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Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2
Developmental Brain Research 110 Ž1998. 159–174
Interactive report
Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2 II. Expression in neural crest derivatives and their target sites in the rat Stefan R. Hansson a
a,2
´ Mezey , Eva
b,)
, Beth J. Hoffman
1
a
Unit on Molecular Pharmacology, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA b Basic Neuroscience Program, National Institute of Neurological Disease and Stroke, Bldg. 36, 3D10, Bethesda, MD 20892, USA Accepted 4 July 1998
Abstract We used in situ hybridization histochemistry to study the expression of the two vesicular monoamine transporters ŽVMAT1 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 Žand to some extent VMAT1. mRNA is expressed in ganglionic cells of the parasympathetic nervous system and in cranial ganglia Žtrigeminal, 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 ŽE11–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 Žpituitary 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 ŽE8–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. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Biogenic amines; Dorsal root ganglia; Sympathetic ganglia; Heart; Gastrointestinal tract; Endocrine; Placenta
1. Introduction After their release, monoamine neurotransmitters are actively removed from the synaptic cleft by plasma membrane transporters w4,29x. Monoamines such as serotonin Ž5HT., dopamine ŽDA., norepinephrine ŽNE., epinephrine ŽE. and histamine ŽHIS. are stored in intracellular storage and released from these organelles by an exocytotic process w4x. Vesicular uptake and storage vesicles depends on a pH gradient, created by a proton ATPase in the vesicle membrane. Two distinct types of vesicular monoamine transporters ŽVMATs. have been cloned, characterized and named VMAT1 w18x and VMAT2 w8x. The two distinct transporter proteins are encoded by two separate genes w25x. The two VMATs differ in their affinity for substrates
and their interaction with inhibitors such as amphetamine, tetrabenazine and reserpine. In adult humans and rodents VMAT2 is present in monoaminergic neurons of the central nervous system and in sympathetic postganglionic neurons w8,27,34x. VMAT1 is predominantly present in certain endocrine cells such as chromaffin cells of the adrenal medulla and enterochromaffin cells of the intestinal tract w20,21,34x. In this present study we examined the distribution of VMAT1 and 2 in the rat embryo. Both of the gene products were studied by in situ hybridization using highly specific and sensitive riboprobes to detect mRNA expression. 2. Material and methods
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Corresponding author. Tel.: q1-301-435-5635; Fax: q1-301-4355465; E-mail: [email protected] 1 Published on the World Wide Web on 3 August 1998. 2 On leave from the Division of Neurobiology, Wallenberg Neurocenter, University of Lund, Sweden. 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 0 3 - 5
2.1. Section preparation Embryos were collected from timed-pregnant Sprague–Dawley rats on embryonic days ŽE. 8 through 21
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Table 1 Distribution of VMAT1 and VMAT2 mRNA in peripheral tissues during ontogenesis Structure
E8
E9
E10
E11
Yolk sac q membrane Spongiotrophoblast Decidua Neural tube Sympathetic ganglia Dorsal root ganglia Rami communicans Spinal cord dorsal horn Cochlea Heart Aorta Adrenal medulla Thyroid gland Pituitary gland Testis Gastrointestinal tract Pancreas Liver Thymus Skin Tongue Joint capsules Cartilage Periosteum Fascial cartilage
q [ q q
q [ q q
q [
[
q
q q
E12
q q
q
q
q
q
E13
E14
E15
E16
E17
E18
E21
[ q q q q [ [
[ q q q q [ [ [
[ q q [ [ [ [ [
[ q q q [ [ [ [
[ q q q ` [ [ [
q [
q [ [
[ q q [ ` [ [ [ ` [ [ [ [ [ [ [ [ q ` q `
[ q ND ND ` ` y [ ` ` ` [ [ [ ` [ ` y ` y `
q
q
q
q [
[
[
[
[
[
`
` q
` q q
[ q q
[ q [ q q
q q
q q
q [
` VMAT 1. q VMAT 2. [ VMAT 1 q VMAT 2. y: no mRNA detected. blank: structure not readily identifiable or developed. ND: not determined.
ŽE8–E21.. The day on which a vaginal plug was observed was designated E1. The embryos were frozen on dry ice and stored at y808C. Tissue sections Ž12 mm. in the coronal, sagittal or horizontal planes were thaw-mounted onto silanized slides and stored at y808C prior to hybridization. 2.2. RNA probes For VMAT1 ŽGenbank accessiona M97380. two different non-overlapping probes were prepared corresponding to Ž1. the glycosylated loop: nucleotides 399–655; Ž2. the 3X-untranslated portion of the rat VMAT2 mRNA:
nucleotides 1741–2177. To detect VMAT2 mRNA ŽGenbank accessiona L00603. we have designed three non-overlapping probes against regions at the 3X end, in the large glycosylated loop and the 5X end. The probes were: probe 1: nucleotides 233–491; probe 2: nucleotides 1649– 2160, probe 3: nucleotides 2161–2661. DNA templates were generated by polymerase chain reaction ŽPCR. from rat cDNA Ž2. using primer pairs consisting of either a T7 RNA promoter and a downstream gene-specific sequence Žanti-sense. or a T3 RNA promoter and an upstream gene-specific primer Žsense.. Solutions containing 1 ng rat VMAT cDNA, 1 mM primers, 200 mM dNTPs, 3 mM MgCl 2 , 10 mM Tris, pH 8.3, 50 mM KCl, 2.5 units Taq polymerase ŽBoehringer, Mannheim.
Fig. 1. Sympathetic ganglia. Darkfield images showing VMAT2 mRNA expression in sagittal sections through the spinal cord at E13 ŽA. and E17 ŽB., respectively. Arrows in ŽA. point at positive cells in the rami communicans. Arrowheads indicate the dorsal root ganglia. In ŽB. the arrow points at the sympathetic chain on the ventral aspect of the spinal cord. The arrowhead points at the rami communicans. Brightfield ŽC. and darkfield ŽD. images show a high magnification of the sympathetic chain Žarrow. and the dorsal root ganglia Žarrowhead.. Not the absence of signal in the vertebra ŽVt.. In ŽE. VMAT1 mRNA expression in the lumbar sympathetic ganglia Žlg. as well as the sympathetic chain Žsc. at E18 is shown. In adjacent sections, brightfield ŽF. and darkfield ŽG. images show VMAT2 mRNA expression. Note the complete overlap between the distribution of VMAT1 and VMAT2 mRNA in these structures. No signal was detected in the aorta Ža.. VMAT1 ŽH. and VMAT2 ŽI,J. mRNA expression is demonstrated in lumbar ganglion at E21 in adjacent sections. Only one brightfield image ŽI. is shown. Scale bars: A, 260 mm; B, 340 mm; C, 110 mm; F, 220 mm; I, 80 mm.
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were amplified at 958C for 1 min, 558C for 2 min and 728C for 2.5 min for 35 cycles with a final extension at 728C for 7 min. DNA templates were purified from agarose gels using GENECLEAN ŽBio101 Inc.. Complementary RNA ŽcRNA. probes were transcribed from 5 ng of gel-purified DNA template using 35 S-UTP ŽDupont NEN, 1300 Cirmmol. and either T3 or T7 RNA polymerase according to the manufacturer’s instructions ŽAmbion MAXIscript. to generate sense and antisense probes, respectively. 2.3. In situ hybridization histochemistry Frozen tissue sections were fixed in 4% buffered formaldehyde, dehydrated and delipidated as previously rr described w3 x Ž for more details see: http:r r lcmr r snge r 3 .. The sections intramural.nimh.nih.govr were hybridized Ž20–24 h, 558C. with 2 = 10 6 cpm of denatured 35 S-cRNA probe per 80 ml hybridization buffer consisting of 20 mM Tris–HCl ŽpH 7.4., 1 mM EDTA ŽpH 8.0., 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1 = Denhardt’s, 25 mgrml yeast tRNA, 100 mgrml salmon sperm DNA, 250 mgrml total yeast RNA Žfraction XI, Sigma., 100 mM dithiothreitol ŽDTT., 0.1% sodium thiosulfate, and 0.1% SDS. Following washes to remove excess probe, slides were apposed to Kodak Hyperfilm Biomax MR for three days, then coated with nuclear track emulsion ŽNTB-3, Kodak.. After an eight week exposure at 48C, slides were developed in Dektol ŽKodak., fixed and counterstained with a Giemsa solution. 2.4. Immunohistochemistry To confirm that the mRNA that we detected using ISHH is translated into protein we performed immunostaining using specific antibodies to VMAT1 and VMAT2, that have been characterized earlier Žw34x a2.. We chose two timepoints ŽE17 and E19. and the ABC technique ŽABC elite kit; Vector Laboratories, Burlingame, CA.. All rr the details of the procedure can be found at http:r r lcmr r snge r Protocol.html 4 . intramural.nimh.nih.govr
3. Results 3.1. Specificity of cRNA probes Both VMAT1 probes showed a qualitatively identical hybridization pattern in the embryos Ždata not shown.. Since hybridization with the 3X probe resulted in a more intense signal, all figures show hybridization using probe
3 4
http:rrintramural.nimh.nih.govrlcmrrsnger http:rrintramural.nimh.nih.govrlcmrrsngerProtocol.html
2. Sense probes did not show any hybridization signal Ždata not shown.. All three VMAT2 probes showed a qualitatively identical hybridization pattern in the embryos, confirming the specificity of the probes. All of the pictures show hybridization with probe 3 since this probe gave the best signal to noise ratio. None of the sense probes showed any hybridization. Throughout the study, the mRNA expression of the two VMATs was analyzed in adjacent sections. This allowed us to compare the distribution of the two mRNAs in all areas. For each time point examined, four animals were analyzed with similar results. The results are presented in a chronological fashion for each system in which expression was detected. Absence of hybridization signal is usually not mentioned. Results are summarized in Table 1. 3.2. Neural tube Hybridization of VMAT2 mRNA in the neural tube and the developing neural crest was first detected at E10. A few cells showed a weak hybridization with a low number of grains per cell. By E11 a distinct group of labelled cells was present in the developing telencephalon. VMAT1 mRNA was abundantly expressed in all cells of the embryo starting at E10. For details on CNS distribution see w11x. 3.3. Spinal cord VMAT2 mRNA expression was detected in the dorsal horn of the spinal cord by E13. The hybridization signal decreased gradually thereafter. By E17 and E18 the signal was mainly confined to the lumbar spinal cord, where it was probably present in autonomic neurons in the intermediate horn. VMAT1 mRNA was expressed in the dorsal horn starting at E15. The expression was weak and did not change over the time interval studied. VMAT2 mRNA expression in the rami communicans was seen beginning at E13 ŽFig. 1A.. This signal was weaker by E18, and no longer detectable at E21. 3.4. Sensory ganglia 3.4.1. Cranial nerÕes sensory ganglia 3.4.1.1. Trigeminal nerÕe. The sensory ŽGasserian. ganglion of the trigeminal ŽV. nerve is distinguishable and starts to express both VMAT1 and VMAT2 mRNA at E12. The expression of VMAT2 mRNA was very strong and remained high even at E21; VMAT1 mRNA gradually decreased and was no longer detectable at E21. 3.4.1.2. Vestibular and cochlear nerÕe. A weak signal in the ganglionic cells of the vestibular and spiral ganglia of
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the VIIIth cranial nerve was detected for VMAT2 mRNA as early as E13. This pattern remained unchanged through E16, after which no VMAT2 mRNA was detectable. VMAT1 mRNA appeared in the same region at E15 and was still present at E21. The special sensory cells of the
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organ of Corti were also positive for VMAT1 starting on E18. 3.4.1.3. Glossopharyngeal nerÕe. A clearly detectable signal for VMAT2 mRNA was observed in the superior
Fig. 2. Heart. Brightfield ŽA. and darkfield ŽB. images showing VMAT2 mRNA expression in sagittal sections through the heart at E14. In ŽA. the arrow points at positive cells at the root of the aorta Ža.. These are likely to be sympathetic cells, part of the conducting system in the heart. Note the signal in the walls of the atrium ŽAt.. Scattered positive cells were distributed in the ventricular ŽV. wall. ŽC. VMAT1 mRNA expression in the ventricular wall at E21. In an adjacent section, brightfield ŽD. and darkfield ŽE. images of VMAT2 mRNA expression is demonstrated. Note the lack of signal in the atrial wall at this time point. Scale bars: A, 120 mm; D, 230 mm.
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ganglion of the IXth cranial nerve on E18. The expression was still present, but reduced at E21. VMAT1 mRNA was also present at the same time and becomes the dominant VMAT mRNA at E21. 3.4.2. Extracranial sensory ganglia 3.4.2.1. Dorsal root ganglia. VMAT2 mRNA in the differentiating dorsal root ganglia was first detected at E11. The hybridization intensity per cell was very strong. By E13 all cells in the dorsal root ganglia seemed to express VMAT2 mRNA at very high levels ŽFig. 1A.. This signal remained very intense until E14. Starting at E14 fewer cells seemed to be positive though the intensity of the signal per cell did not decrease ŽFig. 1C,D.. Beginning around E18 the amount of VMAT2 mRNA started to decrease and the signal was visibly weaker by E21. No VMAT1 mRNA was detected in the DRG at any timepoint studied. 3.5. Autonomic ganglia 3.5.1. Cranial ganglia E18 was the first time when we could identify the parasympathetic ganglia of the VIIth Žfacial. and IXth Žglossopharyngeal. cranial nerves: the pterygopalatine and the otic ganglia. Both expressed VMAT1 and VMAT2 mRNA at this time at similar intensities. 3.5.2. ParaÕertebral ganglia Both VMAT1 and VMAT2 mRNAs were expressed all along the spinal cord in the cells of the sympathetic ganglia starting at E13 ŽFig. 1.. The signal was very intense in all cells of the ganglia throughout gestation. Since all ganglionic cells were positive, it seems likely that the same cells express both mRNAs ŽFig. 1H–J.. The signal intensity remains very high throughout embryonic development. 3.5.3. PreÕertebral ganglia VMAT2 mRNA first appears in the celiac and mesenteric ganglia at E16 and VMAT1 mRNA was found there two days later. Both mRNAs continue to be expressed through E21. 3.5.4. Intramural ganglia We will describe the expression in ganglionic cells in the different organs as we describe the expression pattern within those organs.
3.6. CardioÕascular system 3.6.1. Heart VMAT2 mRNA expression was first detected in the primitive heart tube at E8. The strongest signal during heart development was observed at E12. At E13, when we were able to make a clear distinction between the atria and the ventricles, it seemed that the VMAT2 mRNA expression was limited to the ventricles. By E14 there was expression in the atrium as well ŽFig. 2A,B.. At this time there was a very strong, clear labeling of the developing atrio-ventricular valves. By E16 mainly the ventricles were positive. Up to this time the number of positive cells remained the same and then both the signal intensity and number of cells gradually decreased. At E21 no signal was detectable. Weak expression of VMAT1 mRNA was first detected in the ventricle by E13. The signal intensity increased by E15. At E18 the atrial signal significantly decreased, but the atrio-ventricular valves became positive. The ventricular signal was still present at E21. From adjacent sections it appears that the same cells expressed VMAT2 and VMAT1 ŽFig. 2C–E.. 3.6.2. Aorta The endothelium of the aorta expressed VMAT2 mRNA from E11 through E13. No signal was detected here at later timepoints. A group of intensely labeled VMAT1 and VMAT2 mRNA positive cells was found at the root of the aorta starting at E13. Most likely these were cells of the sinus node, part of the sympathetic nervous system ŽFig. 2A,B.. After E18, this group of cells was no longer detectable. 3.7. Gastrointestinal tract 3.7.1. Esophagus, stomach, small and large intestines The first VMAT2 mRNA-positive cells in the primitive gut were detected by E12. By E13 these positive cells were confined to the ganglionic cells of both the submucosal ŽMeissner. and myenteric ŽAuerbach. plexi. The number of cells increased up to day E16. The Auerbach Žmyenteric. nerve plexus of the esophagus expressed the VMAT2 mRNA by E16. This signal intensified by E17 and remained unchanged throughout gestation ŽFig. 3I,J.. No VMAT1 mRNA was present in the esophagus. By E18 the hybridization signal became more intense per individual cell and a few very intensely labelled cells also appeared within the epithelia of the upper and lower GI tract. Based
Fig. 3. Intestinal tract. Darkfield ŽA. and brightfield ŽB. image show VMAT1 mRNA expression in a group of cells located in the epithelium of the intestinal wall Žarrowheads. at E18. In an adjacent section ŽC. VMAT2 mRNA expression is viewed. Note that most of the signal is located in the outer nerve plexus Žsmall arrow.. Some positive cells are located in the epithelium Žlarge arrow.. Darkfield ŽD. and brightfield ŽE. image show VMAT1 mRNA expression in a group of cells located in the epithelium of the intestinal wall at E21. In adjacent sections ŽF. VMAT2 mRNA expression is viewed. The signal is located in the outer nerve plexus Žarrowheads.. Additional signal is found in the nerve plexus of the stomach ŽS.. The lumen of the stomach is indicated with a star. Brightfield ŽG. and darkfield ŽH. images show VMAT1 mRNA expression in the epithelium and the inner nerve plexus of the antrum at E21. Brightfield ŽI. and darkfield ŽJ. images demonstrate VMAT2 mRNA expression in a longitudinal section of the esophagus Že.. Arrowheads point at cells innervating the distal part of esophagus. Scale bars: B, 50 mm; E, 340 mm; G, 120 mm; I, 150 mm.
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on their number and distribution these cells were reminescent of the endocrine Ženterochromaffin. cells of the gastrointestinal system. The signal remained the same at E21 ŽFig. 3A–F.. Expression of VMAT1 mRNA was first detected at E15. In contrast to VMAT2 mRNA, VMAT1 mRNA was expressed in cells scattered through the epithelial lining. The signal intensity and the number of positive cells increased by E18. By E21, in addition to scattered intensely positive endocrine cells, many mucosal cells in the small intestine had a moderately intense hybridization signal. The fundic stomach mucosa was negative, but the antral mucosa Žwhere the gastrin producing endocrine cells are located. was very strongly labelled for VMAT1 mRNA starting at E18 ŽFig. 3G,H.. A group of cells were labelled in the ganglia of the submucosal plexus ŽMeissner., but these cells showed a low intensity signal.
stationary macrophages ŽKupffer cells.. The hybridization intensity was even throughout gestation. The number of positive cells increased as the liver grew. After E15, the positive cell population seemed constant ŽFig. 4A,B.. VMAT1 mRNA first appeared also at E13, but the signal was rather low and homogeneously distributed. This pattern remained unchanged throughout development and was still present at E21.
3.7.2. LiÕer Beginning at E13, a population of cells expressed high levels of VMAT2 mRNA. These cells are most likely
3.8.1. Pituitary gland VMAT2 mRNA positive cells were observed in the anterior pituitary by E17. The signal was still present at
3.7.3. Pancreas Starting at E18 scattered exocrine pancreatic cells expressed high levels of both VMAT1 and VMAT2 mRNA. The expression pattern and intensity did not change at E21. 3.8. Endocrine organs
Fig. 4. Liver and thymus. Brightfield ŽA. and darkfieldŽB. images show VMAT2 mRNA expression in the liver at E16. Arrows point at some of the positive cells in the parenchyma. Additional expression is seen in the myenteric plexus of the intestine Žarrowheads.. BrightfieldŽC. and darkfield ŽD. images show VMAT1 mRNA-expressing cells in the thymus Žarrows. at E18. Scale bars: B, 130 mm; D, 40 mm.
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E18, but disappeared by E21. VMAT1 mRNA is also expressed in a group Žmost likely all. of cells in the pituitary anterior lobe starting at E18. By E21 only VMAT1 mRNA is expressed in the pituitary in cells that are evenly distributed throughout the anterior lobe ŽFig. 5A–C.. At E18 and E21 there is also a strong expression of VMAT1 mRNA in the median eminencerpituitary stalk area, that
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might reflect expression in the developing portal vasculature.
3.8.2. Thyroid gland VMAT1 mRNA was seen in a group of cells in the thyroid gland starting at E18, and was still present at E21.
Fig. 5. Endocrine tissues. VMAT1 mRNA is expressed in the pituitary gland at E18 ŽA,B. and E21 ŽC.. At E18 the positive cells are grouped in the posterior part of the anterior lobe Žarrow.. By E21 the VMAT1 mRNA positive cells are distributed evenly in the anterior lobe. Arrows point at some of the positive cells. Note the absence of signal in the posterior lobe Žarrowhead.. The base of the skull is indicated by a star. ŽD. High magnification of VMAT1 mRNA expression in the adrenal medulla at E18. In an adjacent section, brightfield ŽE. and darkfield ŽF. images co-expression of VMAT2 mRNA is indicated by arrows. In a high magnification of the developing testis ŽG. at E18 a group of cells with intense expression of VMAT1 mRNA as indicated by the arrows. VMAT1 mRNA expression is detected in a scattered cell population in the seminal vesicle ŽH. at day E21 Žarrows.. Scale bars: B, 120 mm; E, 75 mm; G, 25 mm; H, 40 mm.
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3.8.3. Testis and oÕary VMAT2 mRNA was detected in the testis on E15. The positive cells were found between the tubuli. The signal intensity was high until E18 and then significantly decreased ŽFig. 5G.. VMAT1 mRNA is expressed in the testis starting on E17, and remains unaltered through E21. VMAT2 is also detected in the differentiating ovary at E18.
labeled cells were detected. The intensity per cell remained unchanged but the number of positive cells increased in the medulla throughout gestation. Expression of VMAT1 mRNA was first detected in the developing adrenal medulla at E13. The signal intensity was strong and remained unchanged through E21. Comparing adjacent sections, it appeared that the same cell population expressed VMAT2 and VMAT1 mRNAs ŽFig. 5D–E..
3.8.4. Seminal Õesicle Cells of the seminal vesicle expressed VMAT1 mRNA at E21. Intensely labeled cells were found in the vesicle ŽFig. 5H..
3.9. Sensory organs
3.8.5. Adrenal VMAT2 mRNA expression was first visible in the primitive adrenal by E12. At this time a few intensely
3.9.1. Skin There was a weak VMAT1 mRNA signal detected in the skin of the fore and hind paw by E13. Starting at E16 both VMAT1 and VMAT2 mRNAs are expressed in the skin. The hybridization signal for VMAT2 was very intense by E15 ŽFig. 6E,F.. From E17 throughout gestation,
Fig. 6. Connective tissue. Brightfield ŽA. and darkfield ŽB. images demonstrate VMAT1 mRNA expression in the trabecules of the developing snout at E21. Arrows point at some of the positive cells. Brightfield ŽC. and darkfield ŽD. images show VMAT1 mRNA expression in the developing palatine shelf. The star indicates the epiphyseal cartilage. The positive cells are located within the trabecles of the primitive bone marrow located on both sides of the epiphysis. Brightfield ŽE. and darkfield ŽF. images show VMAT2 mRNA expression in the skin Žarrowhead. of the hind paw and the central touch dome Žarrow.. Scale bars: A, 80 mm; C, 190 mm; E, 110 mm.
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the signal began to weaken. At E21 a small population of cells in the subcutaneous layer of the tail skin still expressed VMAT2 mRNA. 3.9.2. Tongue A small population of cells within the tongue expressed the VMAT2 mRNA starting at day E14. The signal intensity peaked at E17 and then decreased to a non-detectable level by E21. Based on their location, these cells are likely to be muscle spindles, a modified muscle cell which functions as a propioreceptor ŽFig. 7A,B.. VMAT1 mRNA appears in the tongue at E18 and unlike VMAT2, is still present at E21. 3.10. Other organs 3.10.1. Lung Starting at E16, intramural ganglionic cells around the main bronchus expressed VMAT2 mRNA. The expression pattern did not change through E21. 3.10.2. Cartilage, joints, periosteum VMAT2 mRNA positive cells appear in the limb bud as early as E12. Cells expressing VMAT2 mRNA were found in layers surrounding the joint capsules of the limbs at E15
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ŽFig. 7C,D.. No signal was detected in this location after day E18. Cells surrounding the bones Žperiosteum. express VMAT2 mRNA between E15 and E18. There is VMAT1 mRNA expression in the facial cartilage starting at E13 which was still present at E21. Positive cells are found in the trabeculae of the snout and in the palatine shelf ŽFig. 6.. VMAT2 mRNA is also expressed here, but the expression only starts at day E16, and continues through E21. The signal remained unchanged at E21. 3.10.3. Thymus VMAT2 mRNA appeared in the thymus at E17 when a few cells were weakly labelled. At E18 the labeling of VMAT2 mRNA started to decrease, but VMAT1 mRNA appeared in many more cells throughout the parenchyma of the thymus ŽFig. 4C,D.. These cells are likely to be differentiating T cells. The VMAT2 expression disappears, but VMAT1 mRNA expression remains unchanged at E21. 3.11. Extraembryonic structures 3.11.1. Yolk sac The yolk sac was only analyzed on days E10 and E11. A population of cells in the yolk sac seemed to express both VMAT1 and VMAT2 mRNA Ždata not shown..
Fig. 7. Brightfield ŽA. and darkfield ŽB. images show a sagittal section through the tongue at E17. Arrows point at a group of VMAT2 mRNA expressing cells, located deep in the tongue parenchyma. Based on the location these cells are likely to be muscle spindles of the tongue. Brightfield ŽC. and darkfield ŽD. images show VMAT2 mRNA expression in cells adjacent to the elbow joint capsule Žarrows.. Scale bars: A, 150 mm; C, 110 mm.
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Fig. 8. Brightfield ŽA. and darkfield ŽB. view of a high magnification of VMAT1 mRNA expressing cells in the decidua Žarrowheads. at day E14. Brightfield ŽC. and darkfield ŽD. images show VMAT2 mRNA expression in syncytiotrophoblast cells Žarrows. in the spongiotrophoblast zone at E14. Scale bars: A, 45 mm; C, 45 mm.
3.11.2. Placenta The ectoplacental glycogen cells express large amounts of VMAT2 mRNA at E8 and E9. At E14 a population of cells in the spongiotrophoblast zone expressed VMAT2 mRNA ŽFig. 8C,D.. Weak expression of VMAT1 mRNA was also detected in this region. In the decidua, a population of cells radiating out from the ectoplacental cone
expressed VMAT1 but not VMAT2 mRNA. Based on their size, these cells might be migrating giant cells ŽFig. 8A,B.. The placenta was not studied at later time points. 3.11.3. Controls The control experiments showed that 1. None of the sense probes show any labeling in the embryos; 2. The
Fig. 9. Staining in sagittal sections of E17 ŽA,B,E,F. and E19 ŽC,D,G,H. day old embryos. Immunopositivity in adrenal medullary cells is demonstrated for VMAT1 ŽA. and VMAT2 ŽB.. Positive cells for VMAT1 are also present in the myenteric plexus of the developing GI tract ŽC. and VMAT2 positive cells are also present in some epithelial cells ŽD.. Paravertebral sympathetic ganglia are immunopositive for both VMAT1 ŽE. and VMAT2 ŽF.. G demonstrates immunostaining in the trigeminal and H in the vestibulocochlear Žarrows. and trigeminal Žasterisk. and the trigeminal and acoustic nerves. Bar scales: A,B: 500 mm; C,D: 1 mm; E,F: 200 mm; G: 300 mm and H: 400 mm.
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different, non-overlapping, antisense probes showed an identical mRNA distribution for the individual mRNAs; 3. Immunohistochemical staining for VMAT1 and VMAT2 protein at ages E17 and E19 showed a good correlation with the VMAT1 and VMAT2 mRNA distribution at the same time. In Fig. 9 we demonstrate the immunostaining in the adrenal medulla, in sympathetic and cranial ganglia and in the hippocampus. All the above controls strongly suggest that the mRNA labeling is highly specific.
4. Discussion VMATs store and release monoamines. Monoamines Žmostly serotonin. have been suggested to affect embryonic development Žsee w16,23,35x.. In addition to transporting naturally occurring substrates, the transporters can protect neurons by sequestering neurotoxins in synaptic vesicles w1,26,32x. The two transporters have different affinities for their natural substrates. VMAT2 has a 3 fold higher affinity for 5HT, DA, NE than VMAT1 has, and approximately a 100-fold higher affinity for histamine w2,4x. The pharmacological properties of these transporters are also somewhat different w9,17,19x. The purpose of this study was to determine the developmental pattern of the VMATs. In adult animals the distributions of the two vesicular transporters are partially nonoverlapping w8,9,34x. In adult rats w26x both transporter mRNAs were detected in adrenal chromaffin cells, and sympathetic and enteric parasympathetic ganglionic cells. On the other hand, exclusively VMAT2 seems to be present in the CNS and VMAT1 in certain endocrine cells with no overlapping between them w27,34x. Unlike adults, embryos exhibit a considerable co-expression of the two vesicular transporters. In certain areas or tissues the presence of VMATs ŽmRNA or protein. may persist into adulthood. Most likely certain cells in these tissues are truly monoaminergic, and in such case VMATs are required for monoamine storage and release. These tissues include the adrenal medulla, the gastrointestinal tract, and the heart. The adrenal medulla is considered to be part of the sympathetic nervous system and it is derived from neural crest cells. Recently cells exhibiting all the features of monoaminergic cells were described in the stomach w7,14,20x and in the pancreas w21x of the adult rat and a population of intrinsic adrenergic cells were found in the heart of both the adult rodent and human w13x. These cells might help regulate cardiac function in parallel with the autonomic nervous system. In addition to detecting VMATs in tissues destined to have monoaminergic cells in adult animals we found a transient expression of VMAT1 and VMAT2 mRNAs in many neural crest derived tissues where aminergic cells are not known to exist in the adult: sympathetic nervous system, intrinsic parasympathetic ganglia, dorsal root gan-
glia, cranial nerves ganglia, endocrine organs, thymus and connective tissues. It is possible that the transiently expressed VMAT mRNAs have no functional role in these tissues. Alternatively, these tissues have monoaminergic cells that are only present during a certain period of development. These cells, most of which seem to arise from the neural crest, may release monoamines and affect tissue differentiation. For example, VMAT2 mRNA was present in the developing aorta from E11 until E14. Neural crest cells participate in establishing the branching pattern of the aortic arch arteries, and in dictating the formation of the cardiac septum w15,33x. The transient expression of VMAT2 in the aorta may reflect a local role for monoamines during a critical period of heart development. Sensory neurons also derive from the neural crest w5,6,30x. The dorsal root ganglia are relay stations for sensory afferent fibers. Unlike the sympathetic ganglion cells, dorsal root ganglia express VMAT2 mRNA exclusively. Sympathetic ganglion cells derive from a distinct crest lineage from dorsal root ganglionic cells which might explain the differences in VMAT mRNA expression. The rami communicans ŽRC. are nerve bundles transmitting sensory inputs from the dorsal root ganglia to the spinal cord. Monoaminergic characteristics have previously not been shown in the RC, yet here we demonstrate a distinct expression of VMAT2 mRNA in these nerves. At E13, this expression might be explained by glial precursor cells migrating towards the dorsal root ganglia. VMAT2 mRNA is still expressed after day E14 which is the time point when migration ends and when Schwann cell precursors are first detected w22x. The presence of VMAT2 mRNA around the joints of the developing limbs, and in the tongue, suggests that the expression might be located in muscular spindles and propioreceptors in the capsules of joints and tendons. This part of the sensory system supplies the CNS with information required for properly coordinated movements of limbs and tongue. These findings together with the widespread presence of VMAT mRNAs in sensory brainstem nuclei and in the thalamus during the same time in ontogenesis w11x indicates a role of monoamines in the developing sensory pathways.
5. VMAT mRNAs in the placenta Our data suggest that placental cells can store monoamines. The placenta possesses a highly active serotonin transporter w10x. Dopamine and norepinephrine plasma membrane transporter mRNAs are also expressed in the placenta. The expression of VMAT1 and VMAT2 mRNAs in the syncytiotrophoblasts will enable these cells to metabolize and release monoamines in a controlled fashion. Monoamines are effectively cleared from the maternal circulation and the intra villus space w10x. After uptake, the monoamines are rapidly degraded by
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monoamineoxidase A which is present in the placenta. Thus the syncytiotrophoblasts take up and metabolize monoamines, preventing them from interfering with the developing embryo. Excessive amounts of serotonin have been shown to be lethal during embryogenesis w36x and to cause cell death in the neural crest population when injected intraperitoneally in newborn mice w24x. VMAT1 and VMAT2 mRNAs are expressed in different subpopulations in the placenta. VMAT2 mRNA is expressed in the spongiotrophoblast zone, an area where we also detect the serotonin plasma membrane transporter w12x. VMAT1 mRNA is found in large cells of the decidua. Giant cells that express VMAT1 mRNA are hormonally regulated secretory cells, similarly to the enterochromaffin cells of the GI system, where VMAT1 mRNA is present in the adult. A gradual increase in the levels of monoamines and their metabolites is found in the amniotic fluid in late gestation w28,31x. The high expression of VMAT mRNAs in the placenta suggests that a regulated release of monoamines from the placenta could participate in the induction of labor. We have examined the distribution of VMAT1 and VMAT2 mRNA during embryonic development. Strikingly, neural crest derivatives such as sensory neurons, sympathetic ganglia and enteric ganglia all express VMAT2 mRNA. All these areas are also immunopositive when stained with an antibody that recognizes the VMAT2 protein Ždata not shown., which means that the protein is expressed there. Further studies are necessary to determine if these cells are truly monoaminergic and also express one of the plasma membrane transporters. If this is true, than these cells might be involved in regulating monoamine levels in their target areas during embryonic development. Interestingly, both VMAT mRNAs are expressed in many regions at the beginning of ontogenesis. Later, the expression becomes more distinct and it seems that VMAT2 mRNA is predominantly expressed in neural while VMAT1 is expressed in endocrine tissues. Further studies will be necessary to evaluate the significance of these findings.
Acknowledgements The authors would like to thank Miklos ´ Palkovits for his suggestions and help in putting the data together, Michael Brownstein for his help with editing and Ricardo Dreyfuss for photography.
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S.R. Hansson et al.r DeÕelopmental Brain Research 110 (1998) 159–174 neural crest migration, Proc. Natl. Acad. Sci. USA 92 Ž1995. 7182–7186. A.T. Nozue, Relationships between neural crest cells and serotonin in newborn mice, Anat. Anz. 166 Ž1988. 227–237. D. Peter, J.P. Finn, I. Klisak, Y. Liu, T. Kojis, C. Heinzmann, A. Roghani, R.S. Sparkes, R.H. Edwards, Chromosomal localization of the human vesicular amine transporter genes, Genomics 18 Ž1993. 720–723. D. Peter, Y. Liu, N. Brecha, R.H. Edwards, The transport of neurotransmitters into synaptic vesicles, Prog. Brain Res. 105 Ž1995. 273–281. D. Peter, Y. Liu, C. Sternini, R. de Giorgio, N. Brecha, R.H. Edwards, Differential expression of two vesicular monoamine transporters, J. Neurosci. 15 Ž1995. 6179–6188. H. Schneider, M. Progler, W.H. Ziegler, R. Huch, Biochemical changes in the mother and the fetus during labor and its significance for the management of the second stage, Int. J. Gynaecol. Obstet. 31 Ž1990. 117–126. S. Schuldiner, A molecular glimpse of vesicular monoamine transporters, J. Neurochem. 62 Ž1994. 2067–2078. M. Sieber-Blum, Commitment of neural crest cells to the sensory neuron lineage, Science 243 Ž1989. 1608–1611.
w31x R.J. Sodha, M. Proegler, H. Schneider, Transfer and metabolism of norepinephrine studied from maternal-to-fetal and fetal-to-maternal sides in the in vitro perfused human placental lobe, Am. J. Obstet. Gynecol. 148 Ž1984. 474–481. w32x H. Varoqui, J.D. Erickson, Vesicular neurotransmitter transporters. Potential sites for the regulation of synaptic function, Mol. Neurobiol. 15 Ž1997. 165–191. w33x K.L. Waldo, D.H. Kumiski, M.L. Kirby, Association of the cardiac neural crest with development of the coronary arteries in the chick embryo, Anat. Rec. 239 Ž1994. 315–331. w34x E. Weihe, M.K. Schafer, J.D. Erickson, L.E. Eiden, Localization of vesicular monoamine transporter isoforms ŽVMAT1 and VMAT2. to endocrine cells and neurons in rat, J. Mol. Neurosci. 5 Ž1994. 149–164. w35x P.M. Whitaker-Azmitia, M. Druse, P. Walker, J.M. Lauder, Serotonin as a developmental signal, Behav. Brain Res. 73 Ž1996. 19–29. w36x M.S. Yavarone, D.L. Shuey, T.W. Sadler, J.M. Lauder, Serotonin uptake in the ectoplacental cone and placenta of the mouse, Placenta 14 Ž1993. 149–161.
Interactive report
Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2 II. Expression in neural crest derivatives and their target sites in the rat Stefan R. Hansson a
a,2
´ Mezey , Eva
b,)
, Beth J. Hoffman
1
a
Unit on Molecular Pharmacology, Laboratory of Cellular and Molecular Regulation, National Institute of Mental Health, Bethesda, MD 20892, USA b Basic Neuroscience Program, National Institute of Neurological Disease and Stroke, Bldg. 36, 3D10, Bethesda, MD 20892, USA Accepted 4 July 1998
Abstract We used in situ hybridization histochemistry to study the expression of the two vesicular monoamine transporters ŽVMAT1 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 Žand to some extent VMAT1. mRNA is expressed in ganglionic cells of the parasympathetic nervous system and in cranial ganglia Žtrigeminal, 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 ŽE11–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 Žpituitary 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 ŽE8–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. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Biogenic amines; Dorsal root ganglia; Sympathetic ganglia; Heart; Gastrointestinal tract; Endocrine; Placenta
1. Introduction After their release, monoamine neurotransmitters are actively removed from the synaptic cleft by plasma membrane transporters w4,29x. Monoamines such as serotonin Ž5HT., dopamine ŽDA., norepinephrine ŽNE., epinephrine ŽE. and histamine ŽHIS. are stored in intracellular storage and released from these organelles by an exocytotic process w4x. Vesicular uptake and storage vesicles depends on a pH gradient, created by a proton ATPase in the vesicle membrane. Two distinct types of vesicular monoamine transporters ŽVMATs. have been cloned, characterized and named VMAT1 w18x and VMAT2 w8x. The two distinct transporter proteins are encoded by two separate genes w25x. The two VMATs differ in their affinity for substrates
and their interaction with inhibitors such as amphetamine, tetrabenazine and reserpine. In adult humans and rodents VMAT2 is present in monoaminergic neurons of the central nervous system and in sympathetic postganglionic neurons w8,27,34x. VMAT1 is predominantly present in certain endocrine cells such as chromaffin cells of the adrenal medulla and enterochromaffin cells of the intestinal tract w20,21,34x. In this present study we examined the distribution of VMAT1 and 2 in the rat embryo. Both of the gene products were studied by in situ hybridization using highly specific and sensitive riboprobes to detect mRNA expression. 2. Material and methods
)
Corresponding author. Tel.: q1-301-435-5635; Fax: q1-301-4355465; E-mail: [email protected] 1 Published on the World Wide Web on 3 August 1998. 2 On leave from the Division of Neurobiology, Wallenberg Neurocenter, University of Lund, Sweden. 0165-3806r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 3 8 0 6 Ž 9 8 . 0 0 1 0 3 - 5
2.1. Section preparation Embryos were collected from timed-pregnant Sprague–Dawley rats on embryonic days ŽE. 8 through 21
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Table 1 Distribution of VMAT1 and VMAT2 mRNA in peripheral tissues during ontogenesis Structure
E8
E9
E10
E11
Yolk sac q membrane Spongiotrophoblast Decidua Neural tube Sympathetic ganglia Dorsal root ganglia Rami communicans Spinal cord dorsal horn Cochlea Heart Aorta Adrenal medulla Thyroid gland Pituitary gland Testis Gastrointestinal tract Pancreas Liver Thymus Skin Tongue Joint capsules Cartilage Periosteum Fascial cartilage
q [ q q
q [ q q
q [
[
q
q q
E12
q q
q
q
q
q
E13
E14
E15
E16
E17
E18
E21
[ q q q q [ [
[ q q q q [ [ [
[ q q [ [ [ [ [
[ q q q [ [ [ [
[ q q q ` [ [ [
q [
q [ [
[ q q [ ` [ [ [ ` [ [ [ [ [ [ [ [ q ` q `
[ q ND ND ` ` y [ ` ` ` [ [ [ ` [ ` y ` y `
q
q
q
q [
[
[
[
[
[
`
` q
` q q
[ q q
[ q [ q q
q q
q q
q [
` VMAT 1. q VMAT 2. [ VMAT 1 q VMAT 2. y: no mRNA detected. blank: structure not readily identifiable or developed. ND: not determined.
ŽE8–E21.. The day on which a vaginal plug was observed was designated E1. The embryos were frozen on dry ice and stored at y808C. Tissue sections Ž12 mm. in the coronal, sagittal or horizontal planes were thaw-mounted onto silanized slides and stored at y808C prior to hybridization. 2.2. RNA probes For VMAT1 ŽGenbank accessiona M97380. two different non-overlapping probes were prepared corresponding to Ž1. the glycosylated loop: nucleotides 399–655; Ž2. the 3X-untranslated portion of the rat VMAT2 mRNA:
nucleotides 1741–2177. To detect VMAT2 mRNA ŽGenbank accessiona L00603. we have designed three non-overlapping probes against regions at the 3X end, in the large glycosylated loop and the 5X end. The probes were: probe 1: nucleotides 233–491; probe 2: nucleotides 1649– 2160, probe 3: nucleotides 2161–2661. DNA templates were generated by polymerase chain reaction ŽPCR. from rat cDNA Ž2. using primer pairs consisting of either a T7 RNA promoter and a downstream gene-specific sequence Žanti-sense. or a T3 RNA promoter and an upstream gene-specific primer Žsense.. Solutions containing 1 ng rat VMAT cDNA, 1 mM primers, 200 mM dNTPs, 3 mM MgCl 2 , 10 mM Tris, pH 8.3, 50 mM KCl, 2.5 units Taq polymerase ŽBoehringer, Mannheim.
Fig. 1. Sympathetic ganglia. Darkfield images showing VMAT2 mRNA expression in sagittal sections through the spinal cord at E13 ŽA. and E17 ŽB., respectively. Arrows in ŽA. point at positive cells in the rami communicans. Arrowheads indicate the dorsal root ganglia. In ŽB. the arrow points at the sympathetic chain on the ventral aspect of the spinal cord. The arrowhead points at the rami communicans. Brightfield ŽC. and darkfield ŽD. images show a high magnification of the sympathetic chain Žarrow. and the dorsal root ganglia Žarrowhead.. Not the absence of signal in the vertebra ŽVt.. In ŽE. VMAT1 mRNA expression in the lumbar sympathetic ganglia Žlg. as well as the sympathetic chain Žsc. at E18 is shown. In adjacent sections, brightfield ŽF. and darkfield ŽG. images show VMAT2 mRNA expression. Note the complete overlap between the distribution of VMAT1 and VMAT2 mRNA in these structures. No signal was detected in the aorta Ža.. VMAT1 ŽH. and VMAT2 ŽI,J. mRNA expression is demonstrated in lumbar ganglion at E21 in adjacent sections. Only one brightfield image ŽI. is shown. Scale bars: A, 260 mm; B, 340 mm; C, 110 mm; F, 220 mm; I, 80 mm.
S.R. Hansson et al.r DeÕelopmental Brain Research 110 (1998) 159–174
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were amplified at 958C for 1 min, 558C for 2 min and 728C for 2.5 min for 35 cycles with a final extension at 728C for 7 min. DNA templates were purified from agarose gels using GENECLEAN ŽBio101 Inc.. Complementary RNA ŽcRNA. probes were transcribed from 5 ng of gel-purified DNA template using 35 S-UTP ŽDupont NEN, 1300 Cirmmol. and either T3 or T7 RNA polymerase according to the manufacturer’s instructions ŽAmbion MAXIscript. to generate sense and antisense probes, respectively. 2.3. In situ hybridization histochemistry Frozen tissue sections were fixed in 4% buffered formaldehyde, dehydrated and delipidated as previously rr described w3 x Ž for more details see: http:r r lcmr r snge r 3 .. The sections intramural.nimh.nih.govr were hybridized Ž20–24 h, 558C. with 2 = 10 6 cpm of denatured 35 S-cRNA probe per 80 ml hybridization buffer consisting of 20 mM Tris–HCl ŽpH 7.4., 1 mM EDTA ŽpH 8.0., 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1 = Denhardt’s, 25 mgrml yeast tRNA, 100 mgrml salmon sperm DNA, 250 mgrml total yeast RNA Žfraction XI, Sigma., 100 mM dithiothreitol ŽDTT., 0.1% sodium thiosulfate, and 0.1% SDS. Following washes to remove excess probe, slides were apposed to Kodak Hyperfilm Biomax MR for three days, then coated with nuclear track emulsion ŽNTB-3, Kodak.. After an eight week exposure at 48C, slides were developed in Dektol ŽKodak., fixed and counterstained with a Giemsa solution. 2.4. Immunohistochemistry To confirm that the mRNA that we detected using ISHH is translated into protein we performed immunostaining using specific antibodies to VMAT1 and VMAT2, that have been characterized earlier Žw34x a2.. We chose two timepoints ŽE17 and E19. and the ABC technique ŽABC elite kit; Vector Laboratories, Burlingame, CA.. All rr the details of the procedure can be found at http:r r lcmr r snge r Protocol.html 4 . intramural.nimh.nih.govr
3. Results 3.1. Specificity of cRNA probes Both VMAT1 probes showed a qualitatively identical hybridization pattern in the embryos Ždata not shown.. Since hybridization with the 3X probe resulted in a more intense signal, all figures show hybridization using probe
3 4
http:rrintramural.nimh.nih.govrlcmrrsnger http:rrintramural.nimh.nih.govrlcmrrsngerProtocol.html
2. Sense probes did not show any hybridization signal Ždata not shown.. All three VMAT2 probes showed a qualitatively identical hybridization pattern in the embryos, confirming the specificity of the probes. All of the pictures show hybridization with probe 3 since this probe gave the best signal to noise ratio. None of the sense probes showed any hybridization. Throughout the study, the mRNA expression of the two VMATs was analyzed in adjacent sections. This allowed us to compare the distribution of the two mRNAs in all areas. For each time point examined, four animals were analyzed with similar results. The results are presented in a chronological fashion for each system in which expression was detected. Absence of hybridization signal is usually not mentioned. Results are summarized in Table 1. 3.2. Neural tube Hybridization of VMAT2 mRNA in the neural tube and the developing neural crest was first detected at E10. A few cells showed a weak hybridization with a low number of grains per cell. By E11 a distinct group of labelled cells was present in the developing telencephalon. VMAT1 mRNA was abundantly expressed in all cells of the embryo starting at E10. For details on CNS distribution see w11x. 3.3. Spinal cord VMAT2 mRNA expression was detected in the dorsal horn of the spinal cord by E13. The hybridization signal decreased gradually thereafter. By E17 and E18 the signal was mainly confined to the lumbar spinal cord, where it was probably present in autonomic neurons in the intermediate horn. VMAT1 mRNA was expressed in the dorsal horn starting at E15. The expression was weak and did not change over the time interval studied. VMAT2 mRNA expression in the rami communicans was seen beginning at E13 ŽFig. 1A.. This signal was weaker by E18, and no longer detectable at E21. 3.4. Sensory ganglia 3.4.1. Cranial nerÕes sensory ganglia 3.4.1.1. Trigeminal nerÕe. The sensory ŽGasserian. ganglion of the trigeminal ŽV. nerve is distinguishable and starts to express both VMAT1 and VMAT2 mRNA at E12. The expression of VMAT2 mRNA was very strong and remained high even at E21; VMAT1 mRNA gradually decreased and was no longer detectable at E21. 3.4.1.2. Vestibular and cochlear nerÕe. A weak signal in the ganglionic cells of the vestibular and spiral ganglia of
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the VIIIth cranial nerve was detected for VMAT2 mRNA as early as E13. This pattern remained unchanged through E16, after which no VMAT2 mRNA was detectable. VMAT1 mRNA appeared in the same region at E15 and was still present at E21. The special sensory cells of the
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organ of Corti were also positive for VMAT1 starting on E18. 3.4.1.3. Glossopharyngeal nerÕe. A clearly detectable signal for VMAT2 mRNA was observed in the superior
Fig. 2. Heart. Brightfield ŽA. and darkfield ŽB. images showing VMAT2 mRNA expression in sagittal sections through the heart at E14. In ŽA. the arrow points at positive cells at the root of the aorta Ža.. These are likely to be sympathetic cells, part of the conducting system in the heart. Note the signal in the walls of the atrium ŽAt.. Scattered positive cells were distributed in the ventricular ŽV. wall. ŽC. VMAT1 mRNA expression in the ventricular wall at E21. In an adjacent section, brightfield ŽD. and darkfield ŽE. images of VMAT2 mRNA expression is demonstrated. Note the lack of signal in the atrial wall at this time point. Scale bars: A, 120 mm; D, 230 mm.
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ganglion of the IXth cranial nerve on E18. The expression was still present, but reduced at E21. VMAT1 mRNA was also present at the same time and becomes the dominant VMAT mRNA at E21. 3.4.2. Extracranial sensory ganglia 3.4.2.1. Dorsal root ganglia. VMAT2 mRNA in the differentiating dorsal root ganglia was first detected at E11. The hybridization intensity per cell was very strong. By E13 all cells in the dorsal root ganglia seemed to express VMAT2 mRNA at very high levels ŽFig. 1A.. This signal remained very intense until E14. Starting at E14 fewer cells seemed to be positive though the intensity of the signal per cell did not decrease ŽFig. 1C,D.. Beginning around E18 the amount of VMAT2 mRNA started to decrease and the signal was visibly weaker by E21. No VMAT1 mRNA was detected in the DRG at any timepoint studied. 3.5. Autonomic ganglia 3.5.1. Cranial ganglia E18 was the first time when we could identify the parasympathetic ganglia of the VIIth Žfacial. and IXth Žglossopharyngeal. cranial nerves: the pterygopalatine and the otic ganglia. Both expressed VMAT1 and VMAT2 mRNA at this time at similar intensities. 3.5.2. ParaÕertebral ganglia Both VMAT1 and VMAT2 mRNAs were expressed all along the spinal cord in the cells of the sympathetic ganglia starting at E13 ŽFig. 1.. The signal was very intense in all cells of the ganglia throughout gestation. Since all ganglionic cells were positive, it seems likely that the same cells express both mRNAs ŽFig. 1H–J.. The signal intensity remains very high throughout embryonic development. 3.5.3. PreÕertebral ganglia VMAT2 mRNA first appears in the celiac and mesenteric ganglia at E16 and VMAT1 mRNA was found there two days later. Both mRNAs continue to be expressed through E21. 3.5.4. Intramural ganglia We will describe the expression in ganglionic cells in the different organs as we describe the expression pattern within those organs.
3.6. CardioÕascular system 3.6.1. Heart VMAT2 mRNA expression was first detected in the primitive heart tube at E8. The strongest signal during heart development was observed at E12. At E13, when we were able to make a clear distinction between the atria and the ventricles, it seemed that the VMAT2 mRNA expression was limited to the ventricles. By E14 there was expression in the atrium as well ŽFig. 2A,B.. At this time there was a very strong, clear labeling of the developing atrio-ventricular valves. By E16 mainly the ventricles were positive. Up to this time the number of positive cells remained the same and then both the signal intensity and number of cells gradually decreased. At E21 no signal was detectable. Weak expression of VMAT1 mRNA was first detected in the ventricle by E13. The signal intensity increased by E15. At E18 the atrial signal significantly decreased, but the atrio-ventricular valves became positive. The ventricular signal was still present at E21. From adjacent sections it appears that the same cells expressed VMAT2 and VMAT1 ŽFig. 2C–E.. 3.6.2. Aorta The endothelium of the aorta expressed VMAT2 mRNA from E11 through E13. No signal was detected here at later timepoints. A group of intensely labeled VMAT1 and VMAT2 mRNA positive cells was found at the root of the aorta starting at E13. Most likely these were cells of the sinus node, part of the sympathetic nervous system ŽFig. 2A,B.. After E18, this group of cells was no longer detectable. 3.7. Gastrointestinal tract 3.7.1. Esophagus, stomach, small and large intestines The first VMAT2 mRNA-positive cells in the primitive gut were detected by E12. By E13 these positive cells were confined to the ganglionic cells of both the submucosal ŽMeissner. and myenteric ŽAuerbach. plexi. The number of cells increased up to day E16. The Auerbach Žmyenteric. nerve plexus of the esophagus expressed the VMAT2 mRNA by E16. This signal intensified by E17 and remained unchanged throughout gestation ŽFig. 3I,J.. No VMAT1 mRNA was present in the esophagus. By E18 the hybridization signal became more intense per individual cell and a few very intensely labelled cells also appeared within the epithelia of the upper and lower GI tract. Based
Fig. 3. Intestinal tract. Darkfield ŽA. and brightfield ŽB. image show VMAT1 mRNA expression in a group of cells located in the epithelium of the intestinal wall Žarrowheads. at E18. In an adjacent section ŽC. VMAT2 mRNA expression is viewed. Note that most of the signal is located in the outer nerve plexus Žsmall arrow.. Some positive cells are located in the epithelium Žlarge arrow.. Darkfield ŽD. and brightfield ŽE. image show VMAT1 mRNA expression in a group of cells located in the epithelium of the intestinal wall at E21. In adjacent sections ŽF. VMAT2 mRNA expression is viewed. The signal is located in the outer nerve plexus Žarrowheads.. Additional signal is found in the nerve plexus of the stomach ŽS.. The lumen of the stomach is indicated with a star. Brightfield ŽG. and darkfield ŽH. images show VMAT1 mRNA expression in the epithelium and the inner nerve plexus of the antrum at E21. Brightfield ŽI. and darkfield ŽJ. images demonstrate VMAT2 mRNA expression in a longitudinal section of the esophagus Že.. Arrowheads point at cells innervating the distal part of esophagus. Scale bars: B, 50 mm; E, 340 mm; G, 120 mm; I, 150 mm.
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on their number and distribution these cells were reminescent of the endocrine Ženterochromaffin. cells of the gastrointestinal system. The signal remained the same at E21 ŽFig. 3A–F.. Expression of VMAT1 mRNA was first detected at E15. In contrast to VMAT2 mRNA, VMAT1 mRNA was expressed in cells scattered through the epithelial lining. The signal intensity and the number of positive cells increased by E18. By E21, in addition to scattered intensely positive endocrine cells, many mucosal cells in the small intestine had a moderately intense hybridization signal. The fundic stomach mucosa was negative, but the antral mucosa Žwhere the gastrin producing endocrine cells are located. was very strongly labelled for VMAT1 mRNA starting at E18 ŽFig. 3G,H.. A group of cells were labelled in the ganglia of the submucosal plexus ŽMeissner., but these cells showed a low intensity signal.
stationary macrophages ŽKupffer cells.. The hybridization intensity was even throughout gestation. The number of positive cells increased as the liver grew. After E15, the positive cell population seemed constant ŽFig. 4A,B.. VMAT1 mRNA first appeared also at E13, but the signal was rather low and homogeneously distributed. This pattern remained unchanged throughout development and was still present at E21.
3.7.2. LiÕer Beginning at E13, a population of cells expressed high levels of VMAT2 mRNA. These cells are most likely
3.8.1. Pituitary gland VMAT2 mRNA positive cells were observed in the anterior pituitary by E17. The signal was still present at
3.7.3. Pancreas Starting at E18 scattered exocrine pancreatic cells expressed high levels of both VMAT1 and VMAT2 mRNA. The expression pattern and intensity did not change at E21. 3.8. Endocrine organs
Fig. 4. Liver and thymus. Brightfield ŽA. and darkfieldŽB. images show VMAT2 mRNA expression in the liver at E16. Arrows point at some of the positive cells in the parenchyma. Additional expression is seen in the myenteric plexus of the intestine Žarrowheads.. BrightfieldŽC. and darkfield ŽD. images show VMAT1 mRNA-expressing cells in the thymus Žarrows. at E18. Scale bars: B, 130 mm; D, 40 mm.
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E18, but disappeared by E21. VMAT1 mRNA is also expressed in a group Žmost likely all. of cells in the pituitary anterior lobe starting at E18. By E21 only VMAT1 mRNA is expressed in the pituitary in cells that are evenly distributed throughout the anterior lobe ŽFig. 5A–C.. At E18 and E21 there is also a strong expression of VMAT1 mRNA in the median eminencerpituitary stalk area, that
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might reflect expression in the developing portal vasculature.
3.8.2. Thyroid gland VMAT1 mRNA was seen in a group of cells in the thyroid gland starting at E18, and was still present at E21.
Fig. 5. Endocrine tissues. VMAT1 mRNA is expressed in the pituitary gland at E18 ŽA,B. and E21 ŽC.. At E18 the positive cells are grouped in the posterior part of the anterior lobe Žarrow.. By E21 the VMAT1 mRNA positive cells are distributed evenly in the anterior lobe. Arrows point at some of the positive cells. Note the absence of signal in the posterior lobe Žarrowhead.. The base of the skull is indicated by a star. ŽD. High magnification of VMAT1 mRNA expression in the adrenal medulla at E18. In an adjacent section, brightfield ŽE. and darkfield ŽF. images co-expression of VMAT2 mRNA is indicated by arrows. In a high magnification of the developing testis ŽG. at E18 a group of cells with intense expression of VMAT1 mRNA as indicated by the arrows. VMAT1 mRNA expression is detected in a scattered cell population in the seminal vesicle ŽH. at day E21 Žarrows.. Scale bars: B, 120 mm; E, 75 mm; G, 25 mm; H, 40 mm.
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3.8.3. Testis and oÕary VMAT2 mRNA was detected in the testis on E15. The positive cells were found between the tubuli. The signal intensity was high until E18 and then significantly decreased ŽFig. 5G.. VMAT1 mRNA is expressed in the testis starting on E17, and remains unaltered through E21. VMAT2 is also detected in the differentiating ovary at E18.
labeled cells were detected. The intensity per cell remained unchanged but the number of positive cells increased in the medulla throughout gestation. Expression of VMAT1 mRNA was first detected in the developing adrenal medulla at E13. The signal intensity was strong and remained unchanged through E21. Comparing adjacent sections, it appeared that the same cell population expressed VMAT2 and VMAT1 mRNAs ŽFig. 5D–E..
3.8.4. Seminal Õesicle Cells of the seminal vesicle expressed VMAT1 mRNA at E21. Intensely labeled cells were found in the vesicle ŽFig. 5H..
3.9. Sensory organs
3.8.5. Adrenal VMAT2 mRNA expression was first visible in the primitive adrenal by E12. At this time a few intensely
3.9.1. Skin There was a weak VMAT1 mRNA signal detected in the skin of the fore and hind paw by E13. Starting at E16 both VMAT1 and VMAT2 mRNAs are expressed in the skin. The hybridization signal for VMAT2 was very intense by E15 ŽFig. 6E,F.. From E17 throughout gestation,
Fig. 6. Connective tissue. Brightfield ŽA. and darkfield ŽB. images demonstrate VMAT1 mRNA expression in the trabecules of the developing snout at E21. Arrows point at some of the positive cells. Brightfield ŽC. and darkfield ŽD. images show VMAT1 mRNA expression in the developing palatine shelf. The star indicates the epiphyseal cartilage. The positive cells are located within the trabecles of the primitive bone marrow located on both sides of the epiphysis. Brightfield ŽE. and darkfield ŽF. images show VMAT2 mRNA expression in the skin Žarrowhead. of the hind paw and the central touch dome Žarrow.. Scale bars: A, 80 mm; C, 190 mm; E, 110 mm.
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the signal began to weaken. At E21 a small population of cells in the subcutaneous layer of the tail skin still expressed VMAT2 mRNA. 3.9.2. Tongue A small population of cells within the tongue expressed the VMAT2 mRNA starting at day E14. The signal intensity peaked at E17 and then decreased to a non-detectable level by E21. Based on their location, these cells are likely to be muscle spindles, a modified muscle cell which functions as a propioreceptor ŽFig. 7A,B.. VMAT1 mRNA appears in the tongue at E18 and unlike VMAT2, is still present at E21. 3.10. Other organs 3.10.1. Lung Starting at E16, intramural ganglionic cells around the main bronchus expressed VMAT2 mRNA. The expression pattern did not change through E21. 3.10.2. Cartilage, joints, periosteum VMAT2 mRNA positive cells appear in the limb bud as early as E12. Cells expressing VMAT2 mRNA were found in layers surrounding the joint capsules of the limbs at E15
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ŽFig. 7C,D.. No signal was detected in this location after day E18. Cells surrounding the bones Žperiosteum. express VMAT2 mRNA between E15 and E18. There is VMAT1 mRNA expression in the facial cartilage starting at E13 which was still present at E21. Positive cells are found in the trabeculae of the snout and in the palatine shelf ŽFig. 6.. VMAT2 mRNA is also expressed here, but the expression only starts at day E16, and continues through E21. The signal remained unchanged at E21. 3.10.3. Thymus VMAT2 mRNA appeared in the thymus at E17 when a few cells were weakly labelled. At E18 the labeling of VMAT2 mRNA started to decrease, but VMAT1 mRNA appeared in many more cells throughout the parenchyma of the thymus ŽFig. 4C,D.. These cells are likely to be differentiating T cells. The VMAT2 expression disappears, but VMAT1 mRNA expression remains unchanged at E21. 3.11. Extraembryonic structures 3.11.1. Yolk sac The yolk sac was only analyzed on days E10 and E11. A population of cells in the yolk sac seemed to express both VMAT1 and VMAT2 mRNA Ždata not shown..
Fig. 7. Brightfield ŽA. and darkfield ŽB. images show a sagittal section through the tongue at E17. Arrows point at a group of VMAT2 mRNA expressing cells, located deep in the tongue parenchyma. Based on the location these cells are likely to be muscle spindles of the tongue. Brightfield ŽC. and darkfield ŽD. images show VMAT2 mRNA expression in cells adjacent to the elbow joint capsule Žarrows.. Scale bars: A, 150 mm; C, 110 mm.
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Fig. 8. Brightfield ŽA. and darkfield ŽB. view of a high magnification of VMAT1 mRNA expressing cells in the decidua Žarrowheads. at day E14. Brightfield ŽC. and darkfield ŽD. images show VMAT2 mRNA expression in syncytiotrophoblast cells Žarrows. in the spongiotrophoblast zone at E14. Scale bars: A, 45 mm; C, 45 mm.
3.11.2. Placenta The ectoplacental glycogen cells express large amounts of VMAT2 mRNA at E8 and E9. At E14 a population of cells in the spongiotrophoblast zone expressed VMAT2 mRNA ŽFig. 8C,D.. Weak expression of VMAT1 mRNA was also detected in this region. In the decidua, a population of cells radiating out from the ectoplacental cone
expressed VMAT1 but not VMAT2 mRNA. Based on their size, these cells might be migrating giant cells ŽFig. 8A,B.. The placenta was not studied at later time points. 3.11.3. Controls The control experiments showed that 1. None of the sense probes show any labeling in the embryos; 2. The
Fig. 9. Staining in sagittal sections of E17 ŽA,B,E,F. and E19 ŽC,D,G,H. day old embryos. Immunopositivity in adrenal medullary cells is demonstrated for VMAT1 ŽA. and VMAT2 ŽB.. Positive cells for VMAT1 are also present in the myenteric plexus of the developing GI tract ŽC. and VMAT2 positive cells are also present in some epithelial cells ŽD.. Paravertebral sympathetic ganglia are immunopositive for both VMAT1 ŽE. and VMAT2 ŽF.. G demonstrates immunostaining in the trigeminal and H in the vestibulocochlear Žarrows. and trigeminal Žasterisk. and the trigeminal and acoustic nerves. Bar scales: A,B: 500 mm; C,D: 1 mm; E,F: 200 mm; G: 300 mm and H: 400 mm.
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different, non-overlapping, antisense probes showed an identical mRNA distribution for the individual mRNAs; 3. Immunohistochemical staining for VMAT1 and VMAT2 protein at ages E17 and E19 showed a good correlation with the VMAT1 and VMAT2 mRNA distribution at the same time. In Fig. 9 we demonstrate the immunostaining in the adrenal medulla, in sympathetic and cranial ganglia and in the hippocampus. All the above controls strongly suggest that the mRNA labeling is highly specific.
4. Discussion VMATs store and release monoamines. Monoamines Žmostly serotonin. have been suggested to affect embryonic development Žsee w16,23,35x.. In addition to transporting naturally occurring substrates, the transporters can protect neurons by sequestering neurotoxins in synaptic vesicles w1,26,32x. The two transporters have different affinities for their natural substrates. VMAT2 has a 3 fold higher affinity for 5HT, DA, NE than VMAT1 has, and approximately a 100-fold higher affinity for histamine w2,4x. The pharmacological properties of these transporters are also somewhat different w9,17,19x. The purpose of this study was to determine the developmental pattern of the VMATs. In adult animals the distributions of the two vesicular transporters are partially nonoverlapping w8,9,34x. In adult rats w26x both transporter mRNAs were detected in adrenal chromaffin cells, and sympathetic and enteric parasympathetic ganglionic cells. On the other hand, exclusively VMAT2 seems to be present in the CNS and VMAT1 in certain endocrine cells with no overlapping between them w27,34x. Unlike adults, embryos exhibit a considerable co-expression of the two vesicular transporters. In certain areas or tissues the presence of VMATs ŽmRNA or protein. may persist into adulthood. Most likely certain cells in these tissues are truly monoaminergic, and in such case VMATs are required for monoamine storage and release. These tissues include the adrenal medulla, the gastrointestinal tract, and the heart. The adrenal medulla is considered to be part of the sympathetic nervous system and it is derived from neural crest cells. Recently cells exhibiting all the features of monoaminergic cells were described in the stomach w7,14,20x and in the pancreas w21x of the adult rat and a population of intrinsic adrenergic cells were found in the heart of both the adult rodent and human w13x. These cells might help regulate cardiac function in parallel with the autonomic nervous system. In addition to detecting VMATs in tissues destined to have monoaminergic cells in adult animals we found a transient expression of VMAT1 and VMAT2 mRNAs in many neural crest derived tissues where aminergic cells are not known to exist in the adult: sympathetic nervous system, intrinsic parasympathetic ganglia, dorsal root gan-
glia, cranial nerves ganglia, endocrine organs, thymus and connective tissues. It is possible that the transiently expressed VMAT mRNAs have no functional role in these tissues. Alternatively, these tissues have monoaminergic cells that are only present during a certain period of development. These cells, most of which seem to arise from the neural crest, may release monoamines and affect tissue differentiation. For example, VMAT2 mRNA was present in the developing aorta from E11 until E14. Neural crest cells participate in establishing the branching pattern of the aortic arch arteries, and in dictating the formation of the cardiac septum w15,33x. The transient expression of VMAT2 in the aorta may reflect a local role for monoamines during a critical period of heart development. Sensory neurons also derive from the neural crest w5,6,30x. The dorsal root ganglia are relay stations for sensory afferent fibers. Unlike the sympathetic ganglion cells, dorsal root ganglia express VMAT2 mRNA exclusively. Sympathetic ganglion cells derive from a distinct crest lineage from dorsal root ganglionic cells which might explain the differences in VMAT mRNA expression. The rami communicans ŽRC. are nerve bundles transmitting sensory inputs from the dorsal root ganglia to the spinal cord. Monoaminergic characteristics have previously not been shown in the RC, yet here we demonstrate a distinct expression of VMAT2 mRNA in these nerves. At E13, this expression might be explained by glial precursor cells migrating towards the dorsal root ganglia. VMAT2 mRNA is still expressed after day E14 which is the time point when migration ends and when Schwann cell precursors are first detected w22x. The presence of VMAT2 mRNA around the joints of the developing limbs, and in the tongue, suggests that the expression might be located in muscular spindles and propioreceptors in the capsules of joints and tendons. This part of the sensory system supplies the CNS with information required for properly coordinated movements of limbs and tongue. These findings together with the widespread presence of VMAT mRNAs in sensory brainstem nuclei and in the thalamus during the same time in ontogenesis w11x indicates a role of monoamines in the developing sensory pathways.
5. VMAT mRNAs in the placenta Our data suggest that placental cells can store monoamines. The placenta possesses a highly active serotonin transporter w10x. Dopamine and norepinephrine plasma membrane transporter mRNAs are also expressed in the placenta. The expression of VMAT1 and VMAT2 mRNAs in the syncytiotrophoblasts will enable these cells to metabolize and release monoamines in a controlled fashion. Monoamines are effectively cleared from the maternal circulation and the intra villus space w10x. After uptake, the monoamines are rapidly degraded by
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monoamineoxidase A which is present in the placenta. Thus the syncytiotrophoblasts take up and metabolize monoamines, preventing them from interfering with the developing embryo. Excessive amounts of serotonin have been shown to be lethal during embryogenesis w36x and to cause cell death in the neural crest population when injected intraperitoneally in newborn mice w24x. VMAT1 and VMAT2 mRNAs are expressed in different subpopulations in the placenta. VMAT2 mRNA is expressed in the spongiotrophoblast zone, an area where we also detect the serotonin plasma membrane transporter w12x. VMAT1 mRNA is found in large cells of the decidua. Giant cells that express VMAT1 mRNA are hormonally regulated secretory cells, similarly to the enterochromaffin cells of the GI system, where VMAT1 mRNA is present in the adult. A gradual increase in the levels of monoamines and their metabolites is found in the amniotic fluid in late gestation w28,31x. The high expression of VMAT mRNAs in the placenta suggests that a regulated release of monoamines from the placenta could participate in the induction of labor. We have examined the distribution of VMAT1 and VMAT2 mRNA during embryonic development. Strikingly, neural crest derivatives such as sensory neurons, sympathetic ganglia and enteric ganglia all express VMAT2 mRNA. All these areas are also immunopositive when stained with an antibody that recognizes the VMAT2 protein Ždata not shown., which means that the protein is expressed there. Further studies are necessary to determine if these cells are truly monoaminergic and also express one of the plasma membrane transporters. If this is true, than these cells might be involved in regulating monoamine levels in their target areas during embryonic development. Interestingly, both VMAT mRNAs are expressed in many regions at the beginning of ontogenesis. Later, the expression becomes more distinct and it seems that VMAT2 mRNA is predominantly expressed in neural while VMAT1 is expressed in endocrine tissues. Further studies will be necessary to evaluate the significance of these findings.
Acknowledgements The authors would like to thank Miklos ´ Palkovits for his suggestions and help in putting the data together, Michael Brownstein for his help with editing and Ricardo Dreyfuss for photography.
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