Sympathetic neurons and chromaffin cells share a common progenitor in the neural crest in vivo

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Single-cell EP of GFP-DNA into the dorsal neural tube. (A) Diagram showing the two-step procedure for single-cell EP, beginning by plasmid microinjection and followed by application of ventro-dorsally directed electric pulses. Note position of electrodes, injection site, and position of single electroporated cell. (B,C) Live-visualization in ovo at 3.5 h (B) and 24 h (C) after EP. (D) Histological cross section showing confocal analysis of a single GFP-expressing cell in the dorsal NT 3.5 h after EP visualized with an anti-GFP-antibody, combined with DAPI nuclear staining (blue). D, D’, D”, and D”’ show four representative focal planes out of 23 planes from a Z-stack of a 10 μm thick section. Samples were optically screened at 0.35 μm increments. D”” shows orthogonal projections. (E) Summarizes results of 69 experiments, with 71% successful single-cell-EPs verified by immunohistology, and 27.5% of the cases, where two cells were seen. In 1.5% of the cases, GFP was visualized in three cells. Scale bar: 10 μm. NT, neural tube; S, somites.

Figure 2

Analysis of GFP-labelled cells in sympathetic ganglia (A,B) and adrenal gland (C,D) at E6. (A) Sympathetic ganglion (white demarcation) harbours two GFP-positive cells (green). (B) TH antibody staining of the same section as in (A). Arrows mark the two co-labelled cells. (C) Adrenal gland (white demarcation) harbours one GFP-positive cell (green). (D) TH antibody staining of the same section as in (C). Arrows point to the TH+/GFP-positive cells. Note the autofluorescence of red blood cells in dorsal aorta (C,D). (E) Distribution of GFP+/TH+ cells in sympathetic ganglia and adrenal gland at E6, following single cell EP into the dorsal NT at E2. Scale bars: 50 μm. N, notochord; DA, dorsal aorta.

Figure 3

Analysis of GFP-labelled cells derived from a single clone in a sympathetic ganglion (A-C) and adrenal gland (D-F) at E6 using antibodies to GFP (A,D), TH (B,E), and in situ hybridization for NF-M mRNA (C,F). (G) Distribution of GFP+/TH+/NF+ and GFP+/TH+/NF- cells in sympathetic ganglia (SG) and adrenal gland (AG) at E6. Scale bars: 50 μm. N, notochord: DA, dorsal aorta.