Immunocytochemical developmental patterns of

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diaminobenzidine (Frigo et al., 1991). Controls consisted of sequential deletion of the various immunoreagent layers and replacement of the primary polyclonal.
Histology and Histopathology

Histol Histopathol (2005) 20: 383-392

http://www.hh.um.es

Cellular and Molecular Biology

Immunocytochemical developmental patterns of the thoracolumbar sympathetic chain in the chick and a comparison with its adrenal counterpart I. Sánchez-Montesinos1, J.R. Mérida-Velasco2, F. Hita-Contreras1, J. Espín-Ferra1, J.F. Rodríguez-Vázquez2, C. De La Cuadra1, B. Pasini3 and J.A. Mérida-Velasco1 1Human

and Experimental Embriology Research Group, Department of Human Anatomy and Embryology, University of Granada,

Granada, Spain, 2Morphofunctionals and Sports Sciences Institute, Complutense University of Madrid, Madrid, Spain and 3Department

of Genetic, Biology and Biochemistry, University of Torino, Italy

Summary. 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-α 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-α 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-ß-hydroxylase, neuron-specific enolase, vasoactive intestinal polypeptide, and met-enkephalin), differences in immunoreactivity were observed between the medullary and thoracolumbar sympathetic ganglion cells. Key words: Thoracolumbar sympathetic ganglion,

Development, Immunohistochemistry, Neuroendocrine markers Offprint requests to: Prof. Indalecio Sánchez-Montesinos García, Departamento de Anatomía y Embriología Humana, Facultad de Medicina, Avd. de Madrid 11, 18071 Granada, Spain. Fax: +34 958243257. e-mail: [email protected]

Introduction

The immunocytochemical development of the avian thoracolumbar sympathetic ganglion has been described in terms of the expression of catecholaminergic properties (Fauquet and Ziller, 1989). Neural crest cells that migrate to the area dorsolateral to the aorta are known to form thoracolumbar sympathetic ganglionic primordia between days 3 and 4 of incubation (Hammond and Yntema, 1947; Le Douarin and Smith, 1983) and adrenal medulla primordia about 24 h later (Lillie, 1965; Le Douarin and Smith, 1983). Chromaffin cells synthesize, store, and secrete a complex mixture containing amines, structural proteins, enzymes, and neurohormonal polypeptides. The vast majority of studies have been performed on mammals (for a review, see Kondo, 1985; Hacker et al., 1988; Pelto-Huikko, 1989), and only a few recent papers have dealt with avian species, focusing mainly on somatostatin (SMS), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), enkephalin (P-ENK), and galanin (GAL). These studies, among others, provided evidence that the range of expression of neuroactive substances becomes restricted during the development of the cells into mature neurons, with a loss of SMS, PENK, and GAL expression and a gain in the expression of other neuropeptides such as NPY (Fontaine Pérus, 1984; García-Arrarás et al., 1984, 1987, 1992; Maxwell et al., 1984; New and Mudge, 1986; García-Arrarás and Martínez, 1990; Ross et al., 1990; Barreto-Estrada et al., 1997). In the rat, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DßH) were detected in the anlage of the sympathetic ganglion from day 14 onwards (Vogel and Weston, 1990a). This mixture was reported to be modulated by neural and hormonal signals (Unsicker, 1993). Encouraged by the results of our previous research (Sánchez-Montesinos et al., 1996), our group

384

Thoracolumbar sympathetic chain in the chick

immunocytochemically studied the development of the thoracolumbar sympathetic ganglion in the chick embryo with the same panel of antibodies: antibodies to panneuroendocrine markers (chromogranin-A [CgA], chromogranin-B [CgB], secretogranin II [SgII], protein 7B2 [7B2], neuron-specific enolase [NSE], HNK-1 antigen [HNK-1], protein Go-α subunit [Go-α]), catecholamine synthesizing enzymes (TH, DßH, phenylethanolamine N-methyltransferase [PNMT]), proprotein-processing enzymes (proprotein convertases 2 [PC2] and 3 [PC3]), and neuropeptides (SMS, GAL,

NPY, P-ENK, and VIP). Materials and methods

Sixty White Leghorn chick embryos were studied, ten for each stage (days 3.5, 4, 5, 7, 10, and 12 of incubation). Immediately after being removed from the shell, the embryos were fixed in Bouin's solution for 3 to 10 hours, washed, dehydrated in graded ethanols, and embedded in paraffin. Serial 10 µm-thick sections were cut, deparaffinized, and hydrated in 0.05 M Tris-buffered

Table 1. Characteristics of the antibodies to pan-neuroendocrine markers. SERUM/CLONE

DIRECTED AGAINST

IMMUNOGEN

A11

Chromogranin A (CgA)

Chromaffin granules from human pheochromocytoma

Mouse

Biogenesis, Bournemouth, England (see also Pelagi et al., 1989)

B11

Chromogranin B (CgB)

Chromaffin granules from human pheochromocytoma

Mouse

Biogenesis, Bournemouth, England (see also Pelagi et al., 1989)

5A7

Secretogranin II (SgII)

SgII-enriched fraction from bovine anterior pituitary homogenates

Mouse

See Pelagi et al., 1992

SA 1100

Protein 7B2

Porcine 7B2 [23-39] coupled to BSA

Rabbit

Affiniti Res. Prod. Ltd.,Ilkeston, UK

RAP Go

Alpha subunit of Go protein

Purified Go alpha subunit from bovine brain

Rabbit *

see Kato et al., 1987

RAISED IN

SOURCE

BBS/NC/VI-H14

Neuron-specific enolase (NSE)

Purified human brain NSE

Mouse

Dakopatts A/S Glostrup, DK

HNK-1

3-sulfuglucuronic acid

Membrane extract of human lymphoblastoid cell line

Mouse

Becton & Dickinson, CA, USA

* Affinity-purified immunoglobulins

Table 2. Characteristics of the antibodies to proprotein-processing and catecholamine-synthesizing enzymes. SERUM/CLONE

DIRECTED AGAINST

IMMUNOGEN

RAISED IN

SOURCE

2/40/15

Tyrosine hydroxylase (TH)

Purified TH from a rat pheochromocytoma

Mouse

Boehringer Mannheim (D)

DZ 1020

Dopamine beta-hydroxylase (DßH)

Purified DßH from bovine adrenal

Rabbit

Eugene Tech Intern. Inc.

PZ 1040

Phenylethanolamine N-methyltransferase (PNMT)

Purified PNMT from bovine adrenal

Rabbit

Eugene Tech Intern. Inc.

RS20

Proprotein convertase-3 (PC3) coupled to KLH

PreproPC3 Cys[95-108] + preproPC3 [110-122]

Rabbit

See Smeekens et al., 1992

PC2-P4

Proprotein convertase-2 (PC2)

PreproPC2 [611-638] (mouse and human combined)

Rabbit

See Smeekens et al., 1992

Table 3. Characteristics of the antibodies to neuropeptides. SERUM/CLONE

DIRECTED AGAINST

IMMUNOGEN

RAISED IN

SOURCE

CA-08-325

Somatostatin

Cyclic somatostatin-14 coupled to HSA

Rabbit

Cambridge Res. Biochemicals

RAS 7141N

Galanin

Synthetic rat galanin

Rabbit

Peninsula Lab. Inc., CA, USA

NPY02

Neuropeptide tyrosine (NPY)

h NPY [1-36] amide coupled to KLH

Mouse

See Grouzman et al., 1989

PE-25

Pro-enkephalin

h preproenkephalin [1-267] (beta-galactosidase fusion protein)

Mouse

See Spruce et al., 1990

i604/004

Vasoactive intestinal polypeptide (VIP)

Synthetic VIP coupled to bovine thyroglobulin

Rabbit

UCB Biopro ducts, Belgium

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Thoracolumbar sympathetic chain in the chick

saline (TBS), pH 7.4. Endogenous peroxidase activity was suppressed as previously detailed (Frigo et al., 1991). Incubation with the primary antibodies (Tables 13) was carried out overnight at 4 ºC and was followed by the peroxidase-labeled avidin-biotin complex (ABC) system, with development in plain or silver-enhanced diaminobenzidine (Frigo et al., 1991). Controls consisted of sequential deletion of the various immunoreagent layers and replacement of the primary polyclonal (serum) antibodies either with the flow-through of affinity-purified serum or with serum pre-absorbed with an excess of the respective immunogens. The reactivity of the monoclonal antibodies was checked by substitution with unrelated isotype-matched immunoglobulins at the same concentration. Results

Developmental patterns of the thoracolumbar sympathetic ganglion of the chick from days 3.5 to 12 of incubation were compared with those of its adrenal counterpart, previously studied by our group (see Sánchez-Montesinos et al., 1996). Results of the qualitative analyses are summarized in Tables 5-7. At day 3.5 of incubation, cells from the thoracolumbar sympathetic chain began to migrate ventromedially, reaching the adrenal cortical primordia. At this time, the two primordia were clearly separated. At day 4 of incubation, the thoracolumbar sympathetic ganglion cells, which continued to migrate

ventromedially, were strongly immunoreactive for CgA, CgB, Go-α, HNK-1, TH, and GAL. VIP presented a weak immunoreactivity, whereas 7B2 and SMS presented a moderate immunoreactivity. No immunoreactivity for SgII, NSE, PC2, PC3, DßH, PNMT, NPY, or P-ENK was found in the thoracolumbar sympathetic chain at this stage. At day 5, thoracolumbar sympathetic ganglion cells were strongly immunoreactive for Go-α (Fig. 1.A,B), TH (Fig. 2.A,B), SMS, and GAL (Fig. 3A,B), whereas they showed moderate immunoreactivity for CgA, CgB, 7B2, HNK-1, and DßH, and a weak immunoreactivity for PC2 and VIP. No immunoreactivity for SgII, NSE, PC3, PNMT, P-ENK and NPY was detected in these cells at this stage. At day 7, immunoreactivity for CgA, CgB, NSE, Go-α (Fig. 1C,D), HNK-1, TH (Fig. 2C,D), SMS, GAL (Fig. 3C,D) and VIP was very similar to that at day 5 except for DßH, which presented no immunoreactive cells in thoracolumbar sympathetic ganglion. The immunoreactivity was moderate for SgII and PC2 and strong for 7B2. No immunoreactivity for PC3, PNMT, NPY, or P-ENK was found in these cells at this stage. At day 10, the immunoreactivity of thoracolumbar sympathetic ganglion cells followed the trend observed at day 7, with a strong immunoreactivity for 7B2, Go-α, TH, and GAL. NSE immunoreactivity was not found in thoracolumbar sympathetic ganglion cells but appeared within the nervous system in the neuronal bodies of the spinal cord (mostly in the anterior horns). No

Table 4. Developmental phases of adrenal and thracolumbar sympathetic gangiion cells.

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Thoracolumbar sympathetic chain in the chick

Fig. 1. Immunoreactivity patterns for pan-neuroendocrine markers in chick thoracolumbar sympathetic ganglion: protein Go-α subunit [Go-α]: A and B, on day 5 of incubation; C and D, on day 7; E and F, on day 12. s: spinal cord, v: vertebral body, n: notochord, t: thoracolumbar sympathetic ganglion, a: dorsal aorta. A, C, E, x 10; B, D, F, x 40

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Thoracolumbar sympathetic chain in the chick

immunoreactivity for DßH, PNMT, NPY and P-ENK was found at this stage in any of the structures present in the examined sections. Thoracolumbar sympathetic ganglion cells showed a strong immunoreactivity for HNK-1, SgII, PC2, and a moderate one for PC3, CgA, CgB, and SMS. Finally, no VIP immunoreactivity was detected in the adrenal medulla, although it was already present at day 4, with a weak immunoreactivity in thoracolumbar sympathetic ganglion cells, peripheral nerves, and intestinal plexuses. At day 12 (Figs. 1E,F, 2E,F, 3E,F), the immunoreactivity in thoracolumbar sympathetic ganglion was similar to that at day 10, except that no immunoreactivity for CgA, CgB and VIP was detected and the immunoreactivity for 7B2 decreased to moderate. The PC3 enzyme was also observed at day 12 in gut epithelial cells. No NSE immunoreactivity was observed in thoracolumbar sympathetic ganglion cells, whereas NSE-immunoreactive cell bodies in the spinal cord increased. No VIP immunoreactivity was found in the thoracolumbar sympathetic ganglion at this stage, although it remained present in peripheral nerves and

Table 5. Developmental patterns of immunoreactivities for panneuroendocrine markers (chromogranin-A [CgA], chromogranin-B [CgB], secretogranin II [SgII], protein 7B2 [7B2], neuron-specific enolase [NSE], HNK-1 antigen [HNK-1], and protein Go-α subunit [Go-α]) in chick thoracolumbar sympathetic ganglion at days 4, 5, 7, 10 and 12 of incubation.

CgA-B SgII 7B2 NSE Go-a HNK-1

4

5

7

10

12

+++ ++ +++ +++

++ ++ +++ ++

++ ++ +++ +++ ++

++ +++ +++ +++ +++

+++ ++ +++ +++

Expression: Negative (-), Weak (+), Moderate (++), Strong (+++).

Table 6. Developmental patterns of immunoreactivities for proproteinprocessing enzymes (proprotein convertases 2 [PC2] and 3 [PC3]) and catecholamine-synthesizing enzymes (tyrosine hydroxylase [TH], dopamine beta-hydroxylase [DßH], and phenylethanolamine Nmethyltransferase [PNMT]) in chick thoracolumbar sympathetic ganglion at days 4, 5, 7, 10, and 12 of incubation.

PC2 PC3 TH DßH PNMT

4

5

7

10

12

+++ -

+ +++ ++ -

++ +++ -

+++ +++ +++ -

+++ +++ +++ -

Expression: Negative (-), Weak (+), Moderate (++), Strong (+++).

intestinal plexuses. Discussion

Immunocytochemical techniques were applied to study the time of appearance, the distribution, and the developmental pattern of 17 separate antigens in the thoracolumbar sympathetic ganglion. Some of the antigens studied were strongly expressed from the first days of development, while others displayed a widely variable immunoreactive expression at the different stages. In the chick, cells of the sympathetic chains at the thoracolumbar level are derived from the neural crest (Hammond and Yntema, 1947; Weston, 1970; Le Douarin, 1982; Le Douarin and Smith, 1983). The sympathetic cells give rise to medullary cells of the adrenal gland (Le Douarin, 1980; Le Douarin and Smith, 1983). In birds, migrating neural crest cells are initially cholinergic, but as the cells coalesce to form the sympathetic ganglion and medullary cells of the adrenal gland, they start to express catecholaminergic properties (Fauquet et al., 1981; Smith et al., 1993). Neurons of the sympathetic ganglion and chromaffin cells of the adrenal medulla are of sympathoadrenal lineage (Grillo, 1966; Eränkö, 1976; Landis and Patterson, 1981; Coupland, 1989; Unsicker et al., 1989; Unsicker, 1993; Stemple and Anderson, 1993). These cell types share several characteristics, including the expression of catecholamines. The results of Vogel and Weston (1990a,b) are consistent with the hypothesis that components of the sympathoadrenal lineage arise in two steps from a subpopulation of crest-derived cells that initially express neuronal traits. Hence, we were able to establish the following developmental pattern of adrenal and thoracolumbar sympathetic ganglion cells (Table 4): a first pluripotential cell phase to day 5 of development; a second phase of divergence between thoracolumbar sympathetic ganglion and adrenal gland development from days 5 to 7; a third phase of differentiation from days 7 to 10; and finally, from day 10 onward, the start of the functional differentiation phase. Thus, day 5 of

Table 7. Developmental patterns of immunoreactivities for neuropeptides (somatostatin [SMS], galanin [GAL], Neuropeptide tyrosine [NPY], Pro-enkephalin [P-ENK], and vasoactive intestinal polypeptide [VIP]) in chick thoracolumbar sympathetic ganglion at days 4, 5, 7, 10, and 12 of incubation.

SMS GAL NPY P-ENK VIP

4

5

7

10

12

++ +++ +

+++ +++ +

+++ +++ +

++ +++ +

++ +++ -

Expression: Negative (-), Weak (+), Moderate (++), Strong (+++).

388

Thoracolumbar sympathetic chain in the chick

Fig. 2. Immunoreactivity patterns for proprotein-processing enzymes in chick thoracolumbar sympathetic ganglion: tyrosine hydroxylase [TH]: A and B, on day 5 of incubation; C and D, on day 7; E and F, on day 12. s: spinal cord, v: vertebral body, n: notochord, t: thoracolumbar sympathetic ganglion. a: dorsal aorta. A, C, E, x 10; B, D, F, x 40

389

Thoracolumbar sympathetic chain in the chick

Fig. 3. Immunoreactivity patterns for neuropeptides in chick thoracolumbar sympathetic ganglion: galanin [GAL]: A and B, on day 5 of incubation; C and D, on day 7; E and F, on day 12. s: spinal cord, v: vertebral body, n: notochord, t: thoracolumbar sympathetic ganglion, a: dorsal aorta. A, C, E, x 10; B, D, F, x 40

390

Thoracolumbar sympathetic chain in the chick

development marks the time when pluripotential cells diverge and are organized for subsequent differentiation (days 7-10) into neurons of the thoracolumbar sympathetic chain (nervous function) or adrenal gland cells (endocrine or neuroendocrine function). In the pluripotential phase, adrenal gland and thoracolumbar sympathetic chain cells show the same immunoreactivity patterns, reflected in the expression of pan-neuroendocrine type neuronal and neuroendocrine markers (CgA, CgB, Go-α, HNK-1), proproteinprocessing enzymes (TH, DßH), and neuropeptides (GAL). Thus, we observed an increase in DßH immunoreactivity in both adrenal gland and thoracolumbar sympathetic ganglion cells at day 5. In the rat, however, adrenal gland DßH was not detected in anlage of adrenal gland or sympathetic ganglion until day 14 (Vogel and Weston, 1990a). In agreement with results in avians published by Iacovitti et al. (1987), Barreto-Estrada et al. (1997), and García-Arrarás and Torres-Avillán (1999), a strong immunoreactivity for TH was observed in all cells in adrenal gland and thoracolumbar sympathetic ganglion from days 4 to 12 of development. On the other hand, Vogel and Weston (1990a) reported that cells in adrenal glands of avian embryo expressed TH early in development and that the proportion of cells in the adrenal gland expressing TH immunoreactivity declined during embryogenesis. In the rat, TH was not detected in the anlage of adrenal gland or thoracolumbar sympathetic ganglion until day 14 (Vogel and Weston, 1990a). In agreement with data from Barreto-Estrada et al. (1997), GAL immunoreactivity was first detected in primary sympathetic chain cells from day 4 of development. We observed a strong immunoreactivity for GAL in cells of the thoracolumbar sympathetic ganglion from days 4 to 12 of development. As pointed out by García-Arrarás and Torres-Avillán (1999), the fact that all cells within the early embryonic sympathetic ganglion express TH and GAL supports the hypothesis that these neuroactive substances are expressed as part of the sympathoblast developmental process and will eventually differentiate into the principal neurons of the adult. We detected no P-ENK immunoreactivity in thoracolumbar sympathetic chain, in contrast to the moderate expression presented by adrenal gland cells, whereas Barreto-Estrada et al. (1997) found that it appeared at day 6, decreased during development, and was weaker than the immunoreactivity for GAL. From days 5 to 7 of development, when the pluripotential cells develop as either neuronal cells of the thoracolumbar sympathetic chain or as endocrine or neuroendocrine cells to constitute the adrenal gland, there is a change in expression of the markers, characterized by a rapid increase in the SgII, 7B2 and PC2 immunoreactivity of thoracolumbar sympathetic ganglion, and an increase in 7B2 and absence of SgII

immunoreactivity in adrenal gland. The pattern differs from day 7 onward, with no DßH immunoreactivity in the thoracolumbar sympathetic ganglion cells and an increase in the adrenal gland cells. We consider, therefore, that the divergence phase ends at day 7 and the differentiation phase begins (days 7-10). Furthermore, Shirley et al. (1996) observed that all neuronal precursors and neurons of chick sympathetic ganglion expressed TH immunoreactivity at day 7 of development. These data are not consistent with the existence of a precursor for pheochromocytes that initially exhibits neuronal traits and then loses them under the influence of environmental conditions within the adrenal gland (Vogel and Weston, 1990a). The functional differentiation phase takes place between days 10 and 12 of development, characterized by a rapid increase in PC3 immunoreactivity in both thoracolumbar sympathetic ganglion cells and adrenal gland cells. We observed a strong immunoreactivity for chromogranins (CgA and CgB) and a moderate one for P-ENK at day 12 in adrenal gland cells, whereas no immunoreactivity for CgA, CgB or P-ENK was detected at this stage in thoracolumbar sympathetic ganglion cells. Chromaffin cells (and their tumor counterparts) synthesize, store, and release a complex palette of peptides, most notably chromogranins and a large number of neuropeptides including P-ENK, SMS, and NPY. This peptide mix can apparently be modulated by neural and hormonal signals (Unsicker, 1993). In the rat adrenal gland, PNMT was not expressed until days 17-18 of gestation. The immunoreactive expression for PNMT (Bohn et al., 1982; Seidl and Unsicker, 1989a) were found to increase between days 17 and 21, and were restricted to sympathoblasts populating the adrenal gland (Vogel and Weston, 1990a). In the present study, no PNMT immunoreactivity was detected in the thoracolumbar sympathetic ganglion cells from days 4 to 12. Therefore, an ontogenetic increase and persistence of PNMT activity in adrenal medullary cells would depend on glucocorticoids (Bohn et al., 1981; Seidl and Unsicker, 1989a,b), although the initial expression of PNMT by embryonic rat pheochromocytes was not dependent on glucocorticoids or pituitary function (Bohn et al., 1981). This conclusion was based on the finding that expression of the adrenergic phenotype cannot be prematurely triggered by high doses of glucocorticoids (Bohn et al., 1981). Thus, Hofmann et al. (1989) emphasized the role of glucocorticoids in the initiation, development, and maintenance of the endocrine chromaffin phenotype, and as the decisive signal for the initial induction of endocrine differentiation. Moreover, high steroid hormone concentrations, present in adrenal medulla but not in thoracolumbar sympathetic chain ganglion , may be a prerequisite for the maturation of chromaffin cells. Our results for SMS and NPY are in disagreement with those of García Arrarás et al. (1984), Maxwell et al. (1984), and Ross et al. (1990), who reported that SMS

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immunoreactivity was present in the early embryo in both adrenal and sympathetic ganglion primordia but disappeared from the sympathetic ganglion during development and was found only in chromaffin cells after hatching. We observed that the SMS immunoreactivity pattern in the thoracolumbar sympathetic ganglion (increasing from day 4 to 7, then decreasing to a plateau between days 10 and 12) was the inverse of that in the glands (full antigenic expression on day 10). García Arrarás et al. (1984), Maxwell et al. (1984) and Ross et al. (1990) described NPY in both sympathetic ganglion and gland cells during embryonic stages, whereas we detected NPY immunoreactivity only in the adrenal gland from day 10 onward. Finally, we established three expression patterns in both the adrenal medulla (Sánchez-Montesinos et al., 1996) and the thoracolumbar sympathetic ganglion. The first pattern pertains to CgA, CgB, Go-α, TH, HNK-1, and GAL; virtually all medullary and thoracolumbar sympathetic ganglion cells were strongly immunostained from day 4 onwards. Except for HNK-1, CgA and CgB, cells immunoreactive for all remaining antigen showed a steady increase up to day 12. The second pattern (7B2, PC2, and SgII) showed full antigenic expression in medullary and thoracolumbar sympathetic ganglion cells by day 10. The third pattern showed differences between some antigens: a) PC3 immunoreactivity was detected in medullary cells from day 12, and full antigenic expression was reached in thoracolumbar sympathetic ganglion cells by day 10; b) the SMS immunoreactivity pattern in thoracolumbar sympathetic ganglion (increasing from day 4 to 7, then decreasing to a plateau between days 10 and 12) was inverse that in adrenal gland (full antigenic expression by day 10); c) DßH immunoreactivity was increased in both adrenal gland and thoracolumbar sympathetic ganglion at day 5, although the pattern differed from day 7 onward, with an increase in adrenal gland and disappearance in thoracolumbar sympathetic ganglion; d) P-ENK immunoreactivity was detected in adrenal medullary gland from days 4 to 7 followed by an increase to a plateau between days 10 and 12, while no P-ENK immunoreactivity was found in the thoracolumbar sympathetic chain. Acknowledgements. This work was supported by a grant (PI02/0492) from the Fondo de Investigaciones Sanitarias (FIS) of the Instituto de Salud Carlos III (Ministerio de Sanidad y Consumo, Spain) and from the European Union (FEDER). The skillful technical assistance of Morena Gobbo and Barbara Vergani is gratefully acknowledged. We are grateful to Richard Davies for assistance with the English version. We are indebted to the following colleagues for the generous gift of antibodies: T. Asano (Department of Biochemistry, Aichi Prefectural Colony, Japan), E. Comoy (Departement de Biologie Clinique, Institut Gustave Roussy, Villejuif, France), M. Pelagi (DIBIT, S. Raffaele Institute, Milan, Italy), B.A. Spruce (Department of Biochemistry, University of Dundee, UK), D.F. Steiner (Department of Biochemistry and Molecular Biology, The Howard Hughes Medical Institute, Chicago, IL, USA).

References Barreto-Estrada J.L., Medina-Vera L., De Jesús-Escobar J.M. and García-Arrarás J.E. (1997). Development of galanin- and enkephalin-like immunoreactivities in the sympathoadrenal linage of the avian embryo: in vivo and in vitro studies. Dev. Neurosci. 19, 328-336. Bohn M.C., Goldstein M. and Black I.B. (1981). Role of glucocorticoids in expression of the adrenergic phenotype in rat embryonic adrenal gland. Dev. Biol. 82, 1-10. Bohn M.C., Goldstein M. and Black I.B. (1982). Expression of phenylethanolamine N-methyltransferase in rat sympathetic ganglion and extradrenal chromaffin tissue. Dev. Biol. 89, 299-308. Coupland R.E. (1989). The natural history of the chromaffin cell-Twentyfive years on the beginning. Arch. Histol. Cytol. 52, 331-341. Eränkö O. (1976). SIF cells. Structure and function of the samall, intensely fluorescent sympathetic cells. Fogarty International Center Proceedings no 30, DHEW Publication No (NIH) pp 76-942. Fauquet M. and Ziller C. (1989). A monoclonal antibody directed against quail tyrosine hidroxylase. Description and use in immunocytochemical studies on differentiation of neural crest cells. J. Histochem. Cytochem. 37, 1197-1205. Fauquet M., Smith J., Ziller C. and Le Douarin N.M. (1981). Differentiation of autonomic neuron precursors in vitro: Cholinergic and adrenergic traits in cultured neural crest cells. J. Neurosci. 1, 478-492. Fontaine-Pérus J. (1984). Development of VIP in the peripheral nervous system of avian embryos. Peptides 5, 195-200. Frigo B., Scopsi L., Patriarca C. and Rilke F. (1991). Silver enhancement of nickel-diaminobenzidine as applied to single and double immunoperoxidase staining. Biotech. Histochem. 1, 159-166. García-Arrarás J.E. and Martínez R. (1990). Developmental expression of serotinin-like immunoreactivity in the sympathoadrenal system of the chicken (Gallus gallus). Cell Tissue Res. 262, 363-372. García-Arrarás J.E. and Torres-Avillán I. (1999). Developmental expression of galanin-like immunoreactivity by members of the avian sympathoadrenal cell linage. Cell Tissue Res. 295, 33-41. García-Arrarás J.E., Chancoine M. and Fontaine-Pérus J. (1984). In vivo and in vitro development of somatostatin-like-immunoreactivity in the peripheral nervous system of quail embryos. J. Neurosci. 4, 1549-1558. García-Arrarás J.E., Chancoine M., Ziller C. and Fauquet M. (1987). In vivo and in vitro expression of vasoactive intestinal polypeptide-like immunoreactivity by neural crest derivates. Dev. Brain. Res. 33, 255-265. García-Arrarás J.E., Lugo-Chinchilla A.M. and Chévere-Colón I. (1992). The expression of neuroptide Y immunoreactivity in the avian sympathoadrenal system conforms with two models of coexpression development for neurons and chromaffin cells. Development 115, 617-627. Grillo M.A. (1966). Electron microscopy of sympathetic tissues. Pharmacol. Rev. 18, 387-399. Grouzman E., Comoy E. and Bohuon C. (1989). Plasma neuropeptide Y concentrations in patients with neuroendocrine tumors. J. Clin. Endocrinol. Metab. 68, 808-813. Hacker G., Bishop A.E., Terenghi G., Varndell I.M., Aghahowa J., Pollard K., Thurner J. and Polak J.M. (1988). Multiple peptide production and presence of general neuroendocrine markers detected in 12 cases of human pheochromocytoma and in

392

Thoracolumbar sympathetic chain in the chick

mammalian adrenal glands. Virchows Arch. (A) 412, 399-411. Hammond W.S. and Yntema C.L. (1947). Depletions in the thoraco lumbar sympathetic system following removal of neural crest in the chick. J. Comp. Neurol. 86, 237-266. Hofmann H.D., Seidl K. and Unsicker K. (1989). Development and plasticity of adrenal chromaffin cells: cues based on in vitro studies. J. Electron. Microsc. Tech. 12, 397-407. Iacovitti L., Teitelman G., Joh T.H. and Reis D.J. (1987). Chick eye extract promotes expression of a cholinergic enzyme in sympathetic ganglion in culture. Dev. Brain Res. 33, 59-65. Kato K., Asano T., Kamiya N., Haimoto H., Hosoda S., Nagasaka A., Ariyoshi Y. and Ishiguro Y. (1987). Production of the alpha subunit of guanine nucleotide-binding protein G by neuroendocrine tumors. Cancer Res. 47, 5800-5805. Kondo H. (1985). Immunohistochemical analysis of the localization of neuropeptides in the adrenal gland. Arch. Histol. Jap. 48, 453-481. Landis S.C. and Patterson P.H. (1981). Neural crest cell lineages. TINS 7, 172-175. Le Douarin N.M. (1980). The ontogeny of the neural crest in avian embryo chimeras. Nature 286, 663-669. Le Douarin N.M. (1982). The Neural Crest. Cambridge: Cambridge University Press. Le Douarin N.M. and Smith J. (1983). Differentiation of avian autonomic ganglion. In: Autonomic ganglion. Elvin L. (ed). Wiley and Sons Ltd. New York. pp 427-452. Lillie F.R. (1965). Lillie's Development of the Chick. An Introduction to Embryology. 3rd ed. Holt, Rinehart and Winston. New York. pp 501504. Maxwell G.D., Sietz P.D. and Chenard P.H. (1984). Development of somatostatin-like immunoreactivity in embryonic sympathetic ganglion. J. Neurosci. 4, 576-584. New H.V. and Mudge A.W. (1986). Distribution and ontogeny of SP, CRP, SOM and VIP in chick sensory and sympathetic ganglion. Dev. Biol. 116, 337-346. Pelagi M., Bisiani C., Gini A., Bonardi M.A., Rosa P., Mare P., Viale G., Cozzi M.G., Salvadore M., Zanini A., Siccardi A.G. and Buffa R. (1989). Preparation and characterization of anti-human chromogranin A and chromogranin B (secretogranin I) monoclonal antibodies. Mol. Cell Probes 3, 87-101. Pelagi M., Zanini A., Gasparri A., Ermellino L., Giudici A.M., Ferrero S., Siccardi A.G. and Buffa R. (1992). Immunodetection of secretogranin II in animal and human tissues by new monoclonal antibodies. Reg. Pept. 39, 201-214. Pelto-Huikko M. (1989). Immunocytochemical localization of neuropeptides in the adrenal medulla. J. Electr. Micr. Tech. 12, 364-379. Ross S., Fischer A. and Unsicker A. (1990). Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure, and quantitative

determinations of catecholamine and somatostatin. Cell Tissue Res. 260, 147-159. Sánchez-Montesinos I., Mérida-Velasco J.A., Espín-Ferra J. and Scopsi L. (1996). Development of the sympathoadrenal system in the chick embryo. An immunocytochemical study with antibodies to panneuroendocrine markers, catecholamine-synthesizing enzymes, proprotein-processing enzymes, and neuropeptides. Anat. Rec. 245, 94-101. Seidl K. and Unsicker K. (1989a). The determination of the adrenal medullary cell fate during embryogenesis. Dev. Biol. 136, 481-490. Seidl K. and Unsicker K. (1989b). Survival and neuritic growth of sympathoadrenal (chromaffin) precursor cells in vitro. Int. J. Dev. Neurosci. 7, 465-473. Shirley M.L., Campbell J.D. and Hall A.K. (1996). Developing sympathetic neurons express a neuronal trait before a catecholaminergic synthetic enzyme in vivo. J. Neurosci. Res. 46, 42-48. Smeekens S.P., Montag A.G., Thomas G., Albiges-Rizo C., Carrol R., Benig M., Phillips L.A., Martin S., Ohagi S., Gardner P., Swift H.H. and Steiner D.F. (1992). Proinsulin processing by the subtilisinrelated proprotein convertases furin, PC2, and PC3. Proc. Natl. Acad. Sci. USA 89, 8822-8826. Smith J., Vyas S. and García-Arrarás J.E. (1993). Selective modulation of cholinergic properties in cultures of avian embryonic sympathetic ganglion. J. Neurosci. Res. 34, 346-356. Spruce B.A., Curtis R., Wilkin G.P. and Glover D.M. (1990). A neuropeptide precursor in cerebellum: proenkephalin exists in subpopulations of both neurons and astrocytes. EMBO J. 9, 17871795. Stemple D.L. and Anderson D.J. (1993). Lineage diversification of the neural crest: In vitro investigations. Dev. Biol. 159, 12-23. Unsicker K. (1993). The chromaffin cell: paradigm in cell, developmental and growth factor biology. J. Anat. 183, 207-221. Unsicker K., Seidl K. and Hofmann H.D. (1989). The neuro-endocrine ambiguity of sympatho adrenal cells. Internat. J. Dev. Neurosci. Special issue. Unsicker K. (ed) “Development and plasticity of sympathoadrenal cells”. pp 413-417. Vogel K.S. and Weston J.A. (1990a). The sympathoadrenal lineage in avian embryos. I. Adrenal chromaffin cells lose neuronal traits during embryogenesis. Dev. Biol. 139, 1-12. Vogel K.S. and Weston J.A. (1990b). The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells. Dev. Biol. 139, 13-23. Weston J.A. (1970). The migration and differentiation of neural crest cells. Adv. Morphol. 8, 41-58. Accepted October 28, 2004