied for 1 h, then sections were washed with PBS. After washing, Cy2TM-conjugated streptavidine (dilution 1:200, Jackson ImmunoResearch) was applied for 1 h. Nuclei were counterstained with 4’,6’-diamidino-2-phenylindole dichloride (DAPI) (dilution 1:10,000 in 1xPBS, Boehringer). Sections were incubated for 5 min with DAPI, then washed in 1 x PBS and mounted with fluorescent-covering medium Fluoromount-G (Southern Biotech).
Data analysis and statistics
The in ovo-images were taken with a Zeiss camera equipped with the AxioVision programme, and images were saved in JPG or TIFF format. If necessary, brightness and contrast were adjusted to the entire image using AxioVision and Photoshop 5.0 (AdobeSystems, USA), merged images were prepared using GIMP 2.0. Analysis of paraffin sections was performed using a Zeiss Axiophot microscope. Confocal scanning was performed on 11 embryos, including Z-stacks and orthogonal projections. A Zeiss confocal microscope with a 364 nm laser, ZEN 2010 software and x63 objective (1.2 W) was used. Within the NT, a single GFP-labelled cell was apparent in one transverse section.
To evaluate a common progenitor for chromaffin cells and sympathetic neurons, as well as neuronal (NF-M-positive) or neuroendocrine (NF-M-negative) cells, the χ2 test was used. Analysis of cell numbers in sympathetic ganglia vs. adrenal gland was performed using the t-test (P = 0.5).
Chaya Kalcheim and Klaus Unsicker are joint senior authors.
We thank Ute Bauer, Günter Frank, Helmut Gerlach, Lidia Koschny, Ulla Hinz, and Ute Lausch for excellent technical assistance. We thank Dr Angela Naumann, Center for Biological Systems Analysis, for help with confocal microscopy. This work was supported by grants from Deutsche Forschungsgemeinschaft to K Unsicker and C Kalcheim (SFB 488 A6, and SFB 592 A23).
- LeDouarin NM, Kalcheim C: The Neural Crest. Cambridge: Cambridge University Press; 1999.View Article
- Huber K, Kalcheim C, Unsicker K: The development of the chromaffin cell lineage from the neural crest. Auton Neurosci 2009,151(1):10–16.PubMedView Article
- Langley K, Grant NJ: Molecular markers of sympathoadrenal cells. Cell Tissue Res 1999,298(2):185–206.PubMedView Article
- Unsicker K: The chromaffin cell: paradigm in cell, developmental and growth factor biology. J Anat 1993,183(Pt 2):207–221.PubMed
- Unsicker K, Huber K, Schütz G, Kalcheim C: The chromaffin cell and its development. Neurochem Res 2005,30(6–7):921–925.PubMedView Article
- Unsicker K, Huber K, Schober A, Kalcheim C: Resolved and open issues in chromaffin cell development. Mech Dev 2013, 130:324–329.PubMedView Article
- Krispin S, Nitzan E, Kalcheim C: The dorsal neural tube: a dynamic setting for cell fate decisions. Dev Neurobiol 2010,70(12):796–812.PubMedView Article
- Serbedzija GN, Bronner-Fraser M, Fraser SE: A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration. Development 1989,106(4):809–816.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(7):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(4):861–870.PubMedView Article
- Ernsberger U, Patzke H, Tissier-Seta J-P, 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(1):125–136.PubMedView Article
- 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(3):463–476.PubMedView Article
- Ernsberger U, Reissmann E, Mason I, Rohrer H: The expression of dopamine beta-hydroxylase, tyrosine hydroxylase, and Phox2 transcription factors in sympathetic neurons: evidence for common regulation during noradrenergic induction and diverging regulation later in development. Mech Dev 2000,92(2):169–177.PubMedView Article
- 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(18):4073–4081.PubMed
- Lucas ME, Muller F, Rudiger R, Henion PD, Rohrer H: The bHLH transcription factor hand2 is essential for noradrenergic differentiation of sympathetic neurons. Development 2006, 133:4015–4024.PubMedView Article
- Pattyn A, Morin X, Cremer H, Goridis C, Brunet JF: Expression and interactions of the two closely related homeobox genes Phox2a and Phox2b during neurogenesis. Development 1997, 124:4065–4075.PubMed
- 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.PubMedView Article
- Tsarovina K, Pattyn A, Stubbusch J, Müller F, Wees J, Schneider C, Brunet JF: Essential role of Gata transcription factors in sympathetic neuron development. Development 2004,131(19):4775–4786.PubMedView Article
- 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(3):473–481.PubMedView Article
- Huber K: The sympathoadrenal cell lineage: specification, diversification, and new perspectives. Dev Biol 2006,298(2):335–343.PubMedView Article
- Rohrer H: Transcriptional control of differentiation and neurogenesis in autonomic ganglia. Eur J Neurosci 2011,34(10):1563–1573.PubMedView Article
- Anderson DJ, Axel R: A bipotential neuroendocrine precursor whose choice of cell fate is determined by NGF and glucocorticoids. Cell 1986,47(6):1079–1090.PubMedView Article
- Michelsohn AM, Anderson DJ: Changes in competence determine the timing of two sequential glucocorticoid effects on sympathoadrenal progenitors. Neuron 1992,8(3):589–604.PubMedView Article
- Unsicker K, Krisch B, Otten U, Thoenen H: Nerve growth factor-induced fiber outgrowth from isolated rat adrenal chromaffin cells: impairment by glucocorticoids. Proc Natl Acad Sci U S A 1978,75(7):3498–3502.PubMedView Article
- Anderson DJ: Cell fate determination in the peripheral nervous system: the sympathoadrenal progenitor. J Neurobiol 1993,24(2):185–198.PubMedView Article
- 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(13):2935–2944.PubMed
- Gut P, Huber K, Lohr J, Brühl 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(20):4611–4619.PubMedView Article
- 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):1–13.PubMedView Article
- Krispin S, Nitzan E, Kassem Y, Kalcheim C: Evidence for a dynamic spatiotemporal fate map and early fate restrictions of premigratory avian neural crest. Development 2010, 137:585–595.PubMedView Article
- LeDouarin NM, Creuzet S, Couly G, Dupin E: Neural crest cell plasticity and its limits. Development 2004,131(19):4637–4650.View Article
- Huber K, Franke A, Brühl B, Krispin S, Ernsberger U, Schober A, Von Bohlen Und Halbach O, Rohrer H, Kalcheim C, Unsicker K: Persistent expression of BMP-4 in embryonic chick adrenal cortical cells and its role in chromaffin cell development. Neural Dev 2008, 3:28.PubMedView Article
- Unsicker K: Fine structure and innervation of the avian adrenal gland. I Fine structure of adrenal chromaffin cells and ganglion cells. Z Zellforsch Mikrosk Anat 1973, 145:389–416.PubMedView Article
- Unsicker K, Habura-Flüh O, Zwarg J: Different types of small granule containing cells and neurons in the guinea pig adrenal medulla. Cell Tissue Res 1978,189(1):109–130.PubMedView Article
- Vogel KS, Weston JA: The sympathoadrenal lineage in avian embryos. I. Adrenal chromaffin cells lose neuronal traits during embryogenesis. Dev Biol 1990,139(1):1–12.PubMedView Article
- Wakamatsu Y, Maynard TM, Weston JA: Fate determination of neural crest cells by NOTCH-mediated lateral inhibition and asymmetrical cell division during gangliogenesis. Development 2000,127(13):2811–2821.PubMed
- Anderson DJ: Genes, lineages and the neural crest: a speculative review. Philos Trans R Soc Lond B Biol Sc 2000,355(1399):953–964.View Article
- Dorsky RI, Moon RT, Raible DW: Environmental signals and cell fate specification in premigratory neural crest. Bioessays 2000,22(8):708–716.PubMedView Article
- Harris ML, Erickson CA: Lineage specification in neural crest cell path finding. Dev Dyn 2007,236(1):1–19.PubMedView Article
- Hass K, Sin WC, Javaherian A, Li Z, Cline HT: Single-cell electroporation for gene transfer in vivo. Neuron 2001, 29:583–591.View Article
- Brown CY, Eom DS, Amarnath S, Agarwala S: A simple technique for early in vivo electroporation of E1 chick embryos. Dev Dyn 2012,241(3):545–552.PubMedView Article
- Hamburger V, Hamilton HL: A series of normal stages in the development of the chick embryo 1951. Dev Dyn 1992,195(4):231–272.PubMedView Article
- Ben-Yair R, Kalcheim C: Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates. Development 2005,132(4):689–701.PubMedView Article
- Momose T, Tonegawa A, Takeuchi J, Ogawa H, Umesono K, Yasuda K: Efficient targeting of gene expression in chick embryos by microelectroporation. Dev Growth Differ 1999, 41:335–344.PubMedView Article
- 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(7):699–708.PubMedView Article
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