Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
« Previous Article | Table of Contents | Next Article »
Circulation Research. 1996;79:930-939

This Article
Right arrow Abstract Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hiltunen, J. O.
Right arrow Articles by Saarma, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hiltunen, J. O.
Right arrow Articles by Saarma, M.
(Circulation Research. 1996;79:930-939.)
© 1996 American Heart Association, Inc.

Articles

Expression of mRNAs for Neurotrophins and Their Receptors in Developing Rat Heart

Jukka O. Hiltunen, Urmas Arumae, Maxim Moshnyakov, Mart Saarma

the Institute of Biotechnology, Laboratory of Molecular Neurobiology, University of Helsinki (Finland).

Correspondence to Jukka O. Hiltunen, Institute of Biotechnology, Laboratory of Molecular Neurobiology, PO Box 56, Viikinkaari 9, FIN-00014, University of Helsinki, Finland. E-mail juhiltun@cc.helsinki.fi.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Because the neurotrophic system has not been systematically studied in developing heart, we studied the expression of mRNAs for neurotrophins and their high- and low-affinity receptors by radioactive in situ hybridization in the rat heart from embryonic day 9 (E9) to parturition. The neurotrophin-3 (NT-3) transcripts were seen in the group of Leu-7 immunoreactive cells in the ventricular region from E11 to parturition, suggesting that NT-3 is expressed in the part of the developing conduction system. mRNAs for truncated trk receptors, trkC.TK- and trkB.T1, were expressed in the outflow tract at E12 and in the walls of developing aorta and pulmonary trunk from E13 to parturition, whereas the mRNA for catalytic trkC.TK+ was revealed in the walls of aorta and pulmonary trunk from E13 to parturition and in the cardiac ganglion neurons from E14 to adult stage. Transcripts for low-affinity neurotrophin receptor (p75) were transiently seen in the distal outflow tract from E11 to E13, declining by E14. At E18, p75 transcripts were also seen in the cardiac ganglia. Transcripts for nerve growth factor, neurotrophin-4/5, trkA, or trkB.TK+ were not detected. Expression of NT-3 mRNA in the developing conduction system and of trkC.TK+ mRNA in the cardiac neurons suggests a role for NT-3 in the innervation of the conduction system. Expression of trkC.TK+ in the wall of aorta and pulmonary trunk suggests that NT-3 also may affect the development of the smooth muscle cells.


Key Words: neurotrophin • embryonic heart • in situ hybridization • conduction system • smooth muscle


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Neurotrophins are proteins regulating neuronal differentiation, maturation, and survival. To date, four NTFs have been described: NGF, BDNF, NT-3, and NT-4/5.1 2 NT-6 has been described from platyfish Xiphophorus, but its mammalian homologue has not been reported.3 NTFs specifically bind to and activate trk tyrosine kinase receptors.4 Thus, NGF activates trkA, BDNF and NT-4/5 activate trkB, and NT-3 is a ligand for trkC (NT-3 is also a less efficient ligand for trkA and trkB).

In addition to full-length catalytic forms of the trk receptors, there are also several alternatively spliced trk isoforms. The mRNA for full-length catalytic trkB (trkB.TK+) is alternatively spliced into two truncated isoforms (trkB.T1 and trkB.T2).5 6 Also, four truncated forms are described for trkC, in addition to other isoforms with inserts in the tyrosine kinase domain.7 8 9 All truncated isoforms of trkB and trkC contain unique amino acid sequences in their cytoplasmic domain that are devoid of distinctive structural motifs. trkB.T1 mRNA is expressed in several regions of rat brain.5 The biological functions of truncated trk receptors are basically unknown. However, in a recent study, leptomeningeal cells of chick embryo were shown to internalize BDNF by truncated trkB, thus restricting the availability of BDNF for certain neuronal populations only.10 The in vivo expressions of trkB.T2 as well as of four truncated trkC isoforms are not described.

LANR (p75) belongs to the tumor necrosis factor receptor family. All NTFs bind to it with low-affinity kinetics, but its functions are poorly defined.11 12 Only recently, NGF binding to LANR has specifically been shown to activate nuclear factor-{kappa}B transcription factor, demonstrating that LANR have independent functions not related to trk receptors.13 LANR transcripts and proteins are widely expressed in the nervous system, in both neuronal and glial cells, and in the nonneuronal tissues.14 15 16

The heart is an interesting model for studying the developmental roles of NTFs because it is innervated by sensory, sympathetic, and parasympathetic nerve fibers. Also, the conduction system of the heart expresses some neuronal markers, suggesting a potential role of NTFs in its development.17 18 However, the data about the NTF system in developing heart are fragmentary. All four NTFs have been detected from developing rat hearts by Northern blotting or RNase protection,19 20 21 22 23 whereas NGF, BDNF, and NT-3 were localized by in situ hybridization in rat hearts.24 trkB.TK+ and trkC.TK+ have been reported in human or rat hearts with Northern blotting or RNase protection.8 25 trkC was localized to the pericardium and the wall of aorta by in situ hybridization using a pan-trkC probe.26 However, tissue localization of other trk receptors as well as of their isoforms in the heart is not known. Only a few studies have mentioned the expression of LANR in the rodent heart, but the exact localization remained undetermined, although there is a demonstration of the expression of LANR mRNA in the E8 to E10 chick parasympathetic cardiac neurons.23 27 28

Because the expression of NTFs and their receptors in the developing rat heart has not been studied systematically and because the localization of the truncated isoforms for NTF receptors is not known, we decided to perform a detailed study of the expression of all NTFs and their receptors with radioactive in situ hybridization. Our results suggest a role for NTFs and their receptors in the development of the conduction system and the OT wall.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Reagents
All reagents were purchased from Merck unless otherwise noted.

Animals
Female Sprague-Dawley rats were mated overnight, and the following day was considered to be E0. The somites of embryos younger than E13 were counted to verify the accuracy of staging. For in situ hybridizations, at least three independent embryos were used at each embryonic day. All embryos produced similar results.

Tissue Processing
The female rats were killed with CO2 followed by cervical dislocation. The embryos and, in some cases, the adult hearts were rapidly removed and dissected in ice-cold Dulbecco's modified PBS, pH 7.4, transferred into freshly prepared cold 4% PFA, and fixed overnight at 4°C. For the in situ hybridization and immunohistochemistry, the embryos and the hearts were rinsed in PBS, dehydrated through ascending ethanol series, cleared with toluene, and embedded in paraffin (Histowax, Reichert-Jung). Sagittal, frontal, or transverse sections of 6 µm were cut and mounted onto the silanized slides. The slides were stored desiccated at 4°C until use.

For the in situ hybridization with cryosections, the E14 and E18 rat embryos were immersion-frozen in isopentane cooled by liquid nitrogen and stored at -80°C until sectioned. Sections of 8 µm were cut, air-dried, fixed in 4% PFA, immersed in 70% and 100% ethanol, air-dried, and stored desiccated at -20°C until use.

Probes
The probes for rat NGF, BDNF, NT-3, NT-4/5, trkA, pan-trkB, trkB.TK+, trkB.T1, pan-trkC, and LANR have been described previously.29 30 31 32 The probe specific for the tyrosine kinase domain of rat trkC (trkC.TK+) was prepared by PCR and cloned into pCRII vector (Invitrogen). The probe for the intracellular part of truncated rat trkC (trkC.TK-) was prepared by PCR using primers as described8 and cloned into pCRII vector. This probe recognizes all four truncated forms and corresponds to amino acids 544 to 597.8 trkC.TK+ probe recognizes all four TK domain–containing isoforms and corresponds to rat trkC cDNA sequence nucleotides 2308 to 2552.33 A 167-bp insert specific for trkB.T2 was cloned by PCR into pCRII and encompasses the nucleotides 1343 to 1509.6 The BDNF probe used in our earlier studies traverses a 290-bp fragment (nucleotides 320 to 60934 ) of the rat preproBDNF mRNA. In addition, a different 366-bp BDNF construct was cloned into pGEM3Z (Promega). This fragment traverses nucleotides 517 to 882 of the rat BDNF cDNA sequence34 and localizes in the mature BDNF mRNA, except for the first two nucleotides, which belong to the proBDNF mRNA. This probe is almost identical to that used by Scarisbrick et al.24 The identity of all probes was verified by direct sequencing.

In Situ Hybridization
35S-labeled single-strand antisense and control sense cRNA probes were prepared by using 35S-labeled UTP (Amersham) and appropriate RNA polymerases as described previously.29 30 Hybridization was performed as described35 with slight modifications.30 Both sense and antisense slides were treated similarly. Briefly, paraffin sections were deparaffinized in xylene, rehydrated, fixed in 4% PFA, rinsed in PBS, treated with proteinase K (20 µg/mL) (Sigma Chemical Co) for 5 minutes, postfixed with 4% PFA, acetylated, dehydrated, air-dried, and hybridized overnight at 52°C with final probe concentration of 30 000 cpm/µL. Cryosections were treated as paraffin sections, with the exception of proteinase K treatment (1 µg/mL). Prehybridization (1 to 2 hours at 52°C) was performed using the hybridization mixture lacking the probe for embryos older than E14. After hybridization, the sections were rinsed in high stringency for 10 to 30 minutes and treated with RNase A (Boehringer Mannheim). Then the slides were dehydrated and air-dried, dipped in NTB-2 emulsion (Kodak), exposed for 3 to 4 weeks, and developed. The sections were counterstained with Harris hematoxylin, cleared with xylene, and mounted with Permount (Fisher Scientific). Photomicrographs were taken with Leitz Axiophot or Reichert Jung Polyvar microscopes equipped with dark-field and phase-contrast illumination. Specificity of hybridization was deduced from the labeling pattern of several tissues known to express specifically different NTFs and their receptors.15 29 30 36 37 Hybridization of adjacent sections with probes in sense orientation always resulted in only background labeling with equal grain distribution over the whole slide, except for blood cells, which showed unspecific labeling in both antisense and sense slides. Cryosections were used as controls for NGF, BDNF, and NT-3 probes at E14 and E18, and their hybridization pattern was similar compared with paraffin sections.

Immunohistochemistry
Mouse monoclonal anti–Leu-7 (HNK-1) antibody was obtained from Becton Dickinson Immunocytometry Systems, and rabbit anti-peripherin antiserum and mouse monoclonal anti-SMA antibodies were purchased from Progen and Chemicon International Inc, respectively. Mouse monoclonal antibody 13AA8, recognizing all forms of neurofilament proteins, was provided by Dr I. Virtanen, Department of Anatomy, University of Helsinki. FITC- and TRITC-labeled affinity-purified donkey anti-rabbit IgG and anti-mouse IgG antibodies were obtained from Jackson Laboratory. For Leu-7 staining, paraffin sections were deparaffinized, rehydrated and rinsed in PBS, digested with proteinase K (100 µg/mL) (Sigma) for 5 minutes at room temperature, postfixed with 4% PFA for 15 minutes, rinsed in PBS, and blocked with 0.1% bovine serum albumin (Sigma), 0.05% Triton X-100, and BTP for 15 minutes. After the sections were rinsed in PBS, the slides were incubated with monoclonal mouse anti–Leu-7 antibody diluted at 1:20 in BTP overnight at 4°C. Sections were rinsed in PBS and incubated with FITC-labeled goat anti-mouse IgM (Sera Lab) diluted at 1:50 in PBS for 45 minutes. For peripherin and SMA staining, deparaffinized sections were treated with trypsin (0.5 mg/mL) (Worthington) at 37°C for 4 minutes and rinsed in PBS. The sections for peripherin staining were treated with 0.01% sodium borohydride for 30 minutes to reduce background. After several changes of PBS, the slides were blocked with BTP and incubated with anti-peripherin antibodies diluted at 1:300 in BTP or with anti-SMA antibodies at 1:250 at 4°C overnight, followed by FITC–anti-rabbit or TRITC–anti-mouse antibodies at a dilution of 1:600 in BTP for 45 minutes. The slides were examined with a Leitz Axiophot microscope equipped with epifluorescence. No staining of the sections was seen in controls, where the primary antibodies were omitted.

Image Processing
Dark-field images of NTF and receptor in situ hybridizations were either digitized using a Cohu 4912 CCD camera and a Scion LG-3 frame-grabber card with NIH Image 1.56 software or scanned from photomicrographs with a Hewlett-Packard ScanJet 3C scanner. The illustrations were prepared with Microsoft Paint Shop Pro 3.0 and Microsoft Designer 4.0.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
In the present study the conus, truncus, and aortic sac are considered as an OT at E11 to E12.5. The term cardiac ganglion neurons denotes all intrinsic neurons of the heart regardless of their neurotransmitter phenotype.

Expression of NT-3 mRNA
The first indications of NT-3 expression in the developing rat heart occurred at E11 (23 somites, not shown). The expression was seen in the developing interventricular septum, and this hybridization was more prominent at E12 (34 somites, Fig 1ADown). At that time NT-3 mRNA expression had also spread to the myocardium along the AV canal.



View larger version (106K):
[in this window]
[in a new window]
  		Figure 1. NT-3 mRNA expression in E12 and E14 rat hearts. A through H, Dark-field (A, C through E, and H) and bright-field (B and F) images of the frontal and sagittal sections through the E12 (A and B) and E14 (C through H) rat hearts hybridized with antisense (A, C, E, and H) and sense (D) NT-3 cRNA probe. Panel G shows immunostaining of a section adjacent to that shown in panels E and F stained with monoclonal anti–Leu-7 antibodies showing immunoreactivity in distal interventricular septum. In panel H, NT-3 mRNA hybridization in section more posterior than that in panel E shows silver grains over cells within AV cushion tissue. Labeled are right atrium (ra), left atrium (la), right ventricle (rv), and left ventricle (lv). The arrow in panel D marks the ventral limit of the cells expressing NT-3 antisense mRNA in panel C. Blood cells in panel G show autofluorescence. Bars=100 µm (A) and 200 µm (D).

At E14, intense NT-3 expression was seen in the distal interventricular septum, continuing on both sides of the septum and septal trabeculae (Fig 1C and 1EUpUp). Also, part of the AV junction cushion tissue showed NT-3 transcripts (Fig 1HUp). Leu-7 immunoreactivity (Fig 1GUp) colocalized with NT-3 probe hybridization. At E18 (Fig 2Down), the NT-3 probe hybridization was seen in the distal IV septum, as at E14, and hybridization continued on both sides of the septum as a narrow subendocardial band toward the apex cordis by gradual declining of NT-3 message and was also seen in part of the trabeculae of both ventricles (not shown). These cells most probably correspond to the bundle of His (Figs 1H and 2AUpDown) and the left and right bundle branches (Figs 1E and 2CUpDown).



View larger version (121K):
[in this window]
[in a new window]
  		Figure 2. NT-3 mRNA expression in the E18 rat heart. A through D, Dark-field (A and C) and bright-field (B and D) images of the frontal sections through the E18 rat heart AV region, hybridized with antisense NT-3 cRNA probe. In panel A, hybridization can be seen within AV cushion tissue. In panel C, subendocardial tissue shows NT-3 hybridization. Labeled are right atrium (ra), left atrium (la), right ventricle (rv), and left ventricle (lv). Bar=200 µm.

Leu-7 is a 3-sulfated glucuronyl-substituted lacto-series oligosaccharide epitope that was first detected as a human natural killer-cell marker.38 39 It is expressed on the surfaces of several cells, and it participates in recognition and adhesion processes. Anti–Leu-7 antibody has been used to recognize cardiac conduction system cells in developing rat heart from E9.5 to E18.5.40 41 42 We demonstrated (Fig 1E and 1GUpUp and not shown) that at least at E12 to E14, Leu-7 antigen was colocalized with NT-3 mRNA in the ventricular region of developing rat heart. Moreover, the region of Leu-7/NT-3 mRNA colocalization overlapped topologically with the region of the conduction system described by Viragh and Challice.43 Therefore, we conclude that conduction system cells express mRNA for NT-3 at E11 to parturition.

NT-3 mRNA was observed in the myocardium along both AV canals, valve leaflets of the superior venae cavae, and the sinoatrial region at E12 and E14 (not shown). Since these regions were also partly stained with anti–Leu-7 antibodies (not shown), they most probably are a part of the conduction system.

Expression of NGF, BDNF, and NT-4/5 mRNAs
NGF and NT-4/5 expressions were not detected in embryonic rat hearts by in situ hybridization at any developmental stage examined (not shown). BDNF expression was observed over few scattered cells in the wall of the aorta and pulmonary trunk at E13 and E14 (not shown).

Expression of mRNAs of trk Receptors
The first indications of trk-specific transcripts in the developing rat heart were seen in the distal OT region at E11 with a pan-trkC probe. This most probably represents the trkC.TK- isoform, because the probe detecting truncated isoforms of trkC hybridized to the same region at E12 (not shown). At E14, truncated trkC expression was seen in the walls of aorta and pulmonary trunk (Fig 3BDown). The hybridization was seen throughout the whole wall, except the endothelium, which was not labeled. At E18, the expression of trkC.TK- in the aorta and pulmonary trunk diminished markedly (not shown). Smooth muscle cells were the main cell type expressing trkC.TK- mRNA in the aortic wall as trkC.TK– mRNA hybridization colocalized with SMA immunoreactivity there (not shown). At E18 to parturition, the pan-trkC probe also labeled the proximal coronary arteries (not shown). This labeling was not seen with the trkC.TK– probe.



View larger version (133K):
[in this window]
[in a new window]
  		Figure 3. Expression of trks in E13, E14, E16, and E18 rat hearts. A through G, Dark-field (A through D and F) and bright-field (E) images of the frontal and sagittal sections through E13 (A), E14 (B, C), E16 (D and E), and E18 (F and G) rat hearts hybridized with antisense trkC.TK+ (A and D), trkC.TK- (B), and trkB.T1 (C and F) cRNA probes. Arrowheads in panels B and C denote the semilunar valve region where trkB.T1 hybridization can be seen, but not trkC.TK-. The small arrow in panel D indicates trkC.TK+ labeling in the sympathetic trunk. In panel G, immunostaining of the section adjacent to that shown in panel F with monoclonal antibodies to smooth muscle {alpha}-actin is shown. The labeling of the blood cells in panel F is a hybridization artifact, and the same blood cells show autofluorescence in panel G. Labeled are developing aorta (ao), pulmonary trunk (pt), ventricle (v), semilunar valves (sv), and sympathetic trunk (sy). Bars=400 µm (A) and 200 µm (G).

trkB.T1 transcripts were first detected over scattered cells in the distal OT at E12 (not shown). At E14 and E18, trkB.T1 expression was clearly seen in the aorta and pulmonary trunk (Fig 3C and 3FUpUp), and the labeling continued into the semilunar valve region where mRNAs for trkC were not seen. As revealed in semitransverse sections of the aortic region at E18, trkB.T1 mRNA and SMA immunoreactivity colocalized in most regions, but there were also SMA-positive areas devoid of trkB.T1 transcripts, eg, the developing adventitial layer of aorta (Fig 3F and 3GUpUp). On the contrary, trkB.T1 mRNA but not SMA-immunoreactivity was found in the semilunar valves of pulmonary trunk and aorta (Fig 3F and 3GUpUp).

trkC.TK+ expression started at E13. It was clearly seen in the wall of the aorta and pulmonary trunk (Fig 3AUp), and from E16 up to the birth, the expression was localized to the parts of the tunica media of aorta and pulmonary trunk (Fig 3D and 3EUpUp). trkC.TK+ mRNA expression colocalized with SMA immunostaining as well as with trkC.TK- mRNA in the wall of the aorta and pulmonary trunk at later stages of embryonic development (not shown).

trkC.TK+ probe hybridized to cell groups in the dorsal wall of the right atrium at E14 (not shown), which most probably represent cardiac ganglion neurons. Some expression was also seen over a few cell bodies in the sinus venosus region as early as E11, but the identity of these cells remains unknown. At later stages, trkC.TK+ transcripts were clearly seen over cardiac ganglion neurons situated in the dorsal aspect of the atria (Figs 4Down and 5) and at the entrance of the inferior venae cavae (not shown). These ganglia stained with anti-peripherin antibodies (Fig 4CDown), which recognize the 58-kD neuronal intermediate filament protein.44 The neuronal nature of these cells was further confirmed by staining with anti-neurofilament antibodies (not shown). Transcripts for trkC.TK- were not found in the cardiac ganglion area.



View larger version (117K):
[in this window]
[in a new window]
  		Figure 4. Expression of trkC.TK+ in adult cardiac ganglion. A, B, and D, Bright-field (A and B) and dark-field (D) images of the transverse section at the level of semilunar valves of the aorta through adult rat cardiac ganglion hybridized with antisense trkC.TK+ cRNA probe. C, The section adjacent to the one depicted in panel D immunostained with polyclonal anti-peripherin antibodies. Arrowheads in panel C indicate the neurons, and open arrows indicate the nerve fibers. Labeled are fat pad (fp) and atrial wall (aw). Bars=300 µm (A) and 50 µm (D).

No specific hybridization was observed with trkA, trkB.TK+, or trkB.T2 probes in any cardiac structures at any stages studied.

Expression of LANR mRNA
The first signal of LANR mRNA in the heart was seen over cells dispersed at the subendothelial space of the distal OT at E11 (not shown). The same pattern of expression was observed at E12 (Fig 6A through 6FDown). These cells did not stain with Leu-7 antibody. By E13, expression of LANR mRNA in the walls of aorta and pulmonary trunk had decreased, and at E14 it was not observable. However, at that time, LANR transcripts were seen over a few cells in the developing semilunar valve region and also detected in the distal edges of developing AV valves (Fig 6G through 6IDown).



View larger version (153K):
[in this window]
[in a new window]
  		Figure 6. Expression of LANR in developing rat heart. A through I, Dark-field (A, C, E, G, and I) and bright-field (B, D, F, and H) images of the frontal (A, B, E, and F) and sagittal (C, D, and G through I) sections through E12 (A through F) and E14 (G through I) rat hearts hybridized with antisense LANR cRNA probe. Clear hybridization of LANR probe is seen in the distal outflow tract (ot) between endomyocardium and myocardium (A, C, and E). In panel C, a sagittal section of E12 rat heart shows that LANR hybridization can only be seen in the distal OT. In panel E (at a higher magnification than panel A), cells that probably have been delaminated from endocardium show LANR transcripts. Developing semilunar valve cushion tissue (G through H) and the distal edge of the AV valve show some silver grains (I). Labeled are right atrium (ra), left atrium (la), right ventricle (rv), and left ventricle (lv). Arrowhead in panel H denotes the LANR expression in developing semilunar valve region. Bars=200 µm (A), 100 µm (E), and 200 µm (G).

At E18, LANR mRNA was still occasionally located over a few cells in the distal edges of the developing AV and semilunar valves. At that stage, LANR transcripts were prominently expressed in the cardiac ganglia (Fig 5CDown), where they were sparsely distributed more or less equally over the ganglion area. LANR messages were also found in the area of nerve emanating from the ganglion (Fig 5CDown), where the message is most probably expressed in the Schwann cells. trkC.TK+ transcripts were expressed in the same ganglion (Fig 5ADown). At that stage, it was not possible to identify the cell type expressing trkC.TK+ mRNA. In the adult cardiac ganglia, trkC.TK+ mRNA expression was confined to the large peripherin-positive neurons (Fig 4Up). In neonatal rat heart, LANR mRNA expression was not detectable in cardiac structures but remained in cardiac ganglia and interganglion regions (not shown).



View larger version (83K):
[in this window]
[in a new window]
  		Figure 5. Expression of trkC.TK+ and LANR in E18 cardiac ganglia. A and B, Dark-field (A) and bright-field (B) images of the same sagittal section through E18 rat cardiac ganglion hybridized with antisense trkC.TK+ cRNA probe. C, Section adjacent to the ones depicted in panels A and B hybridized with antisense LANR cRNA probe. Both trk.TK+ and LANR mRNA–positive areas stained with anti-peripherin antibodies (not shown). Labeled are atrial wall (aw) and pulmonary artery (pa). Bar=200 µm.

The expression of NTFs and NTF receptors in developing rat heart is summarized in the TableDown.


View this table:
[in this window]
[in a new window]
  		Table 1. Temporal Expression of NTFs and Their Receptors in Developing Rat Heart


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
NTFs and their receptors participate in the development of sensory and sympathetic neurons and also of several nonneuronal tissues. In developing heart, both neuronal and nonneuronal cells can be found, many of which are derived from the neural crest. Also, in the conducting system of the heart, both neuronal and nonneuronal markers are coexpressed.17 18 Therefore, we chose the heart as an interesting model for studying the expression and role of the NTF system. As a first step, we mapped systematically the expression of mRNAs for NTFs, LANR, and full-length as well as truncated trk receptors in developing rat heart by radioactive in situ hybridization and used antibodies to identify the cell types expressing particular mRNAs.

We found that a group of cardiomyocytes in the ventricular region strongly expressed mRNA for NT-3 starting from E11 and thereafter. We identified this region as the developing conduction system because it was stained by anti–Leu-7, which was used to recognize the conduction system cells,41 42 and because of topological criteria.43 The sinoatrial node, AV node, and the bundle of His of the conduction system are densely innervated by sympathetic and parasympathetic neurons.45 46 47 The developmental stage when the first axons contact the conduction system cells in rats is not known, but in histological sections the first neurites were seen as late as E16 in the AV node area.48 However, the presence of cholinergic neurons in E9 mouse heart49 and acetylcholinesterase activity in the E12 rat heart50 and the ability of exogenous acetylcholine to evoke negative chronotropic response in E12 rat heart51 suggest that by E12 their first contacts between cardiac ganglion neurons and conduction system cells are established. The expression of NT-3 mRNA in the conduction system at E11 and thereafter suggests that NT-3 may function as a chemoattractant as well as a trophic factor for developing cardiac ganglion neurons expressing trkC.TK+ and innervating conduction system cells.

Recently, mutant mice lacking functional genes for NTFs and their receptors have been created.52 Notably, major changes in heart development in these mutant mice have not been reported, but a recent study by Donovan et al53 showed that in NT-3 mutant mice there are defects in embryonic hearts resembling the changes found after cardiac neural crest ablation.54 Our preliminary results with trkC.TK+ knockout animals show markedly decreased innervation of the AV region. NTFs and their receptors have also been shown to have a role in the reparative regeneration of the neointima in adult rat aorta.55 The expression of trkC.TK+ mRNA in the walls of the developing aorta and pulmonary trunk from E13 to parturition suggests that NT-3 can regulate development of the smooth muscle cells of the great vessels. Together, all these findings suggest the essential role for the NT-3/trkC system in heart development.

We were not able to detect messages for NGF and NT-4/5 in the developing rat heart. However, these messages were demonstrated by other techniques, eg, by Northern blotting from E13 to parturition and in E17 hearts.19 20 The discrepancy may result from the low copy numbers of NGF and NT-4/5 mRNAs, which are dispersed throughout the heart and thus remain below the detection limit of in situ hybridization in sections. Expression of NGF in the heart is suggested by its innervation by sympathetic axons known to be NGF dependent. We do not believe that our inability to detect NGF signal in rat heart results from the wrong probe, because we detected NGF mRNA in other structures where its expression has been previously described, eg, the first pharyngeal arch mesenchyme (not shown).29

Strong expression of BDNF mRNA was reported in the walls of the aorta and pulmonary trunk by Scarisbrick et al24 but was not confirmed in the present study. In spite of repeated hybridizations of the adjacent paraffin and cryosections through the whole heart, we always detected only faint BDNF mRNA expression in the E13 and E14 heart, although we used a probe almost identical to that of Scarisbrick et al (see "Materials and Methods").24 Both our probes hybridized to other tissues reported to express BDNF mRNA, eg, the epithelium of the first pharyngeal arch and the sensory epithelia of the inner ear (not shown).29 30 We do not believe that the discrepancy results from differences in the in situ hybridization procedures used by us and Scarisbrick et al, because those were essentially similar. Actually, higher probe concentrations and more stringent washing conditions were used by them. Strong BDNF mRNA levels were detected by our procedure in the inner ear (not shown); thus, there were no upper detection limitations. We are convinced that our data describe correctly the expression level of BDNF mRNA in developing heart and do not know why Scarisbrick et al reported high BDNF expression there. However, the Sprague-Dawley rat strain was used by us, whereas Wistar rats were studied by Scarisbrick et al, and we cannot exclude BDNF expression differences in different rat strains. Interestingly, Timmusk et al19 also demonstrated only very low and decreasing levels of BDNF mRNA compared with that of NT-4 mRNA in developing rat heart at E13 to postnatal day 1 by RNase protection assay. Only in the adult heart did BDNF transcripts show marked increase. Also, results obtained by Maisonpierre et al23 showed no detectable expression of BDNF mRNA in the newborn rat heart, whereas the adult rat heart showed some BDNF mRNA expression.

We found that mRNAs for truncated trkC and trkB are expressed in the developing rat heart in a highly specific pattern, suggesting that they perform also some specific functions during heart development. However, the functions of truncated trks are still obscure. It has been shown that heterodimerization of full-length and truncated trks results in blockage of NTF signal transduction.56 Such dominant negative regulation may occur in the walls of the aorta and pulmonary trunk, where both full-length and truncated trkC transcripts are expressed. However, in some regions (eg, trkB.T1 in the semilunar valve region, Fig 3C and 3FUpUp), truncated trks were expressed alone, implicating other yet unidentified functions for them (eg, regulation of availability of NTFs by internalization).10 Coexpression of trkB.T1 with trkC.TK+ in the walls of aorta and pulmonary trunk leads to the speculation that truncated trkB can also suppress trkC function by trkB.T1/trkC.TK+ heterodimers. Indeed, NT-3 can interact with both trkB and trkC.57 However, trk heterodimers have not been demonstrated.

The LANR mRNA–expressing cells in the subendothelial space of the distal OT at E11 to E13 may be derived from neural crest and further form the muscle layer of the derivatives of the aortic arches. Indeed, LANR has been used as a marker for rat neural crest cells, and when these cells are cultured, they differentiate into smooth muscle cells.58 Alternatively, those cells might also be generated locally by the epithelial-to-mesenchymal transformation and, after this transformation, start to express LANR mRNA, as they do not express another neural crest marker, the HNK-1/Leu-7 antigen (the present study). Furthermore, LANR mRNA expression does not seem to overlap with trkB.T1 and trkC.TK+/-hybridizations in the OT, but in the cardiac ganglia, LANR and tkrC.TK+ are most probably expressed in the same cells (Fig 5Up and not shown). Whatever the origin of LANR mRNA–positive cells in the OT, the distinct spatiotemporal expression of p75 mRNA suggests a trk receptor–independent function for LANR in the development of the OT and semilunar and AV valves.

The in situ hybridization data presented here and what is known about neural crest distribution in the chick embryo led us to propose a model for the function of the neurotrophic system in rat heart development. At E9 to E10, LANR-expressing neural crest cells migrate into the pharyngeal arches and further into the distal OT.59 Alternatively, distal OT endocardial cells undergoing epithelial-to-mesenchymal transformation start to express LANR mRNA. The neural crest cells, committed to become cardiac neurons and glia, migrate into the atrial region. The first cells of the conduction system appear at E11, and the first axons of the cardiac ganglion neurons contact it probably at E12, guided by NT-3 secreted from the cells of the conduction system. After establishment of the axonal contacts, NT-3 maintains the cardiac ganglion neurons trophically. Beginning at E13, the cells within the developing aorta and pulmonary trunk wall start to proliferate and differentiate into smooth muscle cells.60 These processes may be regulated by locally produced NTFs, which, along with receptors, are expressed there by that time.

Conclusions
The conclusions of the present study are as follows: (1) Cardiac ganglion neurons express mRNA for trkC.TK+ and thus can be responsive to NT-3. (2) Expression of NT-3 mRNA in the developing conduction system suggests that NT-3 may have a chemoattractant and/or trophic function for the cardiac ganglion neurons innervating the conduction system. NT-3 mRNA can also be used as a marker for developing conduction system cells. (3) Smooth muscle cells of the aorta and pulmonary trunk express mRNAs for trkC.TK+ and, thus, are potentially regulated by NT-3. (4) mRNAs for trkC.TK- and trkB.T1 are expressed in developing heart, suggesting specific functions for them. (5) LANR (p75) mRNA is expressed transiently in the developing OT and later in developing valves. (6) Transcripts for NTFs and their receptors are expressed in specific patterns in the developing rat heart, which may be needed to accomplish the delicate distribution of innervation, cell proliferation, and possible other functions.


*    Selected Abbreviations and Acronyms
 
AV = atrioventricular
BDNF = brain-derived neurotrophic factor
BTP = 0.1% bovine serum albumin, 0.05% Triton X-100, and 10% horse serum in PBS
E (associated with number) = embryonic day
LANR = low-affinity neurotrophin receptor
NGF = nerve growth factor
NT-3, NT-4/5, NT-6 = neurotrophin-3, -4/5, and -6
NTF = neurotrophin
OT = outflow tract
PCR = polymerase chain reaction
PFA = paraformaldehyde
SMA = smooth muscle {alpha}-actin
trk = receptor protein-tyrosine kinase


*    Acknowledgments
 
This study was supported by grants from the Academy of Finland, University of Helsinki, and S. Juselius Foundation. Anti-neurofilament antibody was kindly provided by Dr I. Virtanen. We are indebted to E. Kujamaki for excellent technical assistance, Dr H. Sariola for helpful discussion, and Prof M. Kirby for critical comments.

Received March 18, 1996; accepted August 9, 1996.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Davies AM. The role of neurotrophins in the developing nervous system. J Neurobiol. 1994;25:1334-1348.[Medline] [Order article via Infotrieve]
  2. Sariola H, Sainio K, Arumae U, Saarma M. Neurotrophins and ciliary neurotrophic factor: their biology and pathology. Ann Med. 1994;26:355-363.[Medline] [Order article via Infotrieve]
  3. Gotz R, Koster R, Winkler C, Raulf F, Lottspeich F, Schartl M, Thoenen H. Neurotrophin-6 is a new member of the nerve growth factor family. Nature. 1994;372:266-269.[Medline] [Order article via Infotrieve]
  4. Barbacid M. Neurotrophic factors and their receptors. Curr Opin Cell Biol. 1995;7:148-155.[Medline] [Order article via Infotrieve]
  5. Klein R, Conway D, Parada LF, Barbacid M. The trkB tyrosine protein kinase gene codes for a second neurogenic receptor that lacks the catalytic kinase domain. Cell. 1990;61:647-656.[Medline] [Order article via Infotrieve]
  6. Middlemas DS, Lindberg R, Hunter T. TrkB, a neural receptor protein tyrosine kinase: evidence for a full-length and two truncated receptors. Mol Cell Biol. 1991;11:143-153.[Abstract/Free Full Text]
  7. Lamballe F, Tapley P, Barbacid M. trkC encodes multiple neurotrophin-3 receptors with distinct biological properties and substrate specificities. EMBO J. 1993;12:3083-3094.[Medline] [Order article via Infotrieve]
  8. Tsoulfas P, Soppet D, Escandon E, Tessarollo L, Mendoza-Ramirez JL, Rosenthal A, Nikolics K, Parada L. The rat trkC locus encodes multiple neurogenic receptors that exhibit differential response to neurotrophin-3 in PC 12 cells. Neuron. 1993;10:975-990.[Medline] [Order article via Infotrieve]
  9. Valenzuela DM, Maisonpierre PC, Glass DJ, Rojas E, Nunez L, Kong Y, Gies D, Stitt TN, Ip NY, Yancopoulos GD. Alternative forms of rat trkC with different functional capabilities. Neuron. 1993;10:963-974.[Medline] [Order article via Infotrieve]
  10. Biffo S, Offenhauser N, Carter BD, Barde YA. Selective binding and internalisation by truncated receptors restrict the availability of BDNF during development. Development. 1995;121:2461-2470.[Abstract]
  11. Chao MV. The p75 neurotrophin receptor. J Neurobiol. 1994;25:1373-1385.[Medline] [Order article via Infotrieve]
  12. Bothwell M. Functional interactions of neurotrophins and neurotrophin receptors. Annu Rev Neurosci. 1995;18:223-253.[Medline] [Order article via Infotrieve]
  13. Carter BD, Kaltschmidt C, Kaltschmidt B, Offenhauser N, Bohmmatthaei R, Baeuerle PA, Barde YA. Selective activation of NF-kappa-B by nerve growth factor through the neurotrophin receptor p75. Science. 1996;272:542-545.[Abstract]
  14. Buck C, Martinez H, Black I, Chao M. Developmentally regulated expression of the nerve growth factor gene in the periphery and brain. Proc Natl Acad Sci U S A. 1987;84:3060-3063.[Abstract/Free Full Text]
  15. Ernfors P, Hallbook F, Ebendal T, Shooter EM, Radeke MJ, Misko TP, Persson H. Development and regional expression of beta-nerve growth factor receptor mRNA in the chicken and rat. Neuron. 1988;1:983-996.[Medline] [Order article via Infotrieve]
  16. Sariola H, Saarma M, Sainio K, Arumae U, Palgi J, Vaahtokari A, Thesleff I, Karavanov A. Dependence of kidney morphogenesis on nerve growth factor receptor expression. Science. 1991;254:571-573.[Abstract/Free Full Text]
  17. Gorza L, Vitadello M. Distribution of conduction system fibers in the developing and adult rabbit heart revealed by an antineurofilament antibody. Circ Res. 1989;65:360-369.[Abstract/Free Full Text]
  18. Gonzalez-Sanchez A, Bader D. Characterization of a myosin heavy chain in the conductive system of the adult and developing chicken heart. J Cell Bio. 1985;100:270-275.[Abstract/Free Full Text]
  19. Timmusk T, Belluardo N, Metsis M, Persson H. Widespread and developmentally regulated expression of neurotrophin-4 mRNA in rat brain and peripheral tissues. Eur J Neurosci. 1993;5:605-613.[Medline] [Order article via Infotrieve]
  20. Clegg D, Large TH, Bodary S, Reichardt LF. Regulation of nerve growth factor mRNA levels in developing rat heart ventricle is not altered by sympathectomy. Dev Biol. 1989;134:30-37.[Medline] [Order article via Infotrieve]
  21. Hohn A, Leibrock J, Bailey K, Barde YA. Identification and characterization of a novel member of the nerve growth factor/brain-derived neurotrophic factor family. Nature. 1990;344:339-341.[Medline] [Order article via Infotrieve]
  22. Selby M, Edwards R, Sharp F, Rutter W. Mouse nerve growth factor gene: structure and expression. Mol Cell Biol. 1987;7:3057-3064.[Abstract/Free Full Text]
  23. Maisonpierre PC, Belluscio L, Friedman B, Alderson RF, Wiegand SJ, Furth ME, Lindsay RM, Yancopoulos GD. NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron. 1990;5:501-509.[Medline] [Order article via Infotrieve]
  24. Scarisbrick I, Jones E, Isackson PJ. Coexpression of mRNAs for NGF, BDNF, and NT-3 in the cardiovascular system of the pre-and postnatal rat. J Neurosci. 1993;13:875-893.[Abstract]
  25. Shelton DL, Sutherland J, Gripp J, Camerato T, Armanini MP, Phillips HS, Carroll K, Spencer SD, Levinson AD. Human trks: molecular cloning, tissue distribution, and expression of extracellular domain immunoadhesins. J Neurosci. 1995;15:477-491.[Abstract]
  26. Lamballe F, Smeyne RJ, Barbacid M. Developmental expression of trkC, the neurotrophin-3 receptor, in the mammalian nervous system. J Neurosci. 1994;14:14-28.[Abstract]
  27. Yan Q, Johnson EM Jr. An immunohistochemical study of the nerve growth factor receptor in developing rats. J Neurosci. 1988;8:3481-3498.[Abstract]
  28. Heuer J, Fatemie-Nainie S, Wheeler EF, Bothwell M. Structure and developmental expression of the chicken NGF receptor. Dev Biol. 1990;137:287-304.[Medline] [Order article via Infotrieve]
  29. Arumae U, Pirvola U, Palgi J, Kiema TR, Palm K, Moshnyakov M, Ylikoski J, Saarma M. Neurotrophins and their receptors in rat peripheral trigeminal system during maxillary nerve growth. J Cell Biol. 1993;122:1053-1065.[Abstract/Free Full Text]
  30. Pirvola U, Ylikoski J, Palgi J, Lehtonen E, Arumae U, Saarma M. Brain derived neurotrophic factor and neurotrophin-3 mRNAs in the peripheral target fields of developing inner ear ganglia. Proc Natl Acad Sci U S A. 1992;89:9915-9919.[Abstract/Free Full Text]
  31. Pirvola U, Arumae U, Moshnyakov M, Palgi J, Saarma M, Ylikoski J. Coordinated expression and function of neurotrophins and their receptors in the rat inner ear during target innervation. Heart Res. 1994;75:131-144.
  32. Ylikoski J, Pirvola U, Moshnyakov M, Palgi J, Arumae U, Saarma M. Expression patterns of neurotrophin and their receptor mRNAs in the rat inner ear. Heart Res. 1993;65:69-78.
  33. Merlio JP, Ernfors P, Jaber M, Persson H. Molecular cloning of rat trkC and identification of cells expressing mRNAs for members of the trk family in the rat central nervous system. Neuroscience. 1992;51:513-532.[Medline] [Order article via Infotrieve]
  34. Maisonpierre PC, Le Beau MM, Espinosa R III, Ip NY, Belluscio L de I, Monte SM, Squinto S, Furth ME, Yancopoulos GD. Human and rat brain-derived neurotrophic factor and neurotrophin-3: gene structures, distributions, and chromosomal localizations. Genomics. 1991;10:558-568.[Medline] [Order article via Infotrieve]
  35. Wilkinson DG, Green J. In situ hybridization and the three-dimensional reconstruction of serial sections. In: Copp AJ, Cockroft DL, eds. Postimplantation in Mammalian Embryos: A Practical Approach. Oxford, England: IRL Press; 1990:151-171.
  36. Ernfors P, Persson H. Developmentally regulated expression of HDNF/NT-3 mRNA in rat spinal cord motoneurons and expression of BDNF mRNA in embryonic dorsal root ganglion. Eur J Neurosci. 1993;3:953-961.
  37. Mu X, Silos-Santiago I, Carroll SL, Snider WD. Neurotrophin receptor genes are expressed in distinct patterns in developing dorsal root ganglia. J Neurosci. 1993;13:4029-4041.[Abstract]
  38. Chou DK, Ilyas AA, Evans JE, Costello C, Quarles RH, Jungalwala FB. Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK-1 antibody and some IgM paraproteins in neuropathy. J Biol Chem. 1986;261:11717-11725.[Abstract/Free Full Text]
  39. Abo T, Balch CM. A differentiation antigen of human NK and K cells identified by a monoclonal antibody (HNK-1). J Immunol. 1981;127:1024-1029.[Abstract]
  40. Ikeda T, Iwasaki K, Shimokawa I, Sakai H, Ito H, Matsuo T. Leu-7 immunoreactivity in human and rat embryonic hearts, with special reference to the development of the conduction tissue. Pediatr Res. 1990;33:43-47.[Medline] [Order article via Infotrieve]
  41. Nakagawa M, Thompson RP, Terracio L, Borg TK. Developmental anatomy of HNK-1 immunoreactivity in the embryonic rat heart: co-distribution with early conduction tissue. Anat Embryol. 1993;187:445-460.[Medline] [Order article via Infotrieve]
  42. Nakamura T, Ikeda T, Shimokawa I, Inoue Y, Suematsu T, Sakai H, Iwasaki K, Matsuo T. Distribution of acetylcholinesterase activity in the rat embryonic heart with reference to HNK-1 immunoreactivity in the conduction tissue. Anat Embryol. 1994;190:367-373.[Medline] [Order article via Infotrieve]
  43. Viragh S, Challice CE. The development of the conduction system in the mouse embryo heart. Dev Biol. 1982;89:25-40.[Medline] [Order article via Infotrieve]
  44. Gorham JD, Baker H, Kegler D, Ziff EB. The expression of the neuronal intermediate filament protein peripherin in the rat embryo. Dev Brain Res. 1990;57:235-248.[Medline] [Order article via Infotrieve]
  45. Thaemert JC. Atrioventricular node innervation in ultrastructural three dimensions. Am J Anat. 1970;128:239-264.[Medline] [Order article via Infotrieve]
  46. Bojsen-Moller F, Tranul-Jensen J. Rabbit heart nodal tissue, sinoatrial ring bundle and atrioventricular connexions identified as a neuromuscular system. J Anat. 1972;112:367-382.[Medline] [Order article via Infotrieve]
  47. Moravec M, Moravec J. Intrinsic innervation of the atrioventricular junction of the rat heart. Am J Anat. 1984;171:307-319.[Medline] [Order article via Infotrieve]
  48. Gomez H. The development of the innervation of the heart in the rat embryo. Anat Rec. 1958;130:53-71.[Medline] [Order article via Infotrieve]
  49. Lane MA, Sastre A, Salpeter MM. Innervation of heart cells in culture by an endogenous source of cholinergic neurons. Proc Natl Acad Sci U S A. 1976;73:4506-4510.[Abstract/Free Full Text]
  50. Lamers WH, te Kortschot A, Los JA, Moorman AF. Acetylcholinesterase in prenatal rat heart: a marker for the early development of the cardiac conductive tissue? Anat Rec. 1987;217:361-370.[Medline] [Order article via Infotrieve]
  51. Nakazawa M, Ohno T, Miyagawa S, Takao A. Hemodynamic effects of acetylcholine in the chick embryo and differences from those in the rat embryo. Teratology. 1989;39:555-561.[Medline] [Order article via Infotrieve]
  52. Snider WD. Functions of the neurotrophins during nervous system development: what the knockouts are teaching us. Cell. 1994;77:627-638.[Medline] [Order article via Infotrieve]
  53. Donovan M, Hahn R, Tessarollo L, Hempstead BL. Neurotrophin 3 is essential for mammalian cardiac development. J Neurochem. 1996;66:S6. Abstract.
  54. Kirby ML, Stewart DE. Neural crest origin of cardiac ganglion cells in the chick embryo: identification and extirpation. Dev Biol. 1983;97:433-443.[Medline] [Order article via Infotrieve]
  55. Donovan MJ, Miranda RC, Kraemer R, McCaffrey TA, Tessarollo L, Mahadeo D, Sharif S, Kaplan DR, Tsoulfas P, Parada L, Toranallerand CD, Hajjar DP, Hempstead BL. Neurotrophin and neurotrophin receptors in vascular smooth muscle cells: regulation of expression in response to injury. Am J Pathol. 1995;147:309-324.[Abstract]
  56. Eide FF, Vining ER, Eide BL, Zang K, Wang XY, Reichardt LF. Naturally occurring truncated trkB receptors have dominant inhibitory effects on brain-derived neurotrophic factor signaling. J Neurosci. 1996;10:3123-3129.
  57. Ip NY, Stitt TN, Tapley P, Klein R, Glass DJ, Fandl J, Greene LA, Barbacid M, Yancopoulos GD. Similarities and differences in the way neurotrophins interact with the Trk receptors in neuronal and nonneuronal cells. Neuron. 1993;10:137-149.[Medline] [Order article via Infotrieve]
  58. Anderson DJ. Stem cells and transcription factors in the development of the mammalian neural crest. FASEB J. 1994;8:707-713.[Abstract]
  59. Le Lievre CS, Le Douarin NM. Mesenchymal derivatives of the neural crest: analysis of chimeric quail and chick embryos. J Embryol Exp Morphol. 1975;34:125-154.[Medline] [Order article via Infotrieve]
  60. Miano JM, Cserjesi P, Ligon KL, Periasamy M, Olson EN. Smooth muscle myosin heavy chain exclusively marks the smooth muscle lineage during mouse embryogenesis. Circ Res. 1994;75:803-812.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 BloodHome page
R. N. Pearse, S. L. Swendeman, Y. Li, D. Rafii, and B. L. Hempstead
A neurotrophin axis in myeloma: TrkB and BDNF promote tumor-cell survival
Blood, June 1, 2005; 105(11): 4429 - 4436.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
A. M. Riazi, H. Lee, C. Hsu, and G. Van Arsdell
CSX/Nkx2.5 Modulates Differentiation of Skeletal Myoblasts and Promotes Differentiation into Neuronal Cells in Vitro
J. Biol. Chem., March 18, 2005; 280(11): 10716 - 10720.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Kim, Q. Li, B. L. Hempstead, and J. A. Madri
Paracrine and Autocrine Functions of Brain-derived Neurotrophic Factor (BDNF) and Nerve Growth Factor (NGF) in Brain-derived Endothelial Cells
J. Biol. Chem., August 6, 2004; 279(32): 33538 - 33546.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
C. Rivera, J. Voipio, J. Thomas-Crusells, H. Li, Z. Emri, S. Sipila, J. A. Payne, L. Minichiello, M. Saarma, and K. Kaila
Mechanism of Activity-Dependent Downregulation of the Neuron-Specific K-Cl Cotransporter KCC2
J. Neurosci., May 12, 2004; 24(19): 4683 - 4691.
[Abstract] [Full Text] [PDF]

Home page
 J. Neurosci.Home page
Z.-Y. Chen, P. D. Patel, G. Sant, C.-X. Meng, K. K. Teng, B. L. Hempstead, and F. S. Lee
Variant Brain-Derived Neurotrophic Factor (BDNF) (Met66) Alters the Intracellular Trafficking and Activity-Dependent Secretion of Wild-Type BDNF in Neurosecretory Cells and Cortical Neurons
J. Neurosci., May 5, 2004; 24(18): 4401 - 4411.
[Abstract] [Full Text] [PDF]

Home page
 J. Cell Biol.Home page
C. Rivera, H. Li, J. Thomas-Crusells, H. Lahtinen, T. Viitanen, A. Nanobashvili, Z. Kokaia, M. S. Airaksinen, J. Voipio, K. Kaila, et al.
BDNF-induced TrkB activation down-regulates the K+-Cl- cotransporter KCC2 and impairs neuronal Cl- extrusion
J. Cell Biol., December 9, 2002; 159(5): 747 - 752.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
M. Donovan, M. Lin, P Wiegn, T Ringstedt, R Kraemer, R Hahn, S Wang, C. Ibanez, S Rafii, and B. Hempstead
Brain derived neurotrophic factor is an endothelial cell survival factor required for intramyocardial vessel stabilization
Development, January 11, 2000; 127(21): 4531 - 4540.
[Abstract] [PDF]

Home page
 J. Neurosci.Home page
M. E. Palko, V. Coppola, and L. Tessarollo
Evidence for a Role of Truncated trkC Receptor Isoforms in Mouse Development
J. Neurosci., January 15, 1999; 19(2): 775 - 782.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Tessarollo, P. Tsoulfas, M. J. Donovan, M. E. Palko, J. Blair-Flynn, B. L. Hempstead, and L. F. Parada
Targeted deletion of all isoforms of the trkC gene suggests the use of alternate receptors by its ligand neurotrophin-3 in neuronal development and implicates trkC in normal cardiogenesis
PNAS, December 23, 1997; 94(26): 14776 - 14781.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
F. Qin, R. S. Vulapalli, S. Y. Stevens, and C.-S. Liang
Loss of cardiac sympathetic neurotransmitters in heart failure and NE infusion is associated with reduced NGF
Am J Physiol Heart Circ Physiol, January 1, 2002; 282(1): H363 - H371.
[Abstract] [Full Text]<