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The molecular mechanisms regulating sympathetic innervation of the heart during embryogenesis and its importance for cardiac development and function remain to be fully elucidated. We generated mice in which conditional knockout (CKO) of the Hif1a gene encoding the transcription factor hypoxia-inducible factor 1α (HIF-1α) is mediated by an Islet1-Cre transgene expressed in the cardiac outflow tract, right ventricle and atrium, pharyngeal mesoderm, peripheral neurons, and hindlimbs. These Hif1aCKO mice demonstrate significantly decreased perinatal survival and impaired left ventricular function. The absence of HIF-1α impaired the survival and proliferation of preganglionic and postganglionic neurons of the sympathetic system, respectively. These defects resulted in hypoplasia of the sympathetic ganglion chain and decreased sympathetic innervation of the Hif1aCKO heart, which was associated with decreased cardiac contractility. The number of chromaffin cells in the adrenal medulla was also decreased, indicating a broad dependence on HIF-1α for development of the sympathetic nervous system., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The X-zone is a transient cortical region enriched in eosinophilic cells located in the cortical-medullary boundary of the mouse adrenal gland. Similar to the X-zone, the fetal zone in human adrenals is also a transient cortical compartment, comprising the majority of the human fetal adrenal gland. During adrenal development, fetal cortical cells are gradually replaced by newly formed adult cortical cells that develop into outer definitive zones. In mice, the regression of this fetal cell population is sexually dimorphic. Many mouse models with mutations associated with endocrine factors have been reported with X-zone phenotypes. Increasing findings indicate that the cell fate of this aged cell population of the adrenal cortex can be manipulated by many hormonal and nonhormonal factors. This review summarizes the current knowledge of this transient adrenocortical zone with an emphasis on genes and signaling pathways that affect X-zone cells.

Adrenal chromaffin cells and sympathetic neurons synthesize and release catecholamines, and both cell types are derived from neural crest precursors. However, they have different developmental histories, with sympathetic neurons derived directly from neural crest precursors while adrenal chromaffin cells arise from neural crest-derived cells that express Schwann cell markers. We have sought to identify the genes, including imprinted genes, which regulate the development of the two cell types in mice. We developed a method of separating the two cell types as early as E12.5, using differences in expression of enhanced yellow fluorescent protein driven from the tyrosine hydroxylase gene, and then used RNA sequencing to confirm the characteristic molecular signatures of the two cell types. We identified genes differentially expressed by adrenal chromaffin cells and sympathetic neurons. Deletion of a gene highly expressed by adrenal chromaffin cells, NIK-related kinase, a gene on the X-chromosome, results in reduced expression of adrenaline-synthesizing enzyme, phenyl-N-methyl transferase, by adrenal chromaffin cells and changes in cell cycle dynamics. Finally, many imprinted genes are up-regulated in chromaffin cells and may play key roles in their development.

The observation of two precursor groups of the early stem cells (Groups I and II) leads to the realization that a first amount of fetal stem cells (Group I) migrate from the AMG (Aortal-Mesonephric-Gonadal)-region into the aorta and its branching vessels. A second group (Group II) gains quite a new significance during human development. This group presents a specific developmental step which is found only in the human. This continuation of the early development along a different way indicates a general alteration of the stem cell biology. This changed process in the stem cell scene dominates the further development of the human stem cells. It remains unclear where this phylogenetic step first appears. By far not all advanced mammals show this second group of stem cells and their axonal migration. Essentially only primates seem to be involved in this special development.

Adrenaline is a fundamental circulating hormone for bodily responses to internal and external stressors. Chromaffin cells of the adrenal medulla (AM) represent the main neuroendocrine adrenergic component and are believed to differentiate from neural crest cells. We demonstrate that large numbers of chromaffin cells arise from peripheral glial stem cells, termed Schwann cell precursors (SCPs). SCPs migrate along the visceral motor nerve to the vicinity of the forming adrenal gland, where they detach from the nerve and form postsynaptic neuroendocrine chromaffin cells. An intricate molecular logic drives two sequential phases of gene expression, one unique for a distinct transient cellular state and another for cell type specification. Subsequently, these programs down-regulate SCP-gene and up-regulate chromaffin cell-gene networks. The AM forms through limited cell expansion and requires the recruitment of numerous SCPs. Thus, peripheral nerves serve as a stem cell niche for neuroendocrine system development., (Copyright © 2017, American Association for the Advancement of Science.)

Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca(2+) and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca(2+) buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca(2+) buffering during cell activation. We found that the clearance of cytosolic Ca(2+) transients ([Ca(2+)]c) elicited by high K(+) was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca(2+)]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca(2+) buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K(+)-elicited [Ca(2+)]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K(+)-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca(2+) and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.

The development of sympathetic neurons and chromaffin cells is differentially controlled at distinct stages by various extrinsic and intrinsic signals. Here we use conditional deletion of Dicer1 in neural crest cells and noradrenergic neuroblasts to identify stage specific functions in sympathoadrenal lineages. Conditional Dicer1 knockout in neural crest cells of Dicer1(Wnt1Cre) mice results in a rapid reduction in the size of developing sympathetic ganglia and adrenal medulla. In contrast, Dicer1 elimination in noradrenergic neuroblasts of Dicer1(DbhiCre) animals affects sympathetic neuron survival starting at late embryonic stages and chromaffin cells persist at least until postnatal week 1. A differential function of Dicer1 signaling for the development of embryonic noradrenergic and cholinergic sympathetic neurons is demonstrated by the selective increase in the expression of Tlx3 and the cholinergic marker genes VAChT and ChAT at E16.5. The number of Dbh, Th and TrkA expressing noradrenergic neurons is strongly decreased in Dicer1-deficient sympathetic ganglia at birth, whereas Tlx3(+)/ Ret(+) cholinergic neurons cells are spared from cell death. The postnatal death of chromaffin cells is preceded by the loss of Ascl1, mir-375 and Pnmt and an increase in the markers Ret and NF-M, which suggests that Dicer1 is required for the maintenance of chromaffin cell differentiation and survival. Taken together, these findings demonstrate distinct stage and lineage specific functions of Dicer1 signaling in differentiation and survival of sympathetic neurons and adrenal chromaffin cells., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The adrenal medulla plays a key role in the physiological responses of developing and mature mammals by releasing catecholamines (CAT) during stress. In rodents and humans, the innervation of CAT-producing, adrenomedullary chromaffin cells (AMCs) is immature or absent during early postnatal life, when these cells possess 'direct' hypoxia- and CO2/H(+)-chemosensing mechanisms. During asphyxial stressors at birth, these mechanisms contribute to a CAT surge that is critical for adaptation to extra-uterine life. These direct chemosensing mechanisms regress postnatally, in parallel with maturation of splanchnic innervation. Here, we review the evidence that neurotransmitters released from the splanchnic nerve during innervation activate signaling cascades that ultimately cause regression of direct AMC chemosensitivity to hypoxia and hypercapnia. In particular, we consider the roles of cholinergic and opioid receptor signaling, given that splanchnic nerves release acetylcholine and opiate peptides onto their respective postsynaptic nicotinic and opioid receptors on AMCs. Recent in vivo and in vitro studies in the rat suggest that interactions involving α7 nicotinic acetylcholine receptors (nAChRs), the hypoxia inducible factor (HIF)-2α signaling pathway, protein kinases and ATP-sensitive K(+) (KATP) channels contribute to the selective suppression of hypoxic chemosensitivity. In contrast, interactions involving μ- and/or δ-opiod receptor signaling pathways contribute to the suppression of both hypoxic and hypercapnic chemosensitivity, via regulation of the expression of KATP channels and carbonic anhydrase (CA I and II), respectively. These data suggest that the ontogeny of O2 and CO2/H(+) chemosensitivity in chromaffin cells can be regulated by the tonic release of presynaptic neurotransmitters.

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.

Rationale: The development of the cardiac outflow tract (OFT) and great vessels is a complex process that involves coordinated regulation of multiple progenitor cell populations. Among these populations, neural crest cells make important contributions to OFT formation and aortic arch remodeling. Although numerous signaling pathways, including Notch, have been implicated in this process, the role of epigenetics in OFT development remains largely unexplored., Objective: Because histone deacetylases (Hdacs) play important roles in the epigenetic regulation of mammalian development, we have investigated the function of Hdac3, a class I Hdac, during cardiac neural crest development in mouse., Methods and Results: Using 2 neural crest drivers, Wnt1-Cre and Pax3(Cre), we show that loss of Hdac3 in neural crest results in perinatal lethality and cardiovascular abnormalities, including interrupted aortic arch type B, aortic arch hypoplasia, double-outlet right ventricle, and ventricular septal defect. Affected embryos are deficient in aortic arch artery smooth muscle during midgestation, despite intact neural crest cell migration and preserved development of other cardiac and truncal neural crest derivatives. The Hdac3-dependent block in smooth muscle differentiation is cell autonomous and is associated with downregulation of the Notch ligand Jagged1, a key driver of smooth muscle differentiation in the aortic arch arteries., Conclusions: These results indicate that Hdac3 plays a critical and specific regulatory role in the neural crest-derived smooth muscle lineage and in formation of the OFT.

Unlabelled: With 14 figures and 3 tables, Summary: Each adrenal gland consisted of cortex and medulla that developed from different embryological origins and presented different cellular organization. One hundred male or female camel embryos or fetuses with crown vertebral rump lengths (CVRL) that ranged from 0.8 to 117 cm were examined. The adrenal cortex, which is derived from intermediate mesoderm, was first observed in the 0.8-cm CVRL camel embryo. The adrenal cortex initially was combined with the gonad as a thickened region of proliferating cells derived from splanchnic intermediate mesoderm. Adrenocortical tissue was first separated from the gonadal tissue in the 2-cm CVRL camel fetus and was observed as a separate dorso-medial mass of cells. At 2.5-cm CVRL, the adrenocortical tissue was surrounded by a capsule of undifferentiated mesenchymal cells, except at its proximal pole, where an invagination was located through which chromaffinoblast cells entered the cortex. The chromaffinoblast cells migrated from the neural crest to form the medulla of the developing adrenal gland. In the 3.5-cm CVRL camel fetus, the adrenocortical cells differentiated into two layers: the inner fetal cortex and the outer definitive cortex. As development proceeded, the fetal cortex degenerated and the definitive cortex formed the zona glomerulosa and zona fasciculata. The zona reticularis did not form until the end of gestation. During prenatal life, the adrenal medulla was much thicker than the cortex., (© 2010 Blackwell Verlag GmbH.)

Chromaffin cells are neuroendocrine cells mainly found in the medulla of the adrenal gland. Most existing knowledge of these cells has been the outcome of extensive research performed in animals, mainly in the cow, cat, mouse and rat. However, some insight into the physiology of this neuroendocrine cell in humans has been gained. This review summarizes the main findings reported in human chromaffin cells under physiological or disease conditions and discusses the clinical implications of these results.

Neural crest-derived cells are believed to migrate into the fetal adrenal cortex from the medially-located hilus. However, there appears to be a paucity of observations of the migration and distribution of medullary cells in humans. In sagittal as well as horizontal sections of human fetuses between 9 and 16 weeks of gestation, we identified chromaffin, ganglion and Schwann-like cells in the developing adrenal gland using immunohistochemistry. Cells showing tyrosine hydroxylase (TH) immunoreactivity (i.e., candidate ganglion cells) entered the fetal cortex mainly from the medial half of the adrenal, but the path of entry also included the ventral, dorsal and caudal aspects. These cells displayed linear arrangements, forming a connection between the peripheral and central areas of the gland. S100 protein-immunoreactive cells (i.e., Schwann-like cells) accompanied most (but not all) of the TH-positive cells. The distribution of chromogranin A-immunoreactive cells (i.e., chromaffin cells) was similar to and overlapped with that of TH-positive cells. Chromogranin A-positive cells were observed around the aorta as well as in the adrenal. The entry of neural crest-derived cells does not appear to be restricted to a hypothetical medial hilus, but occurs widely around the cortex, with or without the accompaniment of Schwann-like cells. These cells advance in lines through the fetal cortex in a cord-like arrangement without destruction of the cortical architecture. Some of the TH-positive cells very likely express chromogranin A before entry into the adrenal.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

Sry-box (Sox)8, Sox9, and Sox10 are all strongly expressed in the neural crest. Here, we studied the influence of these closely related transcription factors on the developing adrenal medulla as one prominent neural crest derivative. Whereas Sox9 was not expressed, both Sox8 and Sox10 occurred widely in neural crest cells migrating to the adrenal gland and in the gland itself, and they were down-regulated in cells expressing catecholaminergic traits. Sox10-deficient mice lacked an adrenal medulla. The adrenal anlage was never colonized by neural crest cells, which failed to specify properly at the dorsal aorta and died apoptotically during migration. Furthermore, mutant neural crest cells did not express Sox8. Strong adrenal phenotypes were also observed when the Sox10 dimerization domain was inactivated or when a transactivation domain in the central portion was deleted. Sox8 in contrast had only minimal influence on adrenal gland development. Phenotypic consequences became only visible in Sox8-deficient mice upon additional deletion of one Sox10 allele. Replacement of Sox10 by Sox8, however, led to significant rescue of the adrenal medulla, indicating that functional differences between the two related Sox proteins contribute less to the different adrenal phenotypes of the null mutants than dependence of Sox8 expression on Sox10.

This article reviews the current status of research about the histogenesis and morphofunctional characteristics of chromaffin cells in the adrenal medulla. First, this study reports the selective migration, transcription and activation factors, and the morphological events of the chromaffin cell precursors during adrenal medulla development. Subsequently, the morphofunctional characteristics of adrenergic and non-adrenergic cells are considered, with particular reference to the characteristics of chromaffin granules and their biological steps, including their formation, traffic (storage, targeting and docking), exocytosis in the strict sense and recapture. Moreover, the relationship of chromaffin cells with other tissue components of the adrenal medulla is also revised, comprising the ganglion cells, sustentacular cells, nerves and connective-vascular tissue.

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca(2+)-activated K(+) current [I (K(Ca))] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K(+) current [I (K(ATP))] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for I (K(ATP)) and I (K(Ca)) was 50 and 29.5%, respectively, whereas at F19, I (K(ATP)) is reduced to 14%, and I (K(Ca)) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of K(ATP) and K(Ca) channels.

Gata3 mutant mice expire of noradrenergic deficiency by embryonic day (E) 11 and can be rescued pharmacologically or, as shown here, by restoring Gata3 function specifically in sympathoadrenal (SA) lineages using the human DBH promoter to direct Gata3 transgenic expression. In Gata3-null embryos, there was significant impairment of SA differentiation and increased apoptosis in adrenal chromaffin cells and sympathetic neurons. Additionally, mRNA analyses of purified chromaffin cells from Gata3 mutants show that levels of Mash1, Hand2 and Phox2b (postulated upstream regulators of Gata3) as well as terminally differentiated SA lineage products (tyrosine hydroxylase, Th, and dopamine beta-hydroxylase, Dbh) are markedly altered. However, SA lineage-specific restoration of Gata3 function in the Gata3 mutant background rescues the expression phenotypes of the downstream, as well as the putative upstream genes. These data not only underscore the hypothesis that Gata3 is essential for the differentiation and survival of SA cells, but also suggest that their differentiation is controlled by mutually reinforcing feedback transcriptional interactions between Gata3, Mash1, Hand2 and Phox2b in the SA lineage.

Adult adrenal chromaffin cells are being utilized for therapeutic transplantation. With the prospect of using fetal chromaffin cells in pain therapy, we studied their phenotype, proliferative power, function, and growth in vitro and in situ in order to determine the optimal time for implantation. Between 7 and 10 gestational weeks (GW), we isolated, in vitro, two types of chromaffin cells with a noradrenergic phenotype akin to that observed, in situ. Among the adherent chromaffin cells first observed in vitro, only a few samples expressed met-enkephalin, whereas almost all the neurosphere-like colonies, which appeared later, expressed it. However, neither of the two types of populations expressed an adrenergic phenotype in line with that observed in situ. At the upper limits of the voluntary abortion period authorized in France, this phenotype (12 GW) and met-enkephalin expression (13 GW) were evidenced in situ. For the first time in man, we demonstrate the secretion of noradrenaline in vitro by the two populations of cells. Consistent with this result, we also noted dopamine beta hydroxylase (DbetaH) mRNA expression in vitro and in situ within this period. These observations on the expression of these biological factors indicate that 9-10 GW would be the best stage for sampling these cells for preclinical transplantation experiments.

Using light, electron microscopy and cytochemistry, the early (embryonic week 4-8) stages of adrenal gland (AG) development were studied in domestic pig. The interrelations between the cells of the fetal cortex (FC) and chromaffin cells (CC) were traced. At week 5, AG primordium is represented by FC, which consists of the epithelioid cells, with the ingrowing neural cords containing CC islets. Starting at the early embryonic period and up to fetal period, CC and interrenal cells of FC are closely interrelated with each other and sinusoidal capillaries. Both cellular types are at different stages of differentiation, including the functionally active elements. At weeks 7-8, FC cells undergo involution, while those ones, left at periphery, form definitive cortex. CC are located in the central part of the organ and form suprarenal tissue. Authors hypothesize, that CC, migrating into AG primordium, initially induce the development of interrenal primordium, and later cause the involution of FC. This, possibly, may be explained by the fact that further antenatal and postnatal development of the organism requires more corticosteroids than the amount produced by FC.

The human adrenal is an endocrine gland located at the superior part of the kidney. Composed of the adrenal cortex of mesoblastic origin and the adrenal medulla of neuroectoblastic origin, the human fetal adrenal grows considerably during the first three months of development. From 12 to 18 weeks of development (WD), the weight of the adrenals increases seven-fold. The gland's weight doubles from 18 to 28 WD and from 28 to 36 WD. At birth, the two adrenals weigh on average 10 g. At the 8th week, two zones are individualized in the adrenal cortex: the definitive zone and the fetal inner zone. At the second trimester, according to ultrastructural and biochemical studies, a third zone, called the transition zone, is individualized between the definitive zone and the fetal inner zone. The definitive zone persists, but the origin of the three zones (glomerular, fascicular and reticular) of adult adrenal cortex is not known. The fetal inner zone regresses from the 5th month of gestation and disappears totally one year after birth. At the 8th week, the immature neuroblasts migrate to the definitive zone, then to the fetal inner zone to compose the adrenal medulla, which develops essentially after birth and during the first year. Before the 10th week, the human fetal adrenal is able to produce steroid hormones, in particular dehydroepiandrosterone sulfate (DHEA-S); the secretion of cortisol remains discussed. The development of the human fetal adrenal is complex and is under the control of hormones (ACTH, LH and betaHCG), growth factors (ACTH essentially) and transcription factors (essentially SF1 and DAX-1). Knowledge of morphological and molecular phenomena of this development permits to understand the pathophisiology of congenital adrenal deficiencies.

In neural crest (NC) cultures cAMP signaling is an instructive signal in catecholaminergic, sympathoadrenal cell development. However, the extracellular signals activating the cAMP pathway during NC cell development have not been identified. We demonstrate that in avian NC cultures, evidenced by tyrosine hydroxylase expression and catecholamine biosynthesis, adenosine and not adrenergic signaling, together with BMP2, promotes sympathoadrenal cell development. In NC cultures, addition of the adenosine receptor agonist NECA in the presence of BMP2 promotes sympathoadrenal cell development, whereas the antagonist CGS 15943 or the adenosine degrading enzyme adenosine deaminase (ADA) suppresses TH expression. Importantly, NC cells express A2A and A2B receptors which couple with Gsalpha increasing intracellular cAMP. Employing the CNS-derived catecholaminergic CAD cell line, we also demonstrate that neuronal differentiation mediated by serum withdrawal is further enhanced by treatment with IBMX, a cAMP-elevating agent, or the adenosine receptor agonist NECA, acting via cAMP. By contrast, the adenosine receptor antagonist CGS 15943 or the adenosine degrading enzyme ADA inhibits CAD cell neuronal differentiation mediated by serum withdrawal. These results support that adenosine is a physiological signal in neuronal differentiation of the CNS-derived catecholaminergic CAD cell line and suggest that adenosine signaling is involved in NC cell development in vivo.

The immunocytochemical development of the thoracolumbar sympathetic ganglion and its adrenal counterpart was studied in the chick from days 3.5 to 12 of incubation, using antibodies to 17 separate antigens, including antibodies to pan-neuroendocrine markers, catecholamine-synthesizing and proprotein-processing enzymes, and neuropeptides. Some of the antigens studied (Go protein-alpha subunit, thyrosine hydroxylase, and galanin) were strongly expressed from the first days of development, whereas others (chromogranin-A, chromogranin-B, 7B2 protein, and somatostatin) showed a diverse immunoreactive expression at different stages. Three different patterns were found in the development of both adrenal medulla and thoracolumbar sympathetic ganglion. In the first (chromogranin-A and B, Go protein-alpha subunit, tyrosine hydroxylase, HNK-1, and galanin), virtually all medullary and thoracolumbar sympathetic ganglion cells were strongly immunostained from day 4 onward. Except for HNK-1, chromogranin-A and B, there was a steady increase in immunoreactive cells for all the remaining antigens up to day 12. In the second (7B2 protein, proprotein convertase 2, and secretogranin II), full antigenic expression was reached in medullary and thoracolumbar sympathetic ganglion cells by day 10. In the third pattern (proprotein convertase 3, somatostatin, dopamine-beta-hydroxylase, neuron-specific enolase, vasoactive intestinal polypeptide, and met-enkephalin), differences in immunoreactivity were observed between the medullary and thoracolumbar sympathetic ganglion cells.

Adrenal medullary cells are derived from the neural crest. To study the formation process of the adrenal medulla in the embryonic period, we visualized chromaffin cells of rat embryos at 13 to 17 days of gestation using anti-tyrosine hydroxylase (TH) antiserum, and created three-dimensional images from serial tissue sections. Between 13 and 15 days of gestation, TH-positive cells (chromaffin cells) migrated from a group of TH-positive cells present dorsal to the adrenal primordium via the medial cranial end of the adrenal primordium into the adrenal primordium. At or after 16 days of gestation, the adrenal capsule was formed except on the ventral aspect of the cranial end of the adrenal gland, from which TH-positive cells penetrated into the adrenal gland. The reconstructed images showed that TH-positive cells were present contiguously from the sympathetic chain ganglia through a group of TH-positive cells ventral to the adrenal gland into the adrenal cortex, and that the group of TH-positive cells ventral to the adrenal gland communicated with the preaortic ganglion present ventral and caudal to the adrenal gland. These results suggest that neural crest cells use the same pathway to migrate to the sympathetic chain ganglia dorsal to the adrenal gland, to the adrenal gland, and to the preaortic ganglion.

Human fetal adrenal development is characterized by rapid growth, high steroidogenic activity, and a distinct morphology, including a unique cortical compartment known as the fetal zone. For most of gestation, the predominant fetal zone accounts for 80-90% of the cortical volume and is the primary site of growth and steroidogenesis, producing 100-200 mg/day of the androgenic steroid, dehydroepiandrosterone sulfate (DHEA-S). The physiological role of this zone during intrauterine life is not well understood. While the glands appear to be capable of DHEA-S synthesis early in gestation (8-10 weeks), we noticed that this event precedes the differentiation of hairs and sebaceous glands. Hairs begin to develop between 9 and 12 weeks and sebaceous glands between 13 and 15 weeks of gestation. Sebaceous glands form an oily secretion - sebum that mixes with desquamated epidermal cells to form vernix caseosa. Vernix caseosa protects the developing skin from constant exposure to amniotic fluid, and hairs helps to hold the vernix caseosa on the skin. We suggest therefore that the human fetal adrenal cortex produces DHEA-S beginning at around 8-10 weeks of gestation in sufficient quantities to influence the growth of hairs and sebaceous glands. Soon after birth, the fetal zone atrophies, and adrenal androgen production decreases to minimal levels. As a consequence, in concordance with the rapid decrease in adrenal androgen levels and in consistent with our hypothesis, fetal hairs are shed and sebaceous glands shrink to small structures. The mechanism that regulates fetal adrenal androgen production is a key unanswered problem in human adrenal biology. Since there exists a close relationship between epinephrine and DHEA-S levels during adrenarche which shows modulatory interactions between adrenal androgen production and adrenomedullary function, we suggest again that adrenomedullary function might play a role in the control of fetal adrenal androgen secretion.

Cellular differentiation is a stepwise process where environmental factors are essential key components to direct cells to their final phenotype. The sympathoadrenal (SA) system is one of the principal models used to study the role of environmental factors in the development of the peripheral nervous system. Two major cell types originate from the SA progenitor: the principal neurons of the sympathetic ganglia and the chromaffin cells of the adrenal medulla. These cells are directed to their final phenotype by a series of environmental factors, of which nerve growth factor (NGF) and glucocorticoids, are the best studied. Previously, we have shown that 11-day embryonic chick sympathetic cell cultures increased their neuropeptide Y (NPY) protein and mRNA levels in the presence of NGF. In contrast, NGF had no such effect in chromaffin cell cultures from the same developmental stage. These results were unexpected since both cells types respond morphologically to NGF. To determine if these cells can gain or lose their capacity to respond to NGF, morphological and biochemical studies were done at earlier stages using immunocytochemical, radioimmunoassay and polymerase chain reaction (PCR) techniques. Interestingly we found that in E-7 chromaffin cells there is a biochemical and morphological response to NGF, while E-7 sympathetic cells lack this response. Our observations show a developmental point of regulation of morphological and biochemical properties by NGF and reveal an age dependent capacity of SA cells to acquire or lose competence to an environmental factor.

Prenatal exposure to nicotine significantly increases enkephalin mRNA levels in the rat adrenal medulla prenatally, and postnatally the normal up-regulation is obliterated. This may lead to a disturbed modulation or regulation of catecholamine release in the adrenal and may be one factor contributing to the attenuated capacity of nicotine-treated pups to survive severe hypoxia. We speculate that this may be part of the mechanism underlying the relation between maternal smoking and sudden infant death syndrome.

Environmental exposures at crucial points in development permanently alter sympathoadrenal function in mammals. The sympathetic innervation of peripheral tissues and the responsiveness of sympathetic nerves and adrenal medulla to standard stimuli are susceptible to modification by exposures in early life, such as environmental temperature, nutrition and stress. Because the sympathetic nervous system is composed of multiple, function-specific subunits, programming of sympathetic functions occurs on a regional rather than a global basis and can aid development of a phenotype adapted to the local environment. Under some circumstances, however, adaptations in early life might prove maladaptive in adulthood and, as a consequence, might provide a basis for developmental origins of pediatric and adult disease, such as sudden infant death syndrome and obesity.

The sympathoadrenal (SA) cell lineage is a derivative of the neural crest (NC), which gives rise to sympathetic neurons and neuroendocrine chromaffin cells. Signals that are important for specification of these two types of cells are largely unknown. MASH1 plays an important role for neuronal as well as catecholaminergic differentiation. Mash1 knockout mice display severe deficits in sympathetic ganglia, yet their adrenal medulla has been reported to be largely normal suggesting that MASH1 is essential for neuronal but not for neuroendocrine differentiation. We show now that MASH1 function is necessary for the development of the vast majority of chromaffin cells. Most adrenal medullary cells in Mash1(-/-) mice identified by Phox2b immunoreactivity, lack the catecholaminergic marker tyrosine hydroxylase. Mash1 mutant and wild-type mice have almost identical numbers of Phox2b-positive cells in their adrenal glands at embryonic day (E) 13.5; however, only one-third of the Phox2b-positive adrenal cell population seen in Mash1(+/+) mice is maintained in Mash1(-/-) mice at birth. Similar to Phox2b, cells expressing Phox2a and Hand2 (dHand) clearly outnumber TH-positive cells. Most cells in the adrenal medulla of Mash1(-/-) mice do not contain chromaffin granules, display a very immature, neuroblast-like phenotype, and, unlike wild-type adrenal chromaffin cells, show prolonged expression of neurofilament and Ret comparable with that observed in wild-type sympathetic ganglia. However, few chromaffin cells in Mash1(-/-) mice become PNMT positive and downregulate neurofilament and Ret expression. Together, these findings suggest that the development of chromaffin cells does depend on MASH1 function not only for catecholaminergic differentiation but also for general chromaffin cell differentiation.

The hypothesis that nitric oxide plays a key role in the regulation of adrenal blood flow and plasma concentrations of cortisol and catecholamines under basal and hypoxaemic conditions in the llama fetus was tested. At 0.6-0.8 of gestation, 11 llama fetuses were surgically prepared for long-term recording under anaesthesia with vascular and amniotic catheters. Following recovery all fetuses underwent an experimental protocol based on 1 h of normoxaemia, 1 h of hypoxaemia and 1 h of recovery. In nine fetuses, the protocol occurred during fetal I.V. infusion with saline and in five fetuses during fetal I.V. treatment with the nitric oxide synthase inhibitor L-NAME. Adrenal blood flow was determined by the radiolabelled microsphere method during each of the experimental periods during saline infusion and treatment with L-NAME. Treatment with L-NAME during normoxaemia led to a marked fall in adrenal blood flow and a pronounced increase in plasma catecholamine concentrations, but it did not affect plasma ACTH or cortisol levels. In saline-infused fetuses, acute hypoxaemia elicited an increase in adrenal blood flow and in plasma ACTH, cortisol, adrenaline and noradrenaline concentrations. Treatment with L-NAME did not affect the increase in fetal plasma ACTH, but prevented the increments in adrenal blood flow and in plasma cortisol and adrenaline concentrations during hypoxaemia in the llama fetus. In contrast, L-NAME further enhanced the increase in fetal plasma noradrenaline. These data support the hypothesis that nitric oxide has important roles in the regulation of adrenal blood flow and adrenal corticomedullary functions during normoxaemia and hypoxaemia functions in the late gestation llama fetus.

Catecholamine release from the adrenal medulla glands plays a vital role in postnatal adaptation. A number of pathologic situations are characterized by oxygen deficiency. The objective of the present study was to determine the influence of long-term prenatal hypoxia on maturation of the adrenal medulla. Pregnant rats were subjected to hypoxia (10% O2) from the fifth to the 20th d of gestation. The offspring were examined on the 19th d of gestation (E19), the day of birth (P0), and at postnatal (P) day of life P3, P7, P14, P21, and P68. The catecholamine content and activity of tyrosine hydroxylase (TH) in vivo were assayed by HPLC with electrochemical detection. Cellular expression of TH and phenylethanolamine N-methyl transferase was evaluated by protein immunohistochemistry and in situ hybridization of the corresponding mRNA species. Exposure to prenatal hypoxia reduced the epinephrine content of the adrenal medulla on E19, P0, P3, and P7 while increasing the norepinephrine content on E19, P0, and P14. Furthermore, the peak epinephrine to norepinephrine ratio appearing between P7 and P10 in the normoxic offspring was absent in the hypoxic offspring. The in vivo TH activity was increased on P3 and P14 and decreased on P68. The percentage of chromaffin cells in the medulla expressing TH and phenylethanolamine N-methyl transferase was lowered on E19, P0, and P7. TH and phenylethanolamine N-methyl transferase mRNA levels were reduced on P7. Clearly prenatal hypoxia results in major changes in adrenal catecholamine stores and synthesis during the perinatal period, which persist into adulthood. The capacity to cope with postnatal stress might be disturbed as a consequence of prenatal hypoxia.

Neuronal nicotinic receptors (nAChRs) are expressed in the brain but also in the peripheral tissues including the adrenal medulla. However, it is unclear which nAChRs are present in the human adrenal medulla. In the study, receptor binding assay, Western blot and RT-PCR have been performed to investigate the expression of nAChRs in adrenal medulla from human, rat and mouse. The results showed that in human adult adrenal medulla, mRNAs for nAChR alpha3, alpha4, alpha5, alpha7, beta2, beta3, and beta4 subunits but not beta2 in the fetal human adrenal medulla were expressed. Saturation binding of [3H]epibatidine showed two binding sites in human aged adrenal medulla. The specific binding of [3H]epibatidine (0.1 nM) was significantly higher in human fetal compared to human aged adrenal medulla. mRNAs for the alpha3, alpha4, alpha5, alpha7, beta2, and beta4 subunits but not the beta3 were detectable in adult rat and mouse adrenal medulla. No differences in gene-expression of the nAChRs were observed between new born, adult and aged rat adrenal medulla. Saturation binding of [3H]epibatidine showed only one binding site in rat adrenal medulla. Lower protein levels for the nAChR subunits were observed in the rat adrenal medulla compared to rat brain. There was lower protein levels of the nAChRs in aged rat adrenal medulla compared to the young rats. Sub-chronic treatment of nicotine to rats did not influence level of the nAChRs in the adrenal medulla. In conclusion, the expression of nAChRs in adrenal medulla is age- related and species dependent.

The aim of this vie is to discuss the general principles of prenatal development of adrenal gland. On the basis of spatial-temporal heterogenity of structural particularites of fetal adrenal cortex, spectrum steroidogenic enzymes and secreting hormones expression in adrenocorticocytes, regulation of proliferation and differentiation processes mechanisms, authors discuss adrenal morphogenesis in three periods of gestation. It was noted the close relationship between placenta development and hypothalamo-pituitary-adrenocortical system formation with specification in each gestation period. Adrenal embryogenesis accompanied by remodeling of structural, functional and biochemical properties of parenchimal-stromal elements of fetal organ. Definitive zonation formation determined by morphogens: ACTH, renal and intraadrenal angiotensin II, estrogens, prostaglandines and other. The action of these factors realization is due to immediately and thought growth factor system (IGF-I, IGF-II, EGF, bFGF), working as paracrine amplificators of morphogenetic signals and activators of transcriptional factors--c-fos and c-jun.

Background: Because of their lack of long-term viability, adrenal tissue transplants have shown limited success in alleviating the motor disturbances associated with experimental and pathologic striatal dopamine denervation. In this study, we examined how the graft placement site influences adrenal medulla transplant survival and its relation with the reduction of motor deficits in rats bearing unilateral 6-OHDA lesion., Methods: One or 5 microL of fetal adrenal medullar tissue was grafted either inside the striatal parenchyma or into the lateral ventricle in contact with the dopamine-denervated striatum. Motor disturbances, as assessed by apomorphine-induced rotation, were correlated to the graft morphologic survival features., Results: Apomorphine-induced rotation showed a marginal reduction of 11% in all groups independently of graft survival features or placement site. Intrastriatal transplants showed limited viability characterized by a substantial loss of graft initial volume as well as fewer and smaller chromaffin cells compared to ventricular grafts, which had a reduced loss of graft initial volume and more and larger chromaffin cells., Conclusions: Although the lateral ventricle may favor adrenal medulla transplant viability, their induced motor outcome is comparable to that induced by less viable intrastriatal grafts, suggesting that the implanted dopamine-producing cells may interact and influence striatal neurons better when placed in close proximity.

Immunocytochemical technique and light microscopy were used to ascertain the relationship between vasoactive intestinal polypeptide (VIP) and tyrosine hydroxylase in fetal sheep and fetal baboon adrenal cortices and medullae at 85% of gestation. VIP immunostaining was extremely robust in fetal sheep adrenal cortical neurofibers and cells while weak in fibers and nonexistent in cells of fetal baboon. Also, tyrosine hydroxylase-immunopositive cells, present throughout the adrenal cortices of both fetal sheep and baboons, were heavily innervated by VIP-immunoreactive neurofibers in fetal sheep, but not in fetal baboons. Adrenal cortical VIP-immunopositive fibers occurred in greater (P<0.05) frequency in fetal sheep than in fetal baboons (14.82+/-3.10 vs. 0.84+/-0.26 fibers/field), were larger in diameter (2.93+/-0.34 vs. 0.93+/-0.07 microm) and ran for longer distances in the plane of section (127.85+/-5.16 vs. 74.53+/-4.93 microm). VIP immunogenicity in cells (ganglion and chromaffin) and fibers was robust in fetal adrenal medulla of sheep while nonexistent in baboons. VIP fibers in fetal sheep medulla were smaller in diameter compared to fetal sheep cortex (1.22+/-0.13 vs. 2.93+/-0.34 microm, P<0.05), but not compared to extrinsic nerve fibers (1.30+/-0.09 microm). We hypothesize that in fetal sheep of this age, medullary neurofibers derive primarily from extrinsic sources while cortical fibers arise from cortical ganglion cells. We conclude that at 85% of gestation the potential for VIP neural control of paracrine (e.g., glucocorticoid/catecholamine) interactions in both adrenal cortex and medulla is much greater in fetal sheep compared to fetal baboons.

The development of the adrenal gland in the lizard Calotes versicolor was studied histologically and histochemically from the day of oviposition (stage 27) to 60 days after hatching. At stage 27, the adrenocortical cells are found in association with the genital ridge (primordial gonad). The separation of adrenocortical cells from the gonad takes place at stage 31. Organization of adrenocortical cells into cords takes place at stage 34. The catecholamine-secreting chromaffin cells can be seen distinctly on the dorsal region of the adrenal at stage 36, indicating the presence of biologically active catecholamines; the noradrenaline-secreting chromaffin cells appear first at stage 36 and the adrenaline-secreting cells appear later at stage 41. The cortico-medullary ratio of 6:1 during early embryonic development decreases with the increase in age and is 3:1 in posthatching lizards. The histochemical localization of Delta(5)-3beta-hydroxysteroid dehydrogenase (3beta-HSD) and glucose-6-phosphate dehydrogenase in the adrenocortical cells as early as at stage 27 (prior to the gonadal differentiation) indicates the capability of these cells to synthesize steroids. The intensity of the enzyme activity is maximum on the day of hatching and remains more or less the same in the posthatching lizards. The localization of 17beta-HSD enzyme activity observed in the adrenocortical cells at stage 34 is suggestive of their ability to synthesize sex steroids during embryonic life. The intense 3beta-HSD activity on the day of hatching in C. versicolor suggests high production of steroids which may be corticoids. The results of the present work also suggest that the onset of steroid secretion occurs prior to catecholamine secretion during embryogenesis of the adrenal gland in C. versicolor. In addition, there is a significant relationship between ontogenic steroidogenesis of the adrenal gland and sexual differentiation of the gonad., (Copyright 2000 Academic Press.)

The expression of phenylethanolamine N-methyl transferase (EC 2. 1.1.2.8, PNMT), the final enzyme in the cascade of catecholamine synthesis, is differentially regulated in adrenergic neurons in the brain and in adrenal chromaffin cells. Using reverse transcription-polymerase chain reaction-based techniques, we detected in the prenatal developing rat brainstem, two species of PNMT mRNA which were produced by a rare alternative splicing mechanism known as intron retention. The spliced, intronless message was downregulated postnatally, while the intron-retained mRNA species continued to be constitutively expressed through adulthood. By contrast in the adrenals, at all stages of development examined, only the intronless message was expressed. In line with previous reports on the failure of glucocorticoids to induce PNMT expression in the brain, the pattern of PNMT splicing in brainstem explants was not affected by the presence of the synthetic glucocorticoid dexamethasone. Undifferentiated sympathoadrenal PC12 pheochromocytoma cells expressed very low basal levels of both mRNA variants, accompanied by a very low basal PNMT enzymatic activity. Exposure of PC12 cells to dexamethasone resulted in the upregulation of only the spliced mRNA variant concomitant with a 3-fold increase in PNMT enzymatic activity. In contrast, treatment of PC 12 cells with nerve growth factor (NGF) enhanced the expression of both the intron-retained and the intronless mRNA species without changes in the basal enzyme activity. This latter result suggests that the translation of the intronless mRNA species may be regulated by the intron-retained mRNA species, which by itself may yield a truncated, yet enzymatically functional translational product. Our data suggest that the tissue-specific regulation of PNMT expression is based on a rare alternative splicing mechanism termed intron retention, and that in the adrenal, but not in the brain, this mechanism is sensitive to regulation by glucocorticoids. Thus, this system is uniquely suited for studying the hormonal control of tissue-specific splicing in the nervous system.

The aims of this study were to determine whether placental restriction (PR) alters the pattern of localization of the catecholamine-synthesizing enzymes, dopamine-beta-hydroxylase and phenylethanolamine N-methyltransferase, and enkephalin (ENK)-containing peptides in the adrenal gland of the midgestation sheep fetus. We also determined the effect of PR on the content and profile of the molecular mass forms of ENK-containing peptides in the fetal adrenal medulla. Placental growth was restricted by removal of most of the placental implantation sites in the uterus before mating. In midgestation, placental and fetal body weight were reduced (p < 0.05) in the PR group (n = 8; 237.9 +/- 39.5 g, 564.7 +/- 41.6 g, respectively) when compared with the control group (n = 9; 479.1 +/- 36.9 g, 721.2 +/- 22.8 g, respectively). However, combined fetal adrenal weight and adrenal cortical and medullary area were similar in the PR and control fetuses. In PR fetuses, distribution of staining for dopamine-beta-hydroxylase, phenylethanolamine N-methyltransferase, and ENK-containing peptides in the adrenal medulla was similar when compared with the control group; however, staining was less intense and not all adrenomedullary cells were stained. The total adrenal content of ENK-containing peptides was also significantly (p < 0.05) less in the PR group (103.4 +/- 18.6 ng/adrenal) than in the control group (243.6 +/- 24.8 ng/adrenal). However, the molecular mass profile of ENK-containing peptides was not altered in the PR fetuses compared with controls. These data suggest that placental restriction in utero may alter the synthesis and/or secretion of catecholamines and ENK-containing peptides from the fetal adrenal medulla from as early as 90 d gestation.

To clarify when the cholinergic receptor-mediated secretion mechanism of developing adrenal chromaffin cells is expressed and becomes functional, morphological changes and intracellular calcium dynamics were studied by immunohistochemistry, electron microscopy, and Fura-2 digital image analysis. From embryonic day 14 to 16, adrenal medullary cells were immunoreactive to noradrenaline-synthesizing enzyme (dopamine beta-hydroxylase) but not to adrenaline-synthesizing enzyme (phenylethanolamine N-methyltransferase). These cells contained either no granules or just a few granules of high electron density. Exocytotic figures were rarely observed in cells of the control or in cells after carbamylcholine stimulation. Nerve fibers in the adrenal medulla contained either no clear vesicles or very few. Neither methacholine nor nicotine caused a change of intracellular Ca2+ in most chromaffin cells. From embryonic day 18 to 20, chromaffin cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase and they contained relatively numerous secretory granules. Exocytotic figures were often seen in cells after carbamylcholine stimulation. The intra-adrenal nerve fibers contained numerous clear vesicles and a few dense-cored vesicles. Methacholine caused no rise of intracellular Ca2+, but nicotine induced a low to relatively high rise in many cells. From postnatal day 2 or 3 to postnatal week 1, numerous cells were immunoreactive to both dopamine beta-hydroxylase and phenylethanolamine N-methyltransferase, whereas some cells were reactive to dopamine beta-hydroxylase alone. Chromaffin cells were divisible into noradrenaline cells and adrenaline cells based on the ultrastructural features of their granules. Methacholine induced a moderate rise of intracellular Ca2+ and nicotine caused a high rise in many chromaffin cells, whereas, in some chromaffin cells, methacholine induced no rise of intracellular Ca2+ and nicotine induced a high rise. These results suggest that morphological changes of the developing cells and the intra-adrenal nerve fibers are related to the expression of a cholinergic receptor-mediated secretion mechanism and that this mechanism via a nicotinic receptor-mediated Ca2+ signaling pathway precedes the muscarinic receptor-mediated one during development.

We used in situ hybridization histochemistry to study the expression of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic development in the rat. In the adult rat VMAT2 is present exclusively in neuronal tissues and VMAT1 is present in the adrenal medulla and in certain intestinal endocrine cells. We found that both transporter molecules are more widely expressed during development. We demonstrate a complete overlap of the two VMAT mRNAs in the sympathetic nervous system between E13 and E21 days. In addition, VMAT2 (and to some extent VMAT1) mRNA is expressed in ganglionic cells of the parasympathetic nervous system and in cranial ganglia (trigeminal, vestibular and spiral ganglia) between E12 and E21. The sensory neurons of the dorsal root ganglia, which are also neural crest derivatives, express VMAT2 mRNA (E11-E21), exclusively. Both VMAT mRNAs are found in the developing GI system, but in different cells. VMAT1 mRNA was detected in organs of the endocrine system (pituitary gland, adrenal gland, testis, seminal vesicle), some connective tissue cells, and the thymus. We observed expression of both VMAT mRNAs in two separate cell groups in the placenta (E8-E10). Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect migration and differentiation of neural crest derivatives., (Copyright 1998 Elsevier Science B.V.)

Using a model of an ectopic corticotrophic tumor transplanted in mouse embryos, we now report several new findings. One relates to fetal adrenocortex-medulla interactions and others to feedback regulation of the pituitary by adrenal products or other factors in mouse embryos.

Over the last decade, great interest has been generated in evaluation of the extent of neural control of the adrenal cortex and in adrenal cortical/medullary paracrine interactions. The purpose of this review is to summarize current knowledge of fetal adrenal cortical innervation and to present an overview of those studies of fetal adrenal function indicating that adrenal innervation plays a functional role in the control of glucocorticoid secretion under basal conditions and in response to a variety of homeostatic challenges. It will be helpful in understanding both the innervation of the adrenal cortex and the role of adrenal innervation in steroidogenesis during fetal development to briefly review experimental studies that have shed light on adrenal steroidogenesis during postnatal life. This is helpful for two reasons: 1) the vast majority of studies of adrenal innervation and its effect on steroidogenesis have utilized postnatal animals and 2) since the fetus is preparing for postnatal life, evaluating the level of function achieved postnatally provides crucial insights into the developmental stages of adrenal innervation and its role in steroidogenesis in preparing the fetus for an independent postnatal existence.

The present article reviews the immunohistochemical findings on the localization of biogenic amines and neuropeptides in adrenal medullary cells of birds. In the chicken, about 70% of medullary cells are adrenaline-containing cells and the rest of cells seem to be noradrenaline-containing cells. The ratio of adrenaline-cells to noradrenaline-cells extremely varies among avian species. Besides adrenaline and noradrenaline, medullary cells of birds contain many kinds of biogenic amines and neuropeptides: serotonin, galanin, cholecystokinin, somatostatin, enkephalin, neuropeptide tyrosine and atrial natriuretic peptide. The existence of these bioactive substances in medullary cells also exhibits interspecies heterogeneity. In the chicken, serotonin and galanin are contained in both adrenaline- and noradrenaline-cells of the adrenal gland. Cholecystokinin- and somatostatin-immunoreactivity is restricted to adrenaline-containing cells. Enkephalin-immunoreactivity is seen in both adrenaline- and noradrenaline-cells, but in about half of medullary cells. Neuropeptide tyrosine-immunoreactivity is found in the adrenal gland of the chick embryo and newly hatched chick, but not in the adult chicken. Serotonin and these neuropeptides may be selectively coreleased with adrenaline and/or noradrenaline from adrenal medullary cells of the chicken.

Background: The class III beta-tubulin isotype (beta III) is present in neurons of the central and peripheral nervous systems at the earliest stages of morphological differentiation (Easter et al., J Neurosci 13:285-299, 1993; Katsetos et al., J Neuropathol Exp Neurol 52:655-666, 1993). The localization of this protein by immunohistochemistry in the different cell types of the developing human adrenal medulla is described., Methods: A mouse monoclonal antibody, TuJ1, was used to localize beta III in formalin-fixed, paraffin-embedded sections from 18 human fetal and adult adrenal glands. Tissue sections were also studied with rabbit antisera recognizing either S-100 protein or glial fibrillary acidic protein (GFAP)., Results: In the developing human adrenal medulla, beta III immunoreactivity was maximal in migrating sympathoadrenal neuroblasts/immature neurons through the end of the second trimester. Clusters of beta III-positive migrating cells, focally forming Homer Wright rosettes, could be identified in a gradient of adrenocortical invasion, i.e., through the permanent cortex and within sinusoids of the fetal cortex en route to the medulla. Outside the adrenal gland, strong beta III staining was observed in peripheral nerve bundles, sympathetic ganglia, and paraganglia at various developmental stages. In adrenal glands from 23 weeks of gestation on, and throughout adult life, all ganglion cells were beta III immunoreactive. In contrast, not all chromaffin cells exhibited beta III staining, but when present, the staining was finely granular. Sustentacular and satellite cells, adrenocortical cells and other mesenchymal elements were betaIII-negative. In sections of fetal and adult adrenal glands, S-100 protein had a sustentacular localization. No GFAP staining was present in sustentacular cells from either fetal or adult adrenals., Conclusions: In the developing human adrenal medulla, there is a peak of beta III expression during the active wave of migration of sympathetic neuroblasts. In the mature medulla, beta III is invariably present in adrenergic neurons. However, not all chromaffin-like cells express beta III, suggesting that the presence or absence of this protein identifies two subpopulations of chromaffin cells.

Reserpine treatment leads to a rapid trans-synaptic increase of the tyrosine hydroxylase (TH) gene transcription rate and mRNA levels in catecholaminergic tissues including the adrenal medulla (AM) and the superior cervical ganglia (SCG). In the AM, the formation of a specific protein complex with the TPA-responsive element located in the proximal region of the TH gene was enhanced between 30 min and 8 hr following the injection. This complex appears to contain a member of the Fos family and an antigenically related Jun protein. Moreover, the prolonged and enhanced expression of the c-Fos protein in the AM and its phosphorylation are likely to contribute to the increased TH transcription following reserpine treatment. Most strikingly, in the SCG, the trans-synaptic induction of TH transcription is transduced by totally different mechanisms, since no AP-1 complex and only minute amounts of c-Fos immunoreactivity were detected. Our study provides the first demonstration that, following the same stimulus, the induced expression of a single gene is mediated by different cis- and trans-acting factors in two distinct tissues sharing the same embryonic origin.

In our previous paper (Bocian-Sobkowska et al., 1997) we demonstrated a striking difference in development of zonation in adrenals of normal and anencephalic human fetuses. The purpose of the present study was to characterize, by means of stereology, the cellular composition of developing adrenals in the same case. Studies were performed on 11 pairs of adrenal glands from normal fetuses and 10 from anencephalic fetuses. In the studied period of development (24 to 39 weeks of intra-uterine life) the average volume of cells in normal glands increased as follows: zona glomerulosa (ZG) from 355 to 870 microns3; zona fasciculata (ZF) from 779 to 1200 microns3; fetal zone (FZ) from 2004 to 2380 microns3: and medulla (M) from 600 to 970 microns3. In anencephalic fetuses, the appropriate values were: ZG-380-680 microns3; ZF-460-680 microns3; FZ-1820-1680 microns3; and M-870-1400 microns3. At the end of the studied period the number of ZG cells in normal fetuses was two fold higher than in anencephalics, ZF cells-6-fold and in FZ-5-fold higher, while in the M the number of cells was nearly equal in both groups. During the whole investigated period of intra-uterine development the total number of adrenocortical cells in normal glands increased ca 2.5-fold, while in anencephalic glands only ca 0.5-fold, reaching at the end ca 40% of normal value. In both normal and anencephalic adrenals the number of ZG and M cells was highly correlated with ZG/M cell ratio, being slightly higher in normal glands. No such relation was demonstrated for cells of the remaining adrenocortical zones.

The differentiation of glial cells in developing, neonatal, adult and neoplastic human adrenal medulla has studied immunohistochemically. From 8 to 28 weeks' gestational age, S-100 protein and its beta-subunit revealed two different glial cell populations in adrenal glands, namely Schwann-like and sustentacular cells. Schwann-like cells were spindle-shaped cells forming a continuous layer around groups of sympathetic neuroblasts, often in contact with Schwann cells of nerve fibres entering neuroblastic groups. Sustentacular cells were round or oval cells with dendritic cytoplasmic processes; they were not associated with nerve fibres and mingled both with sympathetic neuroblasts and differentiating chromaffin cells. The developmental fate of Schwann-like cells was different from that of sustentacular cells. Schwann-like cells disappeared from the 28th week of gestational age, in association with the disappearance of sympathetic neuroblastic groups, and they were rarely found in neonatal and adult adrenal medulla. In contrast, sustentacular cells persisted between medullary chromaffin cells, and their number and dendritic cytoplasmic processes progressively increased from foetus to adult. In eight cases of primitive adrenal neuroblastic tumours of neonatal age (five undifferentiated neuroblastomas and three ganglioneuroblastomas), Schwann-like cells were found at the periphery of tumoral nests with a lobular growth pattern, while rare sustentacular cells were associated with neuroblasts. In two cases of adult phaeochromocytomas, only sustentacular cells were detected between chromaffin tumoral cells. Our findings suggest that the glial cell types and their distribution in primitive adrenal medulla tumours closely resemble those observed during development in the groups of adrenal sympathetic neuroblasts and in the clusters of chromaffin cells.

The ontogenetic expression of chromogranin A (CgA) and its derived peptides, WE-14 and pancreastatin (PST), was studied in the rat neuroendocrine system employing immunohistochemical analysis of fetal and neonatal specimens from 12.5-day embryos (E12.5), to 42-day postnatal (P42) rats. CgA immunostaining was first detected in endocrine cells of the pancreas, stomach, intestine, adrenal gland and thyroid at E13.5, E14.5, E15.5, E15.5 and E18.5, respectively. PST-like immunoreactivity was detected in endocrine cells of the pancreas at E13.5, stomach, intestine at E15.5, adrenal gland at E17.5 and thyroid at E18.5. WE-14 immunoreactivity was first observed in the immature pancreas at E15.5, mucosal cells of the stomach at E15.5, scattered chromaffin cells in the immature adrenal gland and mucosal cells of the intestine at E17.5 and thyroid parafollicular cells at E18.5. These data confirm that the translation of the CgA gene is regulated differentially in various neuroendocrine tissues and, moreover, suggests that the post-translational processing of the molecule is developmentally controlled.