Schwann Cell Precursors Generate the Majority of Chromaffin Cells in Zuckerkandl Organ and Some Sympathetic Neurons in Paraganglia

Abstract

In humans, neurosecretory chromaffin cells control a number of important bodily functions, including those related to stress response. Chromaffin cells appear as a distinct cell type at the beginning of midgestation and are the main cellular source of adrenalin and noradrenalin released into the blood stream. In mammals, two different chromaffin organs emerge at a close distance to each other, the adrenal gland and Zuckerkandl organ (ZO). These two structures are found in close proximity to the kidneys and dorsal aorta, in a region where paraganglioma, pheochromocytoma and neuroblastoma originate in the majority of clinical cases. Recent studies showed that the chromaffin cells comprising the adrenal medulla are largely derived from nerve-associated multipotent Schwann cell precursors (SCPs) arriving at the adrenal anlage with the preganglionic nerve fibers, whereas the migratory neural crest cells provide only minor contribution. However, the embryonic origin of the ZO, which differs from the adrenal medulla in a number of aspects, has not been studied in detail. The ZO is composed of chromaffin cells in direct contact with the dorsal aorta and the intraperitoneal cavity and disappears through an autophagy-mediated mechanism after birth. In contrast, the adrenal medulla remains throughout the entire life and furthermore, is covered by the adrenal cortex. Using a combination of lineage tracing strategies with nerve- and cell type-specific ablations, we reveal that the ZO is largely SCP-derived and forms in synchrony with progressively increasing innervation. Moreover, the ZO develops hand-in-hand with the adjacent sympathetic ganglia that coalesce around the dorsal aorta. Finally, we were able to provide evidence for a SCP-contribution to a small but significant proportion of sympathetic neurons of the posterior paraganglia. Thus, this cellular source complements the neural crest, which acts as a main source of sympathetic neurons. Our discovery of a nerve-dependent origin of chromaffin cells and some sympathoblasts may help to understand the origin of pheochromocytoma, paraganglioma and neuroblastoma, all of which are currently thought to be derived from the neural crest or committed sympathoadrenal precursors.

Keywords: Schwann cell precursors; Zuckerkandl organ; catecholamines; extra-adrenal chromaffin cells; para-aortic sympathetic ganglia; posterior trunk sympathetic ganglia.

FIGURE 1

The sympathetic ganglia at the level of the Zuckerkandl organ and the organ itself have distinct early-defined origin despite the intermingling anatomy. (A) Dorsal view of whole-mount immunofluorescence (left panel) against the sympathetic marker CART, the chromaffin and sympathetic marker TH and NF200 (showing the innervation on the trunk of an E15.5 wild type embryo and schematic (right panel) showing the sympathetic and chromaffin structures in relation to the dorsal aorta. Note that the mesenteric (MG) and suprarenal ganglion (SRG), as well as the sympathetic chain (SC), are CART⁺, while the Zuckerkandl organ (ZO) is composed mainly by TH⁺/CART^- cells. Additionally note that the para-aortic ganglia (PAG) are the continuation of the sympathetic chain that extends along the anteroposterior axis of the embryo trunk just at the dorsal view of the dorsal aorta, at the level of the ZO and MG. (B,C) Immunofluorescence on cryosections against CART, Ret^TOM and TH on tamoxifen-injected (TAM-injected) embryos at E10.5 and E11.5 respectively shows that Ret^TOM specifically delineated the sympathetic compartment when analyzed at E15.5, with clear tracing of the MG and PAG, while only few Ret^TOM+ cells can be seen in the ZO. Note the difference in CART immunofluorescence levels in the MG and PAG. (D) Immunofluorescence on cryosections against ISL1, Ret^TOM and TH on TAM-injected embryos at E10.5 shows Ret^TOM specific expression by the sympathetic ganglion (SG) and SRG when analyzed at E15.5, while almost no Ret^TOM+ cells can be seen in the adrenal medulla (AM). (E) Ventral view of whole-mount immunofluorescence against CART, Ascl1^TOM and TH on embryos with TAM injection at E11.5 and analyzed at E13.5 shows tracing in the chromaffin cells of the ZO, while no tracing in the MG (shown by white arrowheads). Scale bar in (A–E) = 100 μm. A, anterior; P, posterior; AM, adrenal medulla; MG, mesenteric ganglion; ZO, Zuckerkandl organ; DA, dorsal aorta; SC, sympathetic chain; SRG, suprarenal ganglion; AG, adrenal gland; PAG, para-aortic ganglion; SG, sympathetic ganglion; ChCs, chromaffin cells; SNs, sympathetic neurons.)

FIGURE 2

The chromaffin cells of the Zuckerkandl organ are grossly Schwann-cell-precursor-derived, as well as a small portion of the posterior trunk sympathetic ganglia. (A) Side view of whole-mount immunofluorescence against Plp1^YFP, TH and CART on an E12.5 embryo which received a single tamoxifen (TAM) injection at E11.5, showing the sympathoadrenal (SA) primordium area. Note the big accumulation of Plp1^YFP+ cells ventrally to the first TH⁺ cells and the lack of Plp1^YFP+ cells at the more overlying CART⁺ chain corresponding to the sympathoblasts of the sympathetic chain. (B,C,E) Immunofluorescence on cryosections against Plp1^YFP, CART and TH on an E15.5 embryo injected with TAM at E11.5 and analyzed at E15.5. Note the Plp1^YFP+/TH⁺ cells in the Zuckerkandl organ (ZO), sympathetic para-aortic ganglion (PAG, shown by white arrowheads) and adrenal medulla (AM). (D) Upper panel: immunofluorescence against Plp1^YFP, SOX10 and CART on peripheral nerves of E15.5 Plp1^YFP+ embryos injected with TAM at E11.5. Lower: quantification of recombination efficiency in SOX10⁺ cells of peripheral nerves (corresponding to total peripheral glial cells) of embryos TAM-injected on E11.5 and analyzed at E15.5 (69.63 ± 8.69%, N = 4). Filled arrowheads show Plp1^YFP+/SOX10⁺ glial cells and empty arrowheads show Plp1^YFP-/SOX10⁺ glial cells. (F) Quantification of recombination in chromaffin and sympathetic population in embryos TAM-injected on E11.5 and analyzed at E15.5 (for the ZO 44.77 ± 6.44%, anterior PAG 4.84 ± 1.38%, posterior PAG 11 ± 2.80%, AM 37.69 ± 5.46%, in all cases N = 4). Data are presented as mean ± SEM. Note the high percentage of Plp1^YFP+/TH⁺ cells in the chromaffin organs ZO and AM and the difference in percentage of Plp1^YFP+/TH⁺ cells between the posterior and anterior PAG. Scale bar in (A) = 100 μm, in (B–E) = 50 μm. SA primordium, sympathoadrenal primordium; ZO, Zuckerkandl organ; PAG (ant), para-aortic ganglion (anterior); PAG (post), para-aortic ganglion (posterior); AM, adrenal medulla.

FIGURE 3

A chromaffin body located between the suprarenal and mesenteric ganglion is Schwann-cell-precursor-derived. (A) Immunofluorescence on cryosections against Plp1^YFP, CART and TH on an E15.5 embryo injected with tamoxifen (TAM) at E11.5 showing the sympathetic ganglion (SG), suprarenal ganglion (SRG) and mesenteric ganglion (MG). (B) Quantification of recombination in chromaffin and sympathetic population in embryos TAM-injected on E11.5 and analyzed at E15.5 (SG 2.72 ± 0.65%, SRG 2.35 ± 1.04%, MG 1.96 ± 0.81%, TCB 45.09 ± 4.90%, in all cases N = 4). Data are presented as mean ± SEM. In TCB vs. SG P = 0.0004, TCB vs. SRG P = 0.0003 and TCB vs. MG P = 0.0005. (C) Immunofluorescence on cryosections against Plp1^YFP, CART and TH on an E15.5 embryo injected with TAM at E11.5 at the area of transition from the SRG to the MG, showing the “transitional chromaffin body” (TCB). Note the differential CART and TH signal between the SRG and MG, as well as the Plp1^YFP+/TH^high cells in the TCB. Note the high percentage of Plp1^YFP+/TH⁺ cells in the TCB and the absence of Plp1^YFP+/TH⁺ cells in the SG, SRG and MG. Scale bar in (A,C) = 50 μm. SG, sympathetic ganglion; SRG, suprarenal ganglion; MG, mesenteric ganglion; TCB, transitional chromaffin body; ^∗∗∗P ≤ 0.001.

FIGURE 4

Ablation of Schwann cell precursors or the preganglionic nerves that serve as their route toward the dorsal aorta results in a reduction of chromaffin cell numbers of the Zuckerkandl organ. (A,B) Immunofluorescence on cryosections against CHAT (choline acetyltransferase) (upper panels) at the level of the spinal cord shows the absence of CHAT⁺ (cholinergic) preganglionic motorneurons in the gray matter, also seen as absence of ISL1⁺ motorneurons (lower panel). Scale bar = 100 μm. (C,D) Immunofluorescence against CART and TH at the level of the Zuckerkandl organ (ZO) shows a severely abnormal phenotype in the ZO and para-aortic ganglia (PAG) in Hb9^Cre/+;Isl2^DTA/+ E14.5 embryos in comparison to Isl2^DTA/+ control E14.5 embryos. Scale bar = 100 μm. (E) Quantification of TH⁺ cells at E14.5 in control Isl2^DTA/+ and mutant Hb9^Cre/+;Isl2^DTA/+ embryos shows a decrease in the ZO and PAG, while the mesenteric ganglia (MG) remain unaffected. In Isl2^DTA/+ versus Hb9^Cre/+;Isl2^DTA/+ respectively: total TH⁺ cells in ZO = 426.6 ± 52.66 vs. 201.9 ± 27.43 (P = 0.0194), in MG = 178.4 ± 21.44 vs. 198.4 ± 15.34 (P = 0.4889) and in PAG = 133.1 ± 9.57 vs. 83.22 ± 5.79 (P = 0.0112), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t-test. (F) Schematic showing the experimental design for induction of the visceral nerve ablation using the Hb9^Cre;Isl2^DTA strain. (G,H) Immunofluorescence against SOX10, TH and neurofilaments (NF) on cryosections from Sox10^CreERT2/+ and Sox10^CreERT2/+;R26^DTA/DTA E17.5 embryos following tamoxifen (TAM) injection at E11.5 and E12.5 and analysis at E17.5 showing almost complete Schwann cell precursor (SCP)-ablation and significantly abnormal morphology in ZO, PAG and MG. Scale bar = 100 μm. (I) Quantification of SOX10⁺ cells in control Sox10^CreERT2/+ and SCP-ablated Sox10^CreERT2/+;R26^DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10^CreERT2/+ vs. Sox10^CreERT2/+;R26^DTA/DTA respectively: total SOX10⁺ cells in ZO = 205.47 ± 28.96 vs. 5.33 ± 4.43 (P = 0.0024), in MG = 369.33 ± 27.56 vs. 5.55 ± 5.22 (P = 0.0002) and in PAG = 88.11 ± 6.46 vs. 3.24 ± 2.58 (P = 0.0002), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t-test. (J) Quantification of TH+ cells in control Sox10^CreERT2/+ and SCP-ablated Sox10^CreERT2/+;R26^DTA/DTA E17.5 embryos following TAM injection at E11.5 and E12.5 and analysis at E17.5. In Sox10^CreERT2/+ vs. Sox10^CreERT2/+;R26^DTA/DTA respectively: total TH⁺ cells in ZO = 588.08 ± 28.41 vs. 397.22 ± 23.69 (P = 0.0067), in MG = 243.66 ± 22.98 vs. 226.55 ± 15.24 (P = 0.5685) and in PAG = 103.00 ± 7.61 vs. 94.11 ± 8.04 (P = 0.4672), N = 3 per genotype. Data are presented as mean ± SEM and statistical analysis was performed using two-tailed Student t-test. (K) Schematic outlining the TAM injection times and collection time point in the Schwann cell precursor (SCP)-ablation experiment using the Sox10^CreERT2;R26^DTA strain. SC, spinal cord; GM, gray matter; MG, mesenteric ganglion; PAG, para-aortic ganglion; MNs, motorneurons; DA, dorsal aorta; ZO, Zuckerkandl organ; ns, non-significant; ^∗P ≤ 0.05; ^∗∗P ≤ 0.01; ^∗∗∗P ≤ 0.001.

FIGURE 5

Ascl1 is indispensable for differentiation of Schwann cell precursors toward chromaffin cells of the Zuckerkandl organ. (A,B) Immunofluorescence against CART, Ascl1^TOM and TH on cryosections of tamoxifen (TAM)-injected embryos at E10.5 and analyzed at E15.5 shows a decrease in TH⁺/Ascl1^TOM+ cells in the region of the Zuckerkandl organ (ZO) in mutant Ascl1^{CreERT2/CreERT2};R26^TOM/+ embryos in comparison to control Ascl1^CreERT2/+;R26^TOM/+ embryos, as well as increased CART⁺/Ascl1^TOM+ cell numbers (shown by white arrowheads) and absence of the para-aortic ganglia (PAG). (C) Immunofluorescence against S100B, Ascl1^TOM and SOX10 on cryosections of TAM-injected embryos at E10.5 and analyzed at E15.5 shows an accumulation of S100B⁺/SOX10⁺/Ascl1^TOM+ cells in the region of the ZO in mutant Ascl1^{CreERT2/CreERT2};R26^TOM/+ embryos that are absent in control Ascl1^CreERT2/+;R26^TOM/+ embryos. Scale bar in (A) and in upper panel of (C) = 100 μm, in (B) = 50 μm and in magnified insets in (C) = 25 μm. (D,E) Schematic depicting the main findings upon Ascl1 ablation and tracing of the Ascl1-progeny. The Ascl1^TOM lineage traced from E10.5 gives rise mainly to chromaffin cells (TH⁺) and sympathetic neurons (CART⁺) in control embryos. In contrast, in mutant embryos, the majority of Ascl1^TOM cells remains as glia (S100B⁺/SOX10⁺), with a small population of CART⁺ cells that is not seen in the control, and very few chromaffin cells (TH⁺) are produced. PAG, para-aortic ganglion; DA, dorsal aorta; ZO, Zuckerkandl organ; MG, mesenteric ganglion.

FIGURE 6

The development and anatomy of the Zuckerkandl organ and associated paraganglia in relation to the dorsal aorta. (A) Schematic showing the molecular differences and similarities between sympathetic neurons (SNs) and chromaffin cells (ChCs) during development in relation to the expression of CART, (ISL1 and TH. (B) Side-view of whole-mount immunofluorescence against smooth-muscle-actin (SMA, showing the dorsal aorta -DA), TH and CART on an E12.5 wild type embryo. Note the presence of TH⁺ cells at the dorsal part of the DA just posteriorly to its branching into the inferior mesenteric artery (shown by the white arrowhead). (C) Immunofluorescence on E12.5 wild type embryos against SOX10 and TH (left panel) and SOX10, CART and ISL1 (right panel) showing the close proximity of the developing CART^-/ISL1⁺/TH⁺ Zuckerkandl organ (ZO) and CART⁺/ISL1⁺/TH⁺ mesenteric ganglion (MG). (D) Ventral-view of whole-mount immunofluorescence against NF200, TH and CD31 (showing the endothelium of the DA) on an E12.5 wild type embryo. Note the presence of the ZO below the inferior mesenteric artery (shown by the white arrowhead), which is surrounded by NF200⁺ axons. (E) Immunofluorescence on cryosection of an E13.5 wild type embryo against NF200, TH and CART. Note the innervation pattern around the DA and the TH+ cells of the ZO. (F) Ventral-view of whole-mount immunofluorescence against SMA, TH and CART on an E13.5 wild type embryo. Note the close proximity of the MG and ZO just posteriorly to the inferior mesenteric artery and the separation of the two structures based on the CART⁺/TH^low immunoreactivity in the MG in contrast to the TH^high immunoreactivity of the ZO (shown by the white arrowhead). Also note the presence of TH^high chromaffin structures in close proximity to the MG and ZO (shown by yellow arrows). (G) Immunofluorescence against CART, TH and DAPI on cryosections of a wild type E17.5 embryo, clearly showing the composite nature of the ZO at this later developmental stage, with both TH⁺/CART^- and TH⁺/CART⁺ cells. Scale bar = 100 μm. SNs, sympathetic neurons; ChCs, chromaffin cells; SC, sympathetic chain; DA, dorsal aorta; PAG, para-aortic ganglion; ZO, Zuckerkandl organ; AM, adrenal medulla; MG, mesenteric ganglion.)