ANNALS or ANATOMY =========

Generation of cell diversity in the peripheral autonomic nervous system: The sympathoadrenal cell

lineage revisited*

K. Unsicker, s. Finotto, and K. Kriegistein

Department of Anatomy and Cell Biology (Neuroanatomy), University of Heidelberg, 1m Neuenheimer Feld 307, D-69120 Heidelberg, Germany

Summary. Based on recent evidence from in vitro and gene knock-out/knock-in studies this short review sum­marizes the molecular scenario underlying the develop­ment of autonomic neurons from the neural crest. The focus is on the sympathoadrenal (SA) cell lineage. While migrating ventrally precursors of this cell lineage are ex­posed to signals from notochord/ventral neural tube probably including the protein sonic hedgehog. These and signals in the region of the dorsal aorta (members of the family of bone morphogenetic proteins), where SA progenitor cells subsequently assemble, are essential for the induction of the adrenergic phenotype. SA progenitor cells subsequently differentiate into paravertebral and prevertebral sympathetic neurons, intra- and extra-adre­nal chromaffin cells and intermediate SIF (small inten­sely fluorescent ) cells. Based on in vitro studies with isolated SA and chromaffin progenitor cells glucocorti­coids have been claimed as essential for suppressing a neuronal commitment and channeling SA cells towards the chromaffin phenotype. Unexpectedly, mice deficient for a functional glucocorticoid receptor possess the full complement of adrenal chromaffin cells at birth. We pre­sent a hypothetical scenario consistent with these data, in which chromaffin cell development would be the default pathway in the SA cell lineage, while development into a neuronal direction requires specific growth factor signal­ing, which is probably distinct for paravertebral and pre­vertebral sympathetic neurons.

* Main lecture at the Bipartite Meeting of the Anatomische Ge­sellschaft and the Polish Anatomical Society, Olsztyn, Poland, May 24--May 27,1997 Correspondence to: K. Unsicker

Ann Anat (1997) 179: 495-500

© Gustav Fischer Verlag

Key words: Neural crest - Sympathetic neurons - Chro­maffin cells - Glucocorticoids

1. The neural crest - a model for studying the development of neuron diversity

One of the major goals in developmental biology is to un­derstand the mechanisms that generate cell diversity. Sig­nificant progress has been made in the past few years in revealing how extracellular signals and cell-intrinsic de­velopmental programs synergize to create the wide spec­trum of different cell types originating from the vertebrate neural crest. The neural crest, a transient structure that emerges from the dorsal surface of the em­bryonic neural tube, gives rise to a large variety of differ­ent cell types, including peripheral neurons and glial cells, melanocytes, chromaffin cells, thyroid C-cells, vascular smooth muscle and cells forming chondrocytes and bone (Le Douarin 1982; Bronner-Fraser and Fraser 1991). Neu­rons originating from the crest are extremely diverse comprising the distinct phenotypes of sensory and auto­nomic ganglionic neurons as well as the endocrine chro­maffin cells and small intensely fluorescent (SIP) cells of sympathetic ganglia. Thus, the crest also represents an at­tractive system for studying cell-extrinsic and cell-intrin­sic determinants of neuronal identity. From recent molecular and reverse genetic analyses a picture has started to emerge describing the role of genes that are important in the determination of the distinct phenotypes of peripheral neurons (see Groves and Anderson 1996, for a review).

2. The sympathoadrenal cell lineage: signals from notochord/ventral neural tube and dorsal aorta induce specific markers

The sympathoadrenal (SA) cell lineage represents a ma­jor sublineage of autonomic neurons which gives rise to sympathetic neurons, intra- and extra-adrenal chromaffin cells, and SIF cells. The SA cell lineage is one of the most extensively studied lineages in the neural crest (see An­derson 1993; Unsicker 1993, for reviews). The occurrence of both neurons and endocrine cells as well as intermedi­ate neuroendocrine phenotypes in the progeny of the SA cell lineage suggests underlying actions of molecular de­terminants that may be distinct, in part, from molecular cues generating the progeny of other autonomic neuronal sublineages, which lack endocrine phenotypes, as e. g. en­teric and parasympathetic neurons.

While migrating ventrally, precursors of sympathetic neurons and chromaffin cells are exposed to signals from the notochord, ventral neural tube and somitic mesoderm that are essential for adrenergic differentiation. Subse­quently, SA cells settle in the vicinity of the dorsal aorta, which also provides important signals for the induction of adrenergic differentiation. "Adrenergic" literally de­lineates the capacity of SA cells to synthesize adrena­line, but in the present context is meant to define cells synthesizing adrenaline (about 80% of all intra-adre­nal chromaffin cells in mouse and rat) or noradrenaline (the vast majority of postganglionic sympathetic neurons and about 20% of the intraadrenal chromaffin cells as well as SIF cells). The most widely used marker for moni­toring adrenergic differentiation is tyrosine hydroxylase (TH), the enzyme catalyzing the rate-limiting step in ca­techolamine synthesis (cf. Ernsberger et al. 1995). An­other marker that is common to all catecholaminergic cells, is the transcription factor Phox2 (Valarche et al. 1993). The essential role of notochord/ventral neural tube factors for SA adrenergic differentiation has been well documented by in vivo ablation and in vitro studies (Co­hen 1972; Norr 1973; Teillet and Le Douarin 1983; Ho­ward and Bronner-Fraser 1985, 1986). Interestingly, absence of notochord/ventral neural tube does not inter­fere with migration of crest cells to the dorsal aorta , although the acquisition of adrenergic properties, once crest cells have reached the dorsal aorta, is fully pre­vented (Groves et al. 1995). Crest cells condensing at the dorsal aorta also express MASH-1, a mammalian homo­logue of the Drosophila achaete-scute gene complex. In Drosophila these genes code for basic-loop-helix tran­scription factors with roles in specifying a cell lineage from which external sense organs are derived. MASH-1 is transiently expressed in all crest cells destined to be­come autonomic neurons, as they aggregate at sites of ganglionic primordia (Guillemot and Joyner 1993; Guille­mot et al. 1993). Consequently, mice carrying a null muta­tion for MASH-1 show a virtually complete loss of sympathetic (and parasympathetic) neurons (Guillemot

et al. 1993). Interestingly, chromaffin cells do not seem to be affected, although chromaffin cell progenitors both outside and inside the adrenal anlagen also express MASH-1 for a brief period of time. Shortly before the appearance of MASH-1 crest cells adjacent to the dorsal aorta express c-ret (Pachnis et al. 1993), the tyrosine ki­nase receptor for the glial cell line-derived neurotrophic factor (GDNF). c-ret-mediated signaling seems to be es­sential for the development of those sympathetic neurons that form the superior cervical ganglion, as suggested by the c-ret and GDNF knockouts (Schuchardt et al. 1994; Moore et al. 1996; Pichel et al. 1996; Sanchez et al. 1996). Sympathetic neurons in other sympathetic ganglia are ap­parently not affected.

Identification of signaling molecules from notochord! ventral neural tube and dorsal aorta that trigger adrener­gic properties in crest cells has significantly progressed in the past three years (Fig. 1). Sonic hedgehog, a protein in­volved in the formation of motoneurons (Roelink et al. 1994) and midbrain dopaminergic neurons (Hynes et al. 1995), is a likely candidate for the notochord!ventral neural tube "adrenergic" factor. Bone morphogenetic proteins (BMPs) 4 and 7 are signals produced by the dor­sal aorta at critical times during sympathetic neuron (and probably also chromaffin cell) differentiation and have the ability to enhance development of TH-positive crest­derived cells in cultures and when ectopically expressed in the developing embryo (Reissmann et al. 1996; Shah et al. 1996).

3. Factors for making distinct cell phenotypes in the SA cell lineage: Is glucocorticoid signal­ing essential for chromaffin cell development?

It is widely assumed but not proven that cells of the SA cell lineage that have arrived in the vicinity of the dorsal aorta are homogeneous and do not represent distinct phenotypes of sympathetic neurons and chromaffin cells or SIP cells, respectively. What, then, triggers differentia­tion of morphological and chemical features that distin­guish sympathetic neurons from chromaffin cells? In vitro studies dating back to the late 1970ies that used isolated adrenal chromaffin progenitor cells of various develop­mental ages had suggested that glucocorticoid hormones were essential for preventing neuronal differentiation and triggering development of morphological and chemical features that are characteristic for the chromaffin cell phenotype (Unsicker et al. 1978, 1985; Seidl and Unsick­er 1989 a, b; Doupe et al. 1985 a, b). These features in­clude, in particular, the lack of neurites and the presence of large chromaffin secretory granules. Synthesis of adre­naline mediated through the enzyme phenylethanolamine N-methyltransferase (PNMT) occurs in most, but not all chromaffin cells. PNMT expression is not unique for a subpopulation of chromaffin cells, but occurs in subpopu-

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lations of avian sympathetic neurons, too (Teitelman et al. 1984). This suggests that expression of PNMT in a SA cell is not per se indicative of chromaffin cell differentia­tion. This implies that SA cells selected for this marker do not represent the full set of chromaffin cell pheno­types.

Differentiation of sympathetic neurons could be achieved in cultures of prenatal or early postnatal chromaffin cells by stimulating the cells with nerve growth factor (NGF), ciliary neurotrophic factor (CNTF; Unsicker et al. 1985), or fibroblast growth factors (FGF)-l or -2 (Unsicker and Westermann 1992; Claude et al. 1988; Stemple et al. 1988). Thus, it appeared that glucocorticoid hormones, probably provided by the adre­nal cortex, were responsible for the differentiation of SA progenitor cells towards chromaffin cells inside and in the vicinity of the embryonic adrenal gland. In contrast, SA progenitor cells in sympathetic ganglionic anlagen were assumed to be directed towards sympathetic neu­rons by local growth factor, putatively members of the FGF family. Detailed additional analyses carried out in the 1980ies mainly by Paul Patterson's and David Ander­son's groups at Caltech seemed to confirm and extend the concept of the antagonistic actions of glucocorticoids and certain growth factors in triggering differentiation of chromaffin cells and sympathetic neurons, respectively, from a common SA progenitor cell (Doupe et al. 1985 a, b; Michelsohn and Anderson 1992; Anderson 1993; Stem­ple and Anderson 1993). The principal message that ar­ose from these studies was that glucocorticoid hormones were essentially required for two steps in chromaffin cell development, first, to suppress neuronal differentiation, and second, to induce PNMT. Actions of glucocorticoids could be inhibited by glucocorticoid receptor (GR) blockers (Michelsohn and Anderson 1992) suggesting that these actions were specific and mediated by the GR.

Mice generated in Gunther Schutz's laboratory at the German Cancer Research Center in Heidelberg that are deficient for the GR have recently permitted for the first time with an in vivo approach to challenge the dogma of the essential role of glucocorticoid signaling for the dif­ferentiation of chromaffin cells (Cole et al. 1995). GR-/­mice die shortly after birth, probably because of insuffi­cient maturation of their lungs. Their adrenal glands are larger than those of wildtype littermates. This increase in adrenal size is entirely due to an enlargement of the adre­nal cortex, which is reflected by an enormous rise in glu­cocorticoid hormones, ACTH, and increased proliferation of adrenocortical cells. Newborn GR deficient mice have adrenal chromaffin cells, which are ultrastructurally indis­tinguishable from adrenal chromaffin cells of wildtype lit­termates, and clearly distinct from sympathetic neurons. They express TH/Phox2 and lack neuronal markers, as e. g. neurofilament and peripherin. Together, these data suggest that chromaffin cells arise from progenitor cells even in the absence of glucocorticoid signaling. Although chromaffin cells in GR knockouts do not form a central, solid adrenal medulla and appear to be somewhat

dispersed between cortical cells, their numbers, as re­vealed by counting TH-positive and Phox2-positive cells, are not decreased. Their irregular distribution in the cen­ter of the adrenal gland is probably due to the expansion and proliferative activity of the inner layers of the adre­nal cortex. As expected, based on Wurtman's and Axelrod's early studies on the regulation of adrenaline synthesis in mammals (Wurtman and Axelrod 1965) chro­maffin cells of GR-/- mice lack PNMT and adrenaline. Taken together, the GR knockout phenotype suggests that GR-mediated glucocorticoid signaling is not an ab­solute prerequisite for chromaffin cell development. The lack of PNMT in adrenal chromaffin cells of these ani­mals is the only deficit that has been detected so far. In­terestingly, regulation of PNMT in sympathoadrenal cells by glucocorticoids is a feature that seems to be specific for mammals. In many submammalian vertebrates, as e. g. birds and amphibians, PNMT occurs in postganglionic sympathetic neurons as well as in chromaffin cells (Holz­bauer and Sharman 1972; Teitelman et al. 1984). The en­zyme is not down-regulated following hypophysectomy, nor can its activity be enhanced by glucocorticoids (Wurtman et al. 1968). One would therefore predict that adrenal chromaffin cells of lower vertebrates deprived of glucocorticoid signaling would maintain both their mor­phological phenotype that distinguishes them from sym­pathetic neurons and their capacity to synthesize adrenaline.

Nevertheless, careful reviewers might wish to apply the general caveat that applies to all gene knockouts, the possibility that compensations might have occurred. Although this is an important point and can not be abso­lutely excluded, the massive deficits in mice lacking a functional GR, such as the losses of glucocorticoid-de­pendent functions of lung and liver (cf. Cole et al. 1995), suggest that other steroid hormones, as e. g. progesterone, or GR-independent glucocorticoid effects (if they exist) cannot compensate for GR signalling in these mice.

4. A new putative scenario for the develop­ment of the SA ceUlineage

Two important questions must be asked following analy­sis of the GR knockout: (i) what, in terms of data and experimental designs, con­

tributed to the conceptualization of an essential role of glucocorticoids in chromaffin cell development, and

(ii) what may be the correct scenario of molecular cues triggering chromaffin as opposed to sympathetic neu­ron development?

Certainly, the relationship of an adrenal cortex and centrally located chromaffin adrenal medullary tissue found in mammalian adrenal glands has always provided a strong bias towards steroid hormones specifying the

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chromaffin cell phenotype. This bias was established and has continued to exist despite the fact that in mammals as well as in submammalian vertebrates substantial amounts of chromaffin tissue develop outside adrenal glands. Extra-adrenal chromaffin cells are widely distrib­uted in the body of vertebrates (Kohn 1902; Coupland 1965; Burnstock and Costa 1975; Bock 1982). Many are concentrated in the pre-aortic region ("organ of Zucker­kandl"), smaller groups and single cells are associated with sympathetic, parasympathetic, and sensory ganglia, vagus nerve, heart, blood vessels, carotid and pulmonary bodies, or found in the orbital cavity, just to name a few locations. In many species of cartilaginous and bony fish chromaffin tissue is fully separated from interrenal (i. e. the equivalent of adrenocortical) tissue and, despite this separation, synthesizes substantial amounts of adrenaline (Bachmann 1954; Bock 1982). Together, these "experi­ments provided by nature" suggest that glucocorticoids may not represent the essential signal for chromaffin cell development.

In vitro experiments with SA and chromaffin progeni­tor cells of different developmental ages isolated with the help of monoclonal antibodies or mechanically have con­tributed in the past to substantiate the notion of an ess­ential role of glucocorticoids in chromaffin cell development. In our experiments (cf. Seidl and Unsicker 1989 b; Unsicker et al. 1978, 1985), most (i. e. > 95%) chromaffin (progenitor) cells cultured from prenatal (E16 and E20) and postnatal (usually P6-P12) rat adrenal glands maintained the rounded or slightly flattened phe­notype typical of chromaffin cells and the typical ultra­structural features of chromaffin cells including the large dense-cored vesicles. Dexamethasone, a synthetic gluco­corticoid hormone, significantly improved their survival, but was not necessary to suppress a neuronal phenotype, i. e. the outgrowth of neurites. However, when neuritic growth was elicited by stimulation with growth factors, e. g. by treatment with the conditioned medium from C6 • glioma cells, glucocorticoids were required to suppress the neuronal morphology. If this in vitro performance of early chromaffin progenitors (which, admittedly, are not SA progenitor cells any more) reflects the in vivo reality, the following hypothetical scenario for the initial commit­ment of SA progenitor cells towards sympathetic neurons and chromaffin cells, respectively, could be envisaged (Fig. 1).

SA progenitor cells assembling in sympathetic ganglia primordia encounter BMP417 and neuritogenic/neuronal phenotype inducing stimuli (e. g. by members of the FGF family or other growth factors). The same andlor other factors would be implicated in the regulation of their pro­liferation, survival, and induction of NGF dependence. NGF dependence is a feature typical of paravertebral sympathetic neurons. Prevertebral sympathetic neurons and "short-axon" sympathetic neurons, which have their cell bodies located close to their innervation territories, are either less sensitive or not sensitive at all to NGF withdrawal ("immunosympathectomy"; cf. review by

Thoenen 1972) suggesting that distinct factors may regu­late the early differentiation of "long-axon" and "short­axon" sympathetic neurons. Concerning NGF depend­ence chromaffin cells resemble "short-axon" sympathetic neurons. Mice deficient for the NGF receptor trkA have a largely normal adrenal medulla (Schober et al. 1997) consistent with a late beginning of trkA expression by chromaffin cells in vivo after birth.

Neural Tube

Floor Plate

Notochord

SympathetiC Primordia

Dorsal Aorta

Pre-aortic Sympathetic Celis

Adrenal Primordia

Fig. 1. Migratory routes of neural crest cells destined to become sympathoadrenal progenitor cells and the sequential determina­tion of their TH-lPhox2-positive phenotype by factors from no­tochord/ventral neural tube (sonic hedgehog, shh, and possibly additional factors) and dorsal aorta (members of the bone morphogenetic proteins, BMP). Once SA progenitor cells have arrived in the vicinity of the dorsal aorta, distinct factor combi­nations must act to trigger the phenotypic development of the different cellular derivatives. Paravertebral sympathetic neurons are located dorsolaterally of the dorsal aorta and are NGF-de­pendent. Hypothetical factors triggering their phenotype include members of the fibroblast growth factor family (FGF) and possi­bly unknown factors (X). Pre-aortic and "short axon" sympa­thetic neurons are largely NGF-independent (triggered by unknown factors, X). Intra- and extra-adrenal chromaffin cells are NGF-independent for most of their functions. Their pheno­type has been claimed to be essentially dependent on the actions of glucocorticoid hormones. However, this view is hard to be maintained given the presence of normal numbers of differen­tiated adrenal chromaffin cells in mice lacking a functional glu­cocorticoid receptor (see text).

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SA progenitor cells migrating into the adrenal anlagen would initially be exposed to BMP417 thus completing their "adrenergic" commitment, but would subsequently not be triggered by factors inducing a neuronal pheno­type. The adrenal cortex would replace the sympathetic ganglionic environment as a local source of survival promoting factors (which remain to be identified, and as indicated by the GR-knockout, would not be glucocorti­coids). Massive death of extra-adrenal chromaffin cells after birth (cf. Bock 1982) probably reflects their failure to recruit a trophic support. In this scenario, glucocorti­coid hormones would not act as suppressors of a neuronal phenotype. Rather would their role be restricted to the modulation of the activity of particular genes, some of which are associated with the "adrenergic" phenotype, as e. g. PNMT and TH. In such a model, chromaffin cells would represent the default pathway of SA cell develop­ment. A "default status" of chromaffin cells would also be consistent with the fact that mature chromaffin and chromaffin progenitor cells can be induced to a neuronal phenotype when stimulated with adequate growth factors, while, vice versa, sympathetic neurons cannot be switched to a chromaffin phenotype. Certainly, this hypothetical model of SA progenitor development needs to be sup­ported by more experiments, preferably not in vitro.

We consider it to be a likely possibility that in vitro conditions chosen for studying isolated SA progenitor cells have contributed to the possibly erroneous conclu­sion that glucocorticoids are essential for chromaffin de­velopment. If the isolated SA cells had received a neuronal priming prior to isolation and/or were main­tained in vitro in the presence of serum factors with such a capacity, glucocorticoids were in fact necessary in vitro to suppress the neuronal commitment, just as glucocorti­coids are necessary to suppress the phenotypic switch of chromaffin progenitor cells stimulated with neurite growth inducing factors in vitro.

Thus, it appears that some of the basic issues in SA progenitor cell development are still far from being clari­fied, and, maybe, even more uncertain than it seemed 20 years ago.

Acknowledgement. Work from our laboratory referred to in this article was supported by the German Research Foundation, most recently by Sonderforschungsbereich 317.

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Accepted June 27, 1997

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