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The Dorsal Aorta Initiates a Molecular Cascade That Instructs Sympatho-Adrenal Specification

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Science  22 Jun 2012:
Vol. 336, Issue 6088, pp. 1578-1581
DOI: 10.1126/science.1222369

Master Regulator

Sympathetic neurons and the adrenal medulla are part of the autonomic nervous system, which is important in the control of a variety of bodily functions and in responses to stress. Interactions between the nervous system and the vascular system have been poorly explored. During development, sympathetic neurons and adrenal medulla cells are derived from the same precursors—neural crest cells—embryonic cohorts that undergo massive migration in the body. Using blood vessel–specific gene manipulation in chicken embryos, Saito et al. (p. 1578) revealed a role for the dorsal aorta in regulating the early migration of neural crest cells and later in development on the segregation of adrenal medulla and sympathetic neurons. The dorsal aorta expresses multiple soluble morphogenetic and growth factors that regulate complex morphogenesis in a spatiotemporal manner. Furthermore, in mice, the morphogenesis of the adrenal medulla was controlled both by the aorta and the adrenal cortex.

Abstract

The autonomic nervous system, which includes the sympathetic neurons and adrenal medulla, originates from the neural crest. Combining avian blood vessel–specific gene manipulation and mouse genetics, we addressed a long-standing question of how neural crest cells (NCCs) generate sympathetic and medullary lineages during embryogenesis. We found that the dorsal aorta acts as a morphogenetic signaling center that coordinates NCC migration and cell lineage segregation. Bone morphogenetic proteins (BMPs) produced by the dorsal aorta are critical for the production of the chemokine stromal cell–derived factor–1 (SDF -1) and Neuregulin 1 in the para-aortic region, which act as chemoattractants for early migration. Later, BMP signaling is directly involved in the sympatho-medullary segregation. This study provides insights into the complex developmental signaling cascade that instructs one of the earliest events of neurovascular interactions guiding embryonic development.

The autonomic nervous system (ANS) plays a pivotal role in a wide range of physiological functions, including homeostasis and stress defense, and dysfunction of the ANS leads to a variety of diseases (1). However, it remains largely unexplored how the ANS is established during development at the cellular and molecular levels.

The ANS consists of sympathetic and parasympathetic neurons, as well as stress defense tissues, including the adrenal gland. In the trunk of the early developing embryo, progenitors of sympathetic ganglion (Sg) emerge as a derivative of neural crest cells (NCCs), an embryonic progenitor cell population emigrating from the dorsal aspect of the neural tube (2). In a defined region along the anteroposterior (AP) body axis [somite pairs 18 to 24 in chicken embryos (3, 4)], Sg-progenitor cells also give rise to adrenal medulla (Am) cells, which later become associated with the adrenal cortex, a tissue of different origin. Thus, Sg cells and Am cells are produced from common progenitors (SA progenitors). The SA progenitors first migrate ventrally toward the dorsal aorta, the first-formed embryonic blood vessel, and subsequently, these cells segregate into the Sg and Am lineages in the para-aortic region (58). Thus, the dorsal aorta has long been anticipated to play a role in the regulation of sympatho-adrenal lineages (9). However, its mechanism of action remains elusive, mainly because it has been difficult to manipulate the dorsal aorta (blood vessel) and sympatho-adrenal lineages simultaneously and separately in the same embryo. We demonstrate here that the dorsal aorta acts as a morphogenetic center for SA migration and Sg-Am segregation, and bone morphogenetic proteins (BMPs) play critical roles for these activities.

Three cytokine families, BMPs, stromal cell–derived factor–1 (SDF1, a chemokine, also called CXCL12), and Neuregulin 1 [NRG1 of the epidermal growth factor (EGF) family] have been separately implicated in the generation of SA progenitors from naïve NCCs (6, 1012). We therefore asked if and how these factors were related to the function of the dorsal aorta. We found that BMP signals were widely received by the cells located in a para-aortic area. These cells included SA progenitors [NCC marker antibody HNK-1–positive] (Fig. 1A) (13), whose response to BMPs was revealed by their staining with antibody against phosphorylated Smad1, 5, and 8 (pSmad) (14, 15). The BMP ligands (BMP4 and BMP7), produced by the dorsal aorta (fig. S1), were critical for the SA progenitors to be properly positioned, as transfection with the BMP inhibitor Noggin cDNA specifically into the dorsal aorta extinguished the para-aortic pSmad signal and abrogated SA progenitors completely (Fig. 1, B to D, and G to I). This notion was further supported by unilateral overexpression of Noggin, which resulted in a unilateral abrogation of SA progenitors (fig. S2) (16). However, the effects of BMPs on the SA accumulation were not direct, because SA progenitors electroporated with a dominant-negative type of BMP receptor (DN-BMPR1A) (17) migrated almost normally toward the aorta (Fig. 1, L and M, and fig. S3), consistent with a previously reported mouse genetic study (18).

Fig. 1

SDF1 and NRG1, but not BMPs, act directly on migrating SA progenitors. (A) NC-derived sympathetic precursors (HNK-1 positive) were active for BMP signals (pSmad1, 5, and 8 positive). Diagram: DNA-transfection into the dorsal aorta and red fluorochrome (PKH26)–labeling of NCCs of a single embryo. Transfection with EGFP (B to F) or Noggin (G to K). Arrows (EGFP control) and arrowheads (Noggin-transfected) indicate corresponding regions. (L) Electroporation into NC. (M) Quantitative representation of the ratio of electroporated NCCs located in the para-aortic region (square) over the total number of electroporated NCCs. The data were obtained by those shown in fig. S3. Scale bars, 100 μm. NT, neural tube; n, notochord; DA, dorsal aorta.

We therefore investigated the roles of SDF1 and NRG1, previously shown to be distributed in the para-aortic mesenchyme (10, 11) (fig. S4). Our data strongly suggest that these cytokines are the direct mediators for SA-progenitor migration and/or distribution. Functional inhibition of their respective receptors, CXCR4 (fig. S4) or EGF family ErbBs, in NCCs by short hairpin RNA (19) or DN-type constructs (DN-ErbB4, p75KD-ErbB1) (20, 21) profoundly disturbed the SA cell accumulation in the para-aortic region (Fig. 1, L and M, and fig. S3). Simultaneous knockdown of these two receptors enhanced the phenotype (fig. S3). Furthermore, the para-aortic expression of SDF1 and NRG1 was extinguished either bilaterally or unilaterally by Noggin overexpression in the dorsal aorta or a unilateral site, respectively (Fig. 1, E, F, and J to K, and fig. S2). Thus, the aortic BMPs are required for the expression of SDF1 and NRG1 in the para-aortic mesenchyme, which in turn directly regulate SA cell morphogenesis.

Both in vivo and in vitro analyses further revealed that SDF1 and NRG1 act as chemoattractants for the SA progenitors. When an aggregate of NRG1- or SDF1-producing DF1 cells (chicken mesenchymal cell line) was implanted into a somitic area, an ectopic accumulation of neural crest (NC)–derived cells was observed near the implant (Fig. 2, A to F, and fig. S5, A and B). Such ectopic accumulation also occurred when a piece of dorsal aorta was transplanted into a similar region (fig. S6, A to C). In these cases, expression of SDF1 and NRG1 was up-regulated in mesenchymal cells near the graft (fig. S6, D and E). Implantation of BMP-producing DF1 cells resulted in neither up-regulation of SDF1 and NRG1 nor NCC accumulation (fig. S5, C and D). Together, we conclude that the dorsal aorta initiates the expression of SDF1 and NRG1, which in turn attract the SA progenitors. For these events, the aortic BMPs are required.

Fig. 2

SDF1 and NRG1 on SA progenitors act as chemoattractants. (A) SDF1- or NRG1-producing DF1 cells (EGFP-positive) were implanted into a somitic area of a st 13 embryo. (B to E) PKH26, HNK-1, and EGFP signals were visualized in the same histological section of st 23 embryos. Arrows, Ectopic accumulation of NCCs. Scale bar, 100 μm. (F) The number of NCCs found within a 20-μm distance from the implant, assessed with 50 histological sections obtained from 10 embryos for each manipulation (*P < 0.001). Error bars represent SEM. (G and H) In vitro live imaging and chemoattraction analyses of NCCs. Migratory tracks of individual NCCs exposed to a given factor, as shown in fig. S7, are represented by Chemotaxis Index (see supplementary material). Dots represent index of individual cells (*P < 0.05). Red bar, average index.

The direct attraction by SDF1 and NRG1 was further verified by in vitro time-lapse imaging analyses. A clump of NCCs prepared from early embryos was cocultured in Matrigel with an aggregate of SDF1- or NRG1-producing DF1 cells (Fig. 2G). The NCCs exhibited directional migration toward SDF1- or NRG1-producing cells (movies S2 and S3), but not to enhanced green fluorescent protein (EGFP)–positive or BMP4-producing cells (movies S2 and S4), although the NCCs maintained motility (Fig. 2H and fig. S7). Thus, SDF1 and NRG1, but not BMPs, act as chemoattractants on migrating SA progenitors. This work identifies how the dorsal aorta directs the migration of SA progenitors, showing that this morphogenetic event involves multiple temporally and spatially regulated factors.

We further explored the mechanisms underlying the late-occurring event, Sg-Am segregation from SA progenitors. To our surprise, upon reaching the aortic region, SA progenitors turned off the pSmad signal at embryonic stage (st) 21 (Fig. 3, A and B). Subsequently, the pSmad signal reappeared at st 23, but, this time, the pSmad labeling was seen only in the Am cells and not in the Sg cells (Fig. 3C). To determine whether this asymmetric distribution of BMP signaling actually would cause the Am-Sg segregation, we inhibited BMP signals specifically in these Am-Sg cells through tetracycline (tet)–controlled conditional expression (22, 23). Thus, the expression of tet-DN-BMPR1A was turned on at st 20 just before Am-Sg segregation (fig. S8A). Whereas control EGFP-electroporated cells were randomly distributed both in Sg and Am populations, DN-BMPR1A–expressing cells failed to participate in Am production (Fig. 3, D to F), which suggested that the BMP signaling is essential for the Am-Sg segregation. BMP ligands, BMP4 and BMP7, are most likely provided by the dorsal aorta (fig. S1, D and E). In support of this, tet-controlled Noggin expression in the dorsal aorta at st 20 disturbed Am cells, but not Sg cells, seen in both sides of the aorta (Fig. 3, G and H, and fig. S8, D to H). Unilateral transfection further confirmed this notion (fig. S8, I to L).

Fig. 3

BMPs and NRG1 determine the late event of Am-Sg segregation. (A to C) Double staining for NCCs (PHOX2B) and BMP signals. Solid and broken brackets indicate SA/Sg cells and Am cells, respectively. (D and E) Tet-induced expression of DN-BMPR1A into SA lineage (see fig. S8A for experimental design) abrogated Am cells. (F) The number of electroporated cells (EGFP+) located in Sg and Am populations. Error bars, SEM. (G to J) Tet-induced expression of EGFP or Noggin cDNAs transfected into the dorsal aorta (see fig. S8D). (K) NRG1 localization near Am cells at st 23. (L and M) DF1 cells, transfected with tet-inducible EGFP or NRG1 genes (green), were implanted into coelomic mesoderm at st 11 (arrows) (see fig. S8M for experimental design). (K′) and (M′) are magnified fields. Scale bars, 100 μm.

NRG1 was also important for the Am cells. NRG1 was localized along the migration pathway of Am cells, so that these cells were enclosed by NRG1-positive areas (Fig. 3K). NRG1-ErbBs signaling was necessary for proper Am-cell segregation, as SA progenitors expressing tet-DN-ErbB4 or tet-p75KD-ErbB1 failed to differentiate into Am cells (Fig. 3F and fig. S8, B and C).

To determine whether BMPs and NRG1 play distinct roles in Am morphogenesis, we placed a BMP4- or NRG1-producing cell aggregate into an ectopic site ventral to the aorta (fig. S8M) and found that the NRG1-aggregate, but not the BMP4, could attract Am cells to the ectopic site (Fig. 3, L and M, and fig. S8N). Thus, NRG1 and BMP4 exert different effects. It is noteworthy that BMPs are required for the NRG1 distribution, as tet-controlled Noggin cDNA overexpressed in the aorta extinguished the NRG1 signals along the migration pathway of Am cells (Fig. 3, I and J). Together, BMP4 and 7 signaling are required for both the Am segregation and NRG1 patterning, whereas NRG1, in turn, acts as an attractant for Am-cell displacement.

Most previous studies of Am morphogenesis focused on possible interactions between Am cells and adrenal cortex, because these two tissues become associated to constitute the adrenal gland (2426). However, even in the cortex-deficient mice produced by knocking out the gene for transcriptional factor steroid factor 1 (Sf1 or Ad4BP) (27, 28), about half the Am cells are present (29), which leaves the role of cortical cells undefined. Based on our observations (Fig. 3K), we reasoned that even when deprived of the cortex, NRG1 expression would remain in other areas and provide guidance cues for the Am cells. Indeed, in addition to the NRG1-positive cortex (Sf1+/BMP4+) (30), para-aortic mesenchyme was found to express NRG1 (Fig. 4, A and B).

Fig. 4

Am cells are guided by two distinct NRG1-positive domains. (A and B) Transverse views of Am cells near the dorsal aorta of chicken embryos. (A′ and B′) Magnified fields. Yellow arrows indicate cortex precursors (Sf1+/BMP4+). (C to H) Para-aortic regions in mouse wild type- and Sf1 KO embryos (E11.5). Yellow arrows show the areas that correspond to adrenal cortical precursors. White arrowheads and white arrows indicate Am cells and NRG-positive para-aortic mesenchyme, respectively. Scale bars, 100 μm.

To determine whether the NRG1 signal would remain near the Am cells when deprived of the cortex, we examined the para-aortic region of Sf1 knockout (KO) mice (Fig. 4, C and D) (28). The distribution patterns of pSmad proteins and NRG1 in wild-type mouse embryos at the time of Am-cell segregation from SA progenitors, embryonic day 11.5 (E11.5), were virtually the same as in the chicken embryo (Fig. 4, C, E, and G). In Sf1 KO mice at E11.5, a substantial number, although smaller than wild type, of Am cells were detected as previously reported (29). These cells exhibited pSmad1, 5, and 8 (Fig. 4F, white arrowhead). Furthermore, NRG1 was maintained near the Am cells in KO mice; the signal intensity in the para-aortic mesenchymal area (Fig. 4, G and H, white arrow) was comparable between KO and wild type, whereas the area that would normally be occupied by the cortex exhibited little signal in KO mice (Fig. 4, G and H, yellow arrow). These findings thereby explain how Am cells could survive in Sf1 KO mice: The Am cells can receive aortic BMP signals that allow them to be segregated from Sg cells, and these Am cells are guided by the NRG1 that remains in the para-aortic region (fig. S9).

In this study, we have unveiled the molecular and cellular mechanisms by which the sympathetic nervous system and stress defense organ are established (fig. S9). This occurs in the vicinity of the dorsal aorta, where multiple factors are repeatedly used in a manner regulated spatiotemporally by the dorsal aorta. The dorsal aorta controls the migration of early SA progenitors, Sg-Am segregation, and the ventral displacement of Am lineage. For these sequential events to proceed correctly, cells constantly change their responsiveness to factors they encounter (i.e., their BMP responsiveness). The dorsal aorta thus serves as a critical relay point to coordinate the diversification of sympathetic nervous system.

Supplementary Materials

www.sciencemag.org/cgi/content/full/336/6088/1578/DC1

Materials and Methods

Figs. S1 to S9

References (3237)

Movies S1 to S4

References and Notes

  1. Although it was previously shown that a Noggin-soaked bead implanted into a somite partially disturbed SA progenitors, this might be due to an incomplete inhibition of BMPs (31).
  2. Acknowledgments: We thank S. F. Gilbert, L. Niswander, G. Corfas, S. O. Yoon, and Y. Wakamatsu for experimental materials and discussions and K. Morohashi for providing us with Sf1 KO mice. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas, and Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT); the Global Centers of Excellence (GCOE) program (Frontier Biosciences, Nara Institute of Science and Technology) from MEXT, Japan; CREST (JST); The Mitsubishi Foundation; and Takeda Science Foundation. Y. Takase is a fellow of the Japan Society for the Promotion of Science.
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