Synaptic protein and pan-neuronal gene expression and their regulation by Dicer-dependent mechanisms differ between neurons and neuroendocrine cells.

BACKGROUND: Neurons in sympathetic ganglia and neuroendocrine cells in the adrenal medulla share not only their embryonic origin from sympathoadrenal precursors in the neural crest but also a range of functional features. These include the capacity for noradrenaline biosynthesis, vesicular storage and regulated release. Yet the regulation of neuronal properties in early neuroendocrine differentiation is a matter of debate and the developmental expression of the vesicle fusion machinery, which includes components found in both neurons and neuroendocrine cells, is not resolved. RESULTS: Analysis of synaptic protein and pan-neuronal marker mRNA expression during mouse development uncovers profound differences between sympathetic neurons and adrenal chromaffin cells, which result in qualitatively similar but quantitatively divergent transcript profiles. In sympathetic neurons embryonic upregulation of synaptic protein mRNA follows early and persistent induction of pan-neuronal marker transcripts. In adrenal chromaffin cells pan-neuronal marker expression occurs only transiently and synaptic protein messages remain at distinctly low levels throughout embryogenesis. Embryonic induction of synaptotagmin I (Syt1) in sympathetic ganglia and postnatal upregulation of synaptotagmin VII (Syt7) in adrenal medulla results in a cell type-specific difference in isoform prevalence. Dicer 1 inactivation in catecholaminergic cells reduces high neuronal synaptic protein mRNA levels but not their neuroendocrine low level expression. Pan-neuronal marker mRNAs are induced in chromaffin cells to yield a more neuron-like transcript pattern, while ultrastructure is not altered. CONCLUSIONS: Our study demonstrates that remarkably different gene regulatory programs govern the expression of synaptic proteins in the neuronal and neuroendocrine branch of the sympathoadrenal system. They result in overlapping but quantitatively divergent transcript profiles. Dicer 1-dependent regulation is required to establish high neuronal mRNA levels for synaptic proteins and to maintain repression of neurofilament messages in neuroendocrine cells.

The development of the chromaffin cell lineage from the neural crest.

Chromaffin cells are neuroendocrine cells, which are highly specialized for the synthesis and release of multiple hormones. Like sympathetic neurons, which are essential, inter alia, for neural control of vascular tone, they are derivatives of the neural crest, a transient structure at the dorsal surface of the embryonic neural tube. Chromaffin cells and sympathetic neurons have many features in common, but are also distinct in several respects. This review provides a summary of similarities and differences regarding the development of chromaffin cells and sympathetic neurons, viewed from molecular and morphological perspectives. Two major, still not finally settled issues, are whether (1) the two related cell types arise from one common or two separate cell lineages of delaminating neural crest cells, (2) in the former case when does lineage segregation occur, and what are the molecules underlying their phenotypic diversification.

Study of migration of neural crest cells to adrenal medulla by three-dimensional reconstruction.

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.

Development of chromaffin cells depends on MASH1 function.

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.

Neuropeptide Y immunohistochemistry and ultrastructure of developing chromaffin tissue in the cloudy dogfish, Scyliorhinus torazame (Chondrichthyes, Elasmobranchii).

Ontogenetic changes in neuropeptide Y-like immunoreactivity (NPY-LI) were studied in chromaffin tissue of the cloudy dogfish, Scyliorhinus torazame. In adults and post-hatching juveniles, NPY-LI was demonstrated in chromaffin cells, but not in ganglion cells and supporting cells. Immunoreactive fibers were also found in the axillary body (the major chromaffin tissue) of the adult fish. During the embryonic period, NPY-LI was found at first in chromaffin tissue in the 34-mm stage. In this stage, cells in the periphery of the tissue were positive for NPY. Afterwards, changes were not observed in the topography and relative dominance of labelled cells in the tissue. Transmission electron microscopy of chromaffin tissue of the 26-mm stage showed an early phase of histogenesis in rudimental cell clusters composed of agranular cells and a few granular cells, i.e. pheochromoblasts. In the 43-mm stage, differentiation of the chromaffin tissue enabled ultrastructural classification of adrenalin-producing cells, noradrenalin-producing cells, ganglion cells, supporting cells, and unmyelinated nerve fibers. These results suggest that in the dogfish the appearance of NPY-LI in the developing sympathoadrenal system is related to differentiation of chromaffin cells.

Development of the adrenal gland in the tropical lizard Calotes versicolor.

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.

Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell lineage.

The developmental coexpression of galanin-like immunoreactivity with the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH) was studied in the avian embryo sympathoadrenal system using double-labeling immunocytochemistry. Galanin-like immunoreactivity is expressed by various catecholaminergic cell populations, namely sympathoblasts, chromaffin and small intensely fluorescent (SIF) cells, but not by principal neurons of the paravertebral sympathetic ganglia. Both galanin and somatostatin immunoreactivities are coexpressed in the adrenal and sympathetic ganglion primordia by the neural precursors, but the subsequent expression pattern of both peptides differs. Our results support the hypothesis that early sympathoblasts express a large repertoire of neuroactive substances and that the expression of these becomes restricted during further development as the sympathoblasts become principal neurons.

The influence of dexamethasone treatment of pregnant rats on the development of chromaffin tissue in their offspring during the fetal and neonatal period.

The aim of these examinations was to determine the influence of dexamethasone (Dx)-treatment of gravid females, on day 16 of gestation on the development of medullary chromaffin tissue of their fetuses and neonatal offspring. In conducting these investigations we used stereological as well as spectrofluorimetric measurements, in 20-day-old fetuses and 1-, 3-, 5-, 7-, 9-, 11-, 13- and 14-day-old neonatal rats. Single Dx-treatment (1.5 mg/kg bw) of the dams led to a significant decrease in body and adrenal weight of their fetuses and neonatal offspring, and also reduction of the medullary volume and the number of chromaffin cells during the entire period examined as a result of decreased cell proliferation in the fetal and early neonatal period (till the 5th day of age). The proliferative activity of the chromaffin cells was evaluated through the mitotic index after applying the cytostatic vincristine-sulphate. During the second neonatal week the mitotic index showed significantly higher values in comparison with the corresponding controls, which indicates that there is regeneration and recovery of the adrenal gland medulla. Adrenaline content in the adrenal gland tissue of offspring of Dx-treated dams was significantly reduced only on the 1st neonatal day. Thus, the change in blood glucocorticoid level of pregnant females after a single Dx injection during the period critical for development of the hypothalamo-pituitary-adrenal system in fetuses affects the development and kinetics of medullar chromaffin cell division.

Early expression of chromogranin A and tyrosine hydroxylase during prenatal development of the bovine adrenal gland.

The present study was undertaken to define the temporal pattern and distribution of cells positive for chromogranin A (CgA) and tyrosine hydroxylase (TH) in various developmental stages of fetal bovine adrenal gland. CgA is an acidic protein, co-stored and co-released with amines and a variety of peptide hormones and neurotransmitters in dense core vesicles of neural and endocrine cells and can be used as a marker for these cells and their malignant counterparts. TH is the rate-limiting enzyme in catecholamine biosynthesis and reflects noradrenergic differentiation. The expression of CgA and TH was examined by immunohistochemistry. CgA immunoreactivity appears first in 35-day-old bovine fetuses. By the end of the second month, CgA-labelled cells are scattered throughout the entire primordium of the adrenal gland, and at a fetal age of 85-91 days most of these cells concentrate in the developing adrenal medulla. From this stage onwards, immunoreactive cells of the marginal zone of the medulla exhibit significantly stronger CgA immunoreaction than the central area. TH immunoreactivity appeared in the adrenal primordium for the first time at the end of the second month of gestation. The distribution pattern of TH-positive cells was similar to that described for CgA, and no significant differences in topographical arrangement between TH- and CgA-positive cells can be detected. The results show that bovine adrenal chromaffin cells express CgA already during their earliest stages of development and prior to TH. The stronger immunoreaction of marginal adrenal medullary cells suggests an adrenalcortical effect of glucocorticoids on the expression of CgA.

Mammalian achaete-scute homolog 1 is required for the early development of olfactory and autonomic neurons.

The mouse Mash-1 gene, like its Drosophila homologs of the achaete-scute complex (AS-C), encodes a transcription factor expressed in neural precursors. We created a null allele of this gene by homologous recombination in embryonic stem cells. Mice homozygous for the mutation die at birth with apparent breathing and feeding defects. The brain and spinal cord of the mutants appear normal, but their olfactory epithelium and sympathetic, parasympathetic, and enteric ganglia are severely affected. In the olfactory epithelium, neuronal progenitors die at an early stage, whereas the nonneuronal supporting cells are present. In sympathetic ganglia, the mutation arrests the development of neuronal precursors, preventing the generation of sympathetic neurons, but does not affect glial precursor cells. These observations suggest that Mash-1, like its Drosophila homologs of the AS-C, controls a basic operation in development of neuronal progenitors in distinct neural lineages.

Perinatal development of galanin-like immunoreactivity in chromaffin tissues of the rabbit.

We have analyzed the perinatal development of galanin-like immunoreactivity (GAL-LI) and catecholamines (CA) in the paraaortal paraganglia (PGGL) and adrenal glands. In the PGGL, the tissue content of GAL-LI was highest on the day of birth and decreased postnatally. The fetal levels were lower than at birth. In contrast, the content of CA in the PGGL increased with age. In the adrenal glands, the contents of both GAL-LI and CA also increased with age. During the first postnatal week the contents of both GAL-LI and CA in the PGGL were markedly higher than in the adrenal glands. Chromatographic analysis of GAL-LI in extracts of fetal and postnatal rabbit PGGL, respectively, indicated that most of the GAL-LI from both age groups co-eluted with synthetic porcine GAL. An additional, apparently more polar, component was also detected at both ages, which may represent a differently processed form of the peptide. The high content of GAL-LI in the PGGL at birth may reflect an enhanced synthesis associated with birth.

The expression of neuropeptide Y immunoreactivity in the avian sympathoadrenal system conforms with two models of coexpression development for neurons and chromaffin cells.

We have studied the expression and development of neuropeptide Y-like immunoreactivity (NPY-LI) in the sympathoadrenal system of the chicken using single and double immunocytochemical techniques and radioimmunoassay. NPY-LI is expressed by neurons of the paravertebral sympathetic ganglia and by chromaffin cells of the adrenal gland in embryonic and adult chickens. The peptide is coexpressed with catecholaminergic properties in neurons. In chromaffin cells, it is also expressed with immunoreactivity to somatostatin and serotonin. We have used the expression of NPY-LI to analyze how cells that coexpress two or more neuroactive substances arrive at their final phenotype. Our results suggest that the ontogeny of coexpression in neurons of the avian paravertebral sympathetic ganglia occurs in a sequential pattern, where the expression of the peptide follows the initial expression of the "classical neurotransmitter". In contrast, in chromaffin cells, expression of the peptides occurs concomitantly with expression of catecholaminergic properties or soon after. Initially, coexpression of several neuroactive substances occurs, but this is followed by further specialization where the expression of one peptide prevails over the other. We believe that the two models of coexpression shown by our results can be used to describe the ontogeny of coexpression in other cells of the nervous system.

Evidence that enteric neurons may derive from the sympathoadrenal lineage.

The first neurons that differentiate in the embryonic foregut of mammals transiently express catecholamine biosynthetic enzymes and accumulate catecholamine. Since this transmitter is found predominantly in cells of the sympatho-adrenal (SA) lineage, it has been suggested that enteric and sympathetic neurons may derive from the same progenitor. Enteric neurons would then lose the catecholamine phenotype during further development, as the two lineages diverge. We have further investigated this possibility using the SA1 monoclonal antibody that binds selectively to SA progenitor cells in the embryonic rat. We find that SA1 binds to the tyrosine hydroxylase+, neurofilament+, and SCG10+ cells of the Embryonic Day 14.5 (E14.5) rat foregut. We also find that a marker for later neuronal differentiation in the SA lineage, B2, also appears in the myenteric plexus concomitant with the loss of SA1 staining. Thus, at least some enteric neuronal precursors may exhibit the SA1----B2 antigenic switch previously observed in developing sympathetic neurons at E14.5. SA1 staining in the foregut partially overlaps with staining for neuropeptide Y, vasoactive intestinal polypeptide, and serotonin. These results support the hypothesis that enteric and sympathetic neurons derive from a common progenitor and that as the markers for the SA lineage are down-regulated, the many types of enteric neurons begin to differentiate.

Asynchronous appearance and topographic segregation of neuropeptide-containing cells in the developing rat adrenal medulla.

The developmental expression of neuropeptide Y (NPY) and leucine-enkephalin (L-Enk) was examined in embryonic, early postnatal, and adult chromaffin cells with double- and triple-label immunocytochemical techniques and compared to the expression of immunoreactivity for tyrosine hydroxylase (TH) and phenylethanolamine N-methyltransferase (PNMT). In addition, the establishment of preganglionic innervation was assessed by labeling for choline acetyltransferase (ChAT) and L-Enk. NPY-IR was detectable on embryonic (E) day 15 in a clustered subpopulation of TH-IR cells. L-Enk and PNMT-IR cells were initially present on E16 in a separate nonclustered population of TH-IR cells. By late embryonic development, twice as many TH-IR cells expressed NPY and 4 times as many expressed L-Enk as in the adult. In contrast to early embryonic development, NPY-IR was evident in both the clustered and nonclustered subpopulation of TH-IR cells at this time. The proportion of NPY-IR chromaffin cells decreased to adult values during the first postnatal week at the time when obviously clustered TH-IR cells were no longer observed. The embryonic rise in the proportion of L-Enk-IR cells correlates with the developmental increase in glucocorticoid production, while the postnatal decrease corresponds to the appearance of ChAT-IR in the preganglionic innervation of the adrenal medulla. These results indicate that NPY and L-Enk are expressed at different times and in different subpopulations of cells in the embryonic adrenal. Further, the observation that peptide expression by chromaffin cells undergoes marked changes during development raises the possibility that a number of factors including developmental history, environmental signals and impulse activity play a role in the regulation of neuropeptide expression in sympathoadrenal derivatives of the neural crest.

The sympathoadrenal lineage in avian embryos. I. Adrenal chromaffin cells lose neuronal traits during embryogenesis.

Cells of the sympathoadrenal lineage, including sympathetic neurons, adrenal chromaffin cells (pheochromocytes), and small intensely fluorescent (SIF) cells, arise from the neural crest. We have used antisera against catecholamine biosynthesis enzymes in conjunction with the monoclonal antibody A2B5 and an antiserum against the 160-kDa neurofilament (NF) protein, as markers of neuronal differentiation, to characterize the ontogeny of the sympathoadrenal lineage in quail embryos. The precursors of sympathetic neurons and pheochromocytes, present in the primary sympathetic chains, express neuronal traits and tyrosine hydroxylase (TH) early in development. The precursors that enter the developing adrenal gland from the primary sympathetic chain lose neuronal traits and later express the enzyme phenylethanolamine N-methyltransferase (PNMT). No TH+ cells differentiate in cultures of early (E7) embryonic adrenal glands after all A2B5+ cells have been immunoablated. When transplanted onto the neural crest migratory pathway, cells present in older (E13) embryonic adrenal glands can give rise to NF+ cells in the sympathetic ganglia. We conclude that both sympathetic neurons and pheochromocytes in avian embryos arise from a common bipotential precursor that initially expresses neuronal traits.

Early fetal acquisition of the chromaffin and neuronal immunophenotype by human adrenal medullary cells. An immunohistological study using monoclonal antibodies to chromogranin A, synaptophysin, tyrosine hydroxylase, and neuronal cytoskeletal proteins.

The development of chromaffin and neuronal features in the adrenal medulla was studied in normal human fetuses with gestational ages (GAs) of 6-34 weeks. Monoclonal antibodies specific for chromogranin A, synaptophysin, and tyrosine hydroxylase; for different subunits and phosphoisoforms of neurofilament (NF) proteins; and for microtubule-associated proteins were applied. Morphologically, two major cell types could be distinguished, i.e., "large" cells with pale nuclei and ill-defined cytoplasm, which were present from 9 weeks GA on, and clusters of "small," primitive appearing cells, present from 14 weeks GA on. The large cells were immunoreactive for chromogranin A, synaptophysin, tyrosine hydroxylase, and NF proteins, similar to adult chromaffin cells. In contrast, small cells expressed NF proteins and tyrosine hydroxylase, but not chromogranin A or synaptophysin, more resembling ganglion cells in the adult adrenal medulla. At the latest developmental stages large cells were observed in the center of the clusters of "small" cells, which morphologically resembled immature ganglion cells and expressed NF proteins in their perikarya. These observations indicate that chromaffin and ganglion cells establish their immunophenotype early in embryogenesis. They suggest that "large" and "small" cells are progenitors of the chromaffin and the ganglion cells, respectively, of the mature adrenal medulla.

The prenatal development of the organ of Zuckerkandl in rats.

The effect of maternal glucocorticoid depletion upon the fetal development of the organ of Zuckerkandl (OZ) in rats was determined. Maternal hypophysectomy at 13d8h gestation resulted in a fifty percent decrease in plasma corticosterone levels at 18d8h when compared to both sham operated and unoperated controls. No differences in the volume of the OZ among the three groups of animals were found. The chromaffinity of the OZ was decreased in the hypophysectomized and sham operated groups suggesting a stress-induced depletion of catecholamine stores. The data suggests that the OZ participates in fetal sympathoadrenal activity and that its development is independent of maternal corticosterone titers.

Developmental potential of quail dorsal root ganglion cells analyzed in vitro and in vivo.

The cell types present in quail dorsal root ganglia during early development were identified using markers for neurons, glial cells, and fibroblasts (Rohrer et al., 1985). Using the quail-chick transplantation technique, the potential of quail dorsal root ganglion cells to differentiate to adrenergic chromaffin cells, as identified by tyrosine hydroxylase immunostaining, was analyzed. A population of undifferentiated cells, which is present in quail dorsal root ganglia at embryonic day 5, was separated from neurons and glial cells. We show that this population contains cells that differentiate to adrenergic chromaffin cells after back-transplantation into 2-d-old chick embryos. A large proportion of these undifferentiated cells also differentiates to neurons in vitro. Precursors for adrenal chromaffin cells and for neurons are present in dorsal root ganglia in significant numbers only during early development.

Acetylcholinesterase and pseudocholinesterases in developing chick adrenal.

The distribution of acetylcholinesterase (AChE) and pseudocholinesterases (PsChE) in chick adrenal gland during the first phases of organogenesis was studied. Acetylthiocholine iodide and butyrylthiocholine iodide were used as substrates for the two enzymes, respectively, whereas BW284c51 (1,5 bis (4-allyldimethylammonium-phenyl)pentan-3-one-dibromide) and ISO-OMPA (tetraisopropylpyrophosphoramide) were used as respective inhibitors of AchE and PsChE. AchE was present on the plasma membrane, in the perinuclear cisterna and in some cisternae of the rough endoplasmic reticulum of both interrenal and chromaffin cells; moreover enzymatic activity was found in the same sites of ganglion cells and mesenchymatic undifferentiated cells, i.e. on the inside and in the proximity of the glandular anlage. PsChE activity was localized in the perinuclear space and in the rough endoplasmic reticulum of all types of cells in the anlage. It is suggested that these enzymatic activities may be implicated in morphogenetic mechanisms.