Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie, Deutschordenstrasse 46, 60528 Frankfurt/Main, Germany
* Author for correspondence (e-mail: rohrer{at}mpih-frankfurt.mpg.de)
Accepted 29 August 2002
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SUMMARY |
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Key words: Ciliary, Cholinergic, Noradrenergic, dHand, BMP5, BMP7, Autonomic nervous system
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INTRODUCTION |
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While progress has been made in uncovering the signals and mechanisms
involved in the development of noradrenergic sympathetic neurons, less is
known about the strategies by which cholinergic parasympathetic neurons are
generated. Whereas sympathetic ganglia form near the neural tube,
parasympathetic ganglia are generated close to their peripheral target organs
by neural crest cells that migrate for longer distances. In the mature state,
parasympathetic neurons provide a functionally cholinergic innervation to
their peripheral targets, although there is evidence for expression of some
adrenergic characteristics during development
(Teitelman et al., 1985;
Iacovitti et al., 1985
;
Landis et al., 1987
).
Interestingly, Mash1, Phox2a and Phox2b are not only expressed in
parasympathetic ganglia but are also essential for parasympathetic ganglion
development, as revealed by the severe effects in knockout mice
(Guillemot and Joyner, 1993
;
Guillemot et al., 1993
;
Morin et al., 1997
;
Tiveron et al., 1996
;
Hirsch et al., 1998
;
Pattyn et al., 1999
).
These findings brought into question, whether the expression of these genes
and parasympathetic ganglion development are also dependent on BMPs. In
addition, the issue was raised of how the same group of transcription factors
is able to specify different neuronal fates, i.e. noradrenergic neurons in
sympathetic and cholinergic neurons in parasympathetic ganglia. One
possibility would be that different neuronal fates are generated by different
thresholds of the same transcription factor(s). In the neural tube, the
identities of neuronal progenitors are assigned by the graded action of
inductive signals that are secreted by ventral and dorsal signaling centres
(Jessell, 2000;
Anderson, 2001
). BMPs produced
in the roof plate and dorsal neural tube specify different types of dorsal
interneurons in a concentration-dependent manner
(Lee and Jessell, 1999
). The
signaling of BMP family members in embryonic tissues can result in increased
levels of downstream transcription factors that specify different cell fates
at different thresholds (Gurdon et al.,
1998
; Gurdon et al.,
1999
; Shimizu and Gurdon,
1999
). Thus, different levels of BMPs and downstream transcription
factors may produce noradrenergic sympathetic or cholinergic parasympathetic
neurons. There is indeed recent evidence, in vitro, that BMPs affect the
decision of PNS progenitors to acquire a sensory or autonomic neuron phenotype
and induce cholinergic or noradrenergic genes in autonomic precursors in a
concentration-dependent manner (White et
al., 2001
; Lo et al.,
2002
). An alternative possibility would be that different
environmental signals influence the neural crest precursor cells in such a way
that the same network of transcription factors results in different readouts,
depending on the cellular context.
This study examines the role of BMPs and transcriptional control genes for the development of parasympathetic ciliary ganglion neurons in the chick embryo. We report that BMPs are locally expressed at the site of ciliary ganglion formation and are required for the development of this ganglion. Overexpression of BMPs results in the generation of additional ciliary ganglion neurons, but does not alter the transmitter phenotype. During normal ciliary ganglion development Cash1, Phox2b and Phox2a are expressed with a similar timing as in sympathetic ganglia. In addition, noradrenergic genes are expressed before cholinergic marker genes but are subsequently downregulated. The transient expression of noradrenergic marker genes may be due to the absence of dHand expression in ciliary ganglia, since ectopic dHand expression maintains noradrenergic differentiation. Our finding lead to the following conclusions: first, there is a common dependence of sympathetic and parasympathetic ciliary neuron development on BMPs. Second, dHand is probably involved in the maintenance of noradrenergic gene expression in sympathetic neurons. Third, the specification of sympathetic and parasympathetic ciliary neurons involves unknown signals, leading to differential gene expression in their neural crest precursor cells, which determine the pattern of transcription factors induced by BMPs and their subsequent differentiation.
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MATERIALS AND METHODS |
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Injection of noggin-expressing CHO cells, CHO control cells and of
virus-concentrate into chick embryos
Noggin-expressing CHO cells or CHO control cells were collected by
centrifugation and resuspended in 50 µl PBS. RCAS viral stock preparation
was carried out as described previously
(Vogel-Höpker and Rohrer,
2002). Cell suspensions were injected into the mesenchyme caudal
to the developing anlage of the eye of stage 11 embryos, using fine glass
capillaries attached to an aspirator tube (Sigma A-51779). The eggs were
further incubated, staged (Hamburger and
Hamilton, 1951
) and killed by decapitation. The heads were fixed,
sectioned, and the sections were analyzed for expression of Sox10, SCG10,
Phox2b, VAChT and Cash1 by in situ hybridisation. At least three
embryos were analysed for each marker.
In situ hybridization of sections
Nonradioactive in situ hybridization and preparation of digoxigeninor
fluorescein-labeled probes for chick RT, TH, DBH, Phox2a, Phox2b,
ChAT, vesicular acetylcholine transporter (VAChT), chick
high-affinity choline transporter (CHT1), Cash1, SCG10, NF160,
Sox10 were carried out as described previously
(Ernsberger et al., 1997;
Stanke et al., 1999
). As a
probe for chick CHT1, a 1134 bp fragment corresponding to bases
426-1559 of the human sequence was used. The CHT1 fragment was cloned
from E9 chick CG-cDNA, using degenerated PCR primers based on homologous
sequences from nematode CHO-1 and rat CHT1. The resulting
cDNA fragment shows 80% identity to human sequence at nucleotide level and 85%
identity at protein level (EMBL/GenBank/DDBJ accession number AJ11267).
Double in situ hybridization was carried out using DIG- or fluorescein-labeled probes for VAChT and TH. The first colour reaction with Fast Red was stopped by washing in PBS. Photos were taken and the antibody was stripped off by washing two times for 10 minutes with 1 ml 0.1 M glycine pH 1.8. After an additional wash for 1 hour in MABT the second colour reaction with NBT/BCIP was carried out.
Morphometric analysis
The number of TH-positive cells was counted on all sections
infected by the virus indicated by expression of RT mRNA. On
alternate sections the area of Phox2b or ChAT mRNA
expression was quantified morphometrically, using the Metamorph Imaging System
(Version 4.6, Universal Imaging Corporation). The number of
TH-positive cells were counted in relation to the area of
Phox2b-positive cells and expressed as cells/mm2.
To visualize the effect of BMP treatment on ciliary ganglion development, digital images of Phox2b-stained serial sections were aligned and the resulting stacks were used for a 3D-reconstruction, using Metamorph Imaging System Software.
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RESULTS |
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BMPs in parasympathetic ciliary ganglion development
As BMPs control the expression of Cash1 and Phox2a/Phox2b
in sympathetic ganglia (Reissmann et al.,
1996; Shah et al.,
1996
; Schneider et al.,
1999
), we investigated whether BMPs are expressed in the vicinity
of the forming ciliary ganglion. The analysis of BMP4, 5 and
7 expression in the retro-orbital region demonstrates BMP7
expression (Fig. 2) in the
region where the ciliary ganglion forms. Weak, but significant mesenchymal
BMP5 expression, but no BMP4 expression was detected (not
shown). Thus, at the onset of Cash1 and Phox2b expression at
stage 18, both BMP7 and BMP5 could be detected, which is
compatible with the notion that BMPs control the expression of these
transcriptional regulators.
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To see whether the development of ciliary neurons is also dependent on
BMPs, the BMP antagonist noggin was applied in the vicinity of the developing
ciliary ganglion. Cell suspensions of noggin-producing CHO cells or CHO
control cells were applied at stage 10/11 unilaterally into the craniofacial
mesenchyme, close to the optic stalk. The implantation of control cells did
not affect ciliary ganglion development (not shown). In contrast, we observed
in a large proportion of noggin-treated embryos (5 out of 6) a complete lack,
unilaterally, of differentiated ciliary ganglion cells, when the embryos were
analysed for the expression of the generic neuronal marker SCG10, the
autonomic markers Cash1, Phox2b and the cholinergic marker
VAChT at stage 24/25 (Fig.
3A-D). However, a large aggregate of cells expressing the neural
crest marker Sox10 could be observed in the region, where the ciliary
ganglion would form (Fig.
3E,F). In most cases also eye development was severely affected on
the noggin-treated side, resulting in lacking or rudimentary retinae
(Fig. 3A-H). To address the
possibility that the impaired ciliary ganglion development might be caused
indirectly by the absence of the eye, we analysed ciliary ganglion development
after ablation of the optic cup. The gene expression pattern of ciliary
ganglia was completely unaffected by the lack of eye ablation up to stage
24/25 (SCG10, Phox2b see Fig.
3G,H; Cash-1 and VAChT, not shown). This agrees
with previous studies demonstrating that ciliary neuron survival was not
dependent on the peripheral targets up to embryonic day 8
(Landmesser and Pilar, 1974;
Lee et al., 2001
).
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Ectopic cholinergic ciliary ganglion neurons are induced by BMP4
To gain further insight into the role of BMPs in ciliary ganglion
development, BMP4 was ectopically expressed in the ciliary ganglion and its
environment, using the avain retroviral vector RCAS (B)
(Reissmann et al., 1996;
Howard et al., 2000
). The
unilateral implantation of virus-producing chick embryo fibroblast cells
(CEFs) into stage 10/11 chick embryos resulted in the infection and BMP4
production of ciliary ganglion and surrounding mesenchymal cells. BMP4
overexpression resulted in a considerable enlargement of the ciliary ganglion
(Fig. 4A). Using 3D
reconstructions of Phox2b-stained serial sections we observed, in BMP-treated
embryos, ectopic neurons in the oculomotor nerve and in postganglionic ciliary
nerves. The enlarged ganglia were composed of neurons with the same
characteristics as observed during normal development, i.e. many apparent
cholinergic neurons that were positive for ChAT
(Fig. 4A). To investigate
whether BMP overexpression would stimulate adrenergic differentiation in
parasympathetic ciliary ganglia, the number of TH-positive
cells/section (Fig. 4C) and the
areas of ChAT-positive and Phox2b-positive cells/section
(Fig. 4B) were determined in
control and BMP4-treated embryos. Phox2b, ChAT and TH
increased to a similar extent (Fig.
4B,C). Thus, when ChAT and TH expression is
examined in relation to the area of Phox2b expression, i.e. the total area of
ciliary ganglion neurons, neither the proportion of ChAT-expressing
cells (Fig. 4D) nor of
TH-positive cells (Fig.
4E) was changed in response to the BMP4 treatment.
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The control of noradrenergic differentiation in the parasympathetic
ciliary ganglion
While mature parasympathetic neurons display a cholinergic neurotransmitter
phenotype, during development a transient expression of noradrenergic genes
was observed, apparently controlled in part by the same transcription factors
that lead to maintained noradrenergic gene expression in sympathetic ganglia.
What causes the decrease of TH and DBH expression in
parasympathetic ganglia? Two explanations are obvious, the loss of
TH/DBH-positive noradrenergic neurons or a switch in the
neurotransmitter phenotype from noradrenergic to cholinergic. Although ciliary
ganglion cell number apparently does not change during this period
(Landmesser and Pilar, 1974),
recent evidence suggests that considerable cell death does occur and is
compensated by the differentiation of neuron precursor cells present in the
ganglia (Lee et al., 2001
).
The demonstration of ciliary ganglion neurons that co-express TH and
the cholinergic marker gene VAChT
(Fig. 5) is in agreement with
the notion that cells with noradrenergic gene expression acquire a cholinergic
phenotype. Owing to the transient TH and DBH expression and
the graded increase in the expression of cholinergic marker genes, the
relatively low proportion of double-positive cells is in line with the
expectations. With respect to noradrenergic differentiation, these data thus
demonstrate a decrease of TH and DBH as a major difference
between ciliary and sympathetic neurons. This may either be due to the
selective repression/loss of TH/DBH expression in parasympathetic
ganglia or the selective maintenance of noradrenergic gene expression in
sympathetic neurons.
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dHand expression in ciliary ganglia maintains noradrenergic
properties
Differential control of noradrenergic gene expression implies signals that
are selectively expressed in ciliary and sympathetic ganglia, respectively. Of
the transcription factors that have been shown to be directly or indirectly
involved in the control of noradrenergic gene expression, only dHand
is selectively expressed in sympathetic ganglia
(Fig. 1A)
(Howard et al., 1999;
Howard et al., 2000
), whereas
Cash1, Phox2a and Phox2b are pan-autonomic genes. In
sympathetic ganglia, dHand is detectable throughout development and
thus could play a role in the regulation of TH and DBH at
later stages. Thus, the absence of dHand expression in ciliary
ganglia could explain the transient nature of noradrenergic gene
expression.
To test this proposed role, dHand was ectopically expressed in ciliary ganglia using retroviral vectors. Interestingly, a strong increase in the number of TH-expressing (Fig. 6A) and DBH-expressing cells (not shown) was observed in E8 embryos in response to dHand expression (Fig. 6A,B). For quantification, the number of TH-positive cells was determined in relation to the ganglion area (Phox2b-positive) in sections of dHand-RCAS-infected and uninfected contralateral ganglia. The increase in the proportion of TH-positive cells ranged between 1.4- and 12-fold, with a mean increase of 2.6-fold (Fig. 6B), reflecting the variation in ciliary ganglion infection by dHand-RCAS. In contrast to the surrounding mesenchyme, the ciliary ganglion was only partially infected, which may be explained by the inability to infect early postmitotic ciliary neurons. The increased number of TH-expressing cells in response to ectopic dHand expression is compatible with the notion that TH and DBH expression are downregulated in ciliary ganglia because of a lack of dHand which would maintain TH/DBH expression in sympathetic ganglia.
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DISCUSSION |
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Ciliary neurons are derived from neural crest at the
mesencephalic/metencephalic border region
(Hammond and Yntema, 1958;
Noden, 1975
;
Narayanan and Narayanan,
1978
). These cells migrate rostrally towards the optic vesicle and
form the ciliary ganglion primordium behind the eye, close to the optic nerve.
The ganglion was first detectable as an aggregate of Cash-1-positive
cells at stage 18 that was wrapped around the oculomotor nerve. The onset of
Phox2b and Phox2a expression was observed shortly
afterwards, followed by panneuronal and noradrenergic gene expression.
Cholinergic marker gene expression starts after the ganglion has been
generated, i.e. after the expression of Cash1, Phox2a/b and
TH/DBH. A previous study identified ciliary neuron precursors during
migration, using an antibody against a ciliary neuron-specific cell surface
antigen (Barald, 1988
). As
there was evidence to suggest that the antigen may be involved in
high-affinity choline transport (Barald,
1988
), the present analysis also includes the choline transporter.
Our data demonstrate, however, that CHT1
(Okuda et al., 2000
;
Misawa et al., 2001
;
Lips et al., 2002
) is first
expressed together with ChAT and VAChT after the formation
of ciliary ganglion primordia and after a number of other neuronal genes have
started their expression. Although our results do not exclude an earlier
specification of ciliary neurons, they exclude CHT1 as an early
indicator for migrating ciliary neuron precursors.
As sympathetic neuron generation is controlled by BMPs that are expressed
in the dorsal aorta, in close vicinity to the forming primary sympathetic
ganglia (Reissmann et al.,
1996; Shah et al.,
1996
), we asked whether BMPs are also involved in ciliary ganglion
development. BMP5 and BMP7 were found to be expressed in
Rathke's pouch and in the mesenchyme around the ciliary ganglion anlage at
stage 18, albeit at low levels. Whereas the expression in Rathke's pouch seems
to be too far away to represent a significant source of BMPs for the ciliary
neuron precursors, mesenchymal BMP7 (Luo
et al., 1995
; Dudley et al.,
1995
) is most likely involved in ciliary neuron development.
However, the possibility should also be considered that ciliary ganglion
precursors may encounter BMPs during their migration.
Are BMPs important for ciliary neuron development? The interference with
the function of BMPs by the application of the BMP-inhibitor noggin
(Zimmerman et al., 1996;
Schneider et al., 1999
;
Vogel-Höpker and Rohrer,
2002
) resulted in the complete lack of differentiated ciliary
neurons, strongly suggesting that ciliary neuron development requires BMPs.
The presence of aggregates of Sox10-positive cells at the site of the ciliary
ganglion demonstrates that neural crest migration and aggregation are not
affected by noggin. Noggin-treatment also affected the development of the eye,
whereas control embryos, receiving control CHO cells, had normal sized eyes
and the ciliary ganglion was present. To investigate whether the effects on
ciliary neurons are an indirect result of the missing eye, the optic vesicle
was unilaterally extirpated at E2. Although the eye was virtually completely
missing on the operated side, there was no difference with respect to the size
and gene expression pattern of the ciliary ganglion, as expected from previous
studies (Landmesser and Pilar,
1974
; Lee et al.,
2001
). Thus, both ciliary neuron development and the development
of the eye depend on BMPs. Indeed, in the BMP7 knockout mice, eye development
is severely affected (Luo et al.,
1995
; Dudley et al.,
1995
). The fate of the ciliary ganglion has not been analysed in
these mice. Since noggin interferes with the action of several BMP family
members, the identity of the BMP(s) that are essential for ciliary ganglion
development remains unclear. In view of the redundant expression of BMPs and
the stronger phenotype of the BMP5/BMP7 double knockout mice as compared to
single knockout mice (Solloway and
Robertson, 1999
), it is possible that both BMP5 and BMP7 are
involved in ciliary neuron development. The proposed role of BMPs in ciliary
neuron development is strongly supported by BMP overexpression experiments.
Forced expression of BMP4 produced enlarged ciliary ganglia through the
generation of ectopic neurons from neural crest precursor cells. These cells
were generated in the oculomotor nerve and postganglionic ciliary nerve. As
expected from previous BMP overexpression experiments in sympathetic ganglia
and peripheral nerves (Reissmann et al.,
1996
; Howard et al.,
2000
) these cells also expressed Phox2b. Interestingly,
the neurons generated in response to BMPs displayed a cholinergic rather than
a noradrenergic phenotype, as did the neurons in the ciliary ganglion. This is
in contrast to the situation in the trunk, where BMPs produce enlarged
noradrenergic sympathetic ganglia and ectopic noradrenergic neurons in
peripheral nerves (Reissmann et al.,
1996
; Howard et al.,
2000
; Ernsberger et al.,
2000
).
This result raised the question of why noradrenergic gene expression is
regulated differentially in ciliary neurons and nerves as compared to
sympathetic neurons and trunk precursor cells. In view of the similar
expression pattern of Mash1 and Phox2 genes in sympathetic
and parasympathetic ganglia and the Mash1- and
Phox2-dependent development of cranial parasympathetic ganglia
(Hirsch et al., 1998;
Morin et al., 1997
;
Pattyn et al., 1999
) it seems
very likely that Mash1 and Phox2 genes are also involved in the initial onset
of TH and DBH expression in the ciliary ganglion. Although
noradrenergic gene expression in the mouse ciliary ganglion has not been
analysed in Mash1 and Phox2 knockouts, DBH
expression is controlled by Mash1 in the parasympathetic
sphenopalatine ganglion (Hirsch et al.,
1998
). In addition, ciliary ganglion development depends on
Mash1 (Hirsch et al.,
1998
) and likely also on Phox2 genes, as other cranial
parasympathetic ganglia are missing in the Phox2a and Phoxb
knockouts (Morin et al., 1997
;
Pattyn et al., 1999
). However,
whereas TH and DBH are rapidly induced in all
Phox2-positive sympathetic neuron precursors,
TH/DBH-positive cells in ciliary ganglia represent at all stages only
a subpopulation of Phox2-expressing cells. This could be explained by
a lower sensitivity of parasympathetic precursor cells to BMPs, requiring
higher concentrations for TH expression, as compared to the
expression of cholinergic markers like VAChT
(White et al., 2001
). The
observation that BMP overexpression does not increase the proportion of
TH/DBH-expressing cells argues, however, against this notion. The presence of
cells that co-express TH and VAChT at stage 24 suggests that
ciliary neurons transiently express noradrenergic genes before they acquire a
cholinergic transmitter phenotype. The low number of cells that are clearly
double-labeled is expected if noradrenergic and cholinergic gene expression
overlap only during a short time period. Although we cannot exclude that the
disappearance of noradrenergic cells is partly due to the selective death of
these cells (Lee et al.,
2001
), this possibility seems to be unlikely in view of the cells
being in a transitory stage from a noradrenergic to cholinergic phenotype and
the increased TH and DBH expression in response to dHand.
The selective downregulation of TH and DBH expression in
ciliary as compared to sympathetic ganglia may either be due to the presence
of a TH/DBH repressor in ciliary ganglia or due to the lack of a
TH/DBH maintenance signal. Here, we show that the bHLH transcription
factor dHand could be such a maintenance factor. dHand is expressed
in sympathetic neurons under the control of BMPs, downstream of
Phox2b (Howard et al.,
2000). Forced expression of dHand in trunk neural crest precursors
elicits the generation of noradrenergic neurons both in vitro and in vivo
(Howard et al., 1999
;
Howard et al., 2000
). The
increased number of TH- and DBH-positive cells in
dHand-expressing ciliary ganglia now suggests that dHand is also involved in
maintaining noradrenergic differentiation. The effects of forced dHand
expression in neural crest cells are compatible with the notion that dHand
represents a late BMP effector, that is able to act independently of the
upstream factors Cash1, Phox2b, Phox2a. This is in line with the observation
that dHand is able to transactivate DBH reporter constructs (M. Howard,
personal communication). However, dHand overexpression elicits the expression
of both Phox2a and Phox2b genes
(Howard et al., 2000
) and
thus, may act in combination with Phox2 transcription factors. Both
possibilities are compatible with a role for dHand in the maintenance of
TH and DBH expression, a role that still needs to be
confirmed by loss-of-function experiments. The dHand knockout mice
die, however, too early to investigate a role for dHand as a maintenance
signal of DBH and TH expression (Yamagishi
et al., 1999
).
The differential expression of dHand has also important
implications for the understanding of sympathetic and parasympathetic ciliary
neuron development and the role of BMPs in this process. What is the role of
BMPs in the generation of different autonomic neuronal subtypes? In the spinal
cord, different types of dorsal neurons are specified by different levels of
BMPs (Lee and Jessell, 1999)
and our recent work on noradrenergic neuron development in the hindbrain
demonstrated that the generation of noradrenergic locus coeruleus neurons
depends directly or indirectly on the BMP-mediated dorsal patterning of
rhombomere 1 (Vogel-Höpker and
Rohrer, 2002
). As ChAT is induced at lower BMP levels
than TH in cultured peripheral nerve precursor cells it was proposed
that parasympathetic versus sympathetic neuron generation may be specified by
different BMP levels in vivo (White et
al., 2001
). The present demonstration that parasympathetic ciliary
ganglion cells initially also display a noradrenergic phenotype is difficult
to accommodate with this idea. One could argue that TH/DBH would be expressed
in autonomic neurons as long as there is a high-level BMP expression in the
vicinity of autonomic ganglia. Indeed, BMP7 and BMP5 expression in the
retro-orbital mesenchyme decreases between stage 18 and stage 20 (F.
Müller, unpublished observation), whereas BMP4 expression in the dorsal
aorta is maintained up to E9 (U. Ernsberger, personal communication). Thus,
there is a correlation between BMP expression and TH/DBH expression in
autonomic ganglia. However, strong evidence against a BMP-level-dependent
specification of cholinergic parasympathetic and noradrenergic sympathetic
neurons is the observation that BMP overexpression in the ciliary ganglion did
not increase TH/DBH expression, whereas TH/DBH expression
has been shown to be induced in neural crest precursors in trunk peripheral
nerve (Ernsberger et al.,
2000
). This result could also be explained by assuming that BMP
overexpression in ciliary compared with sympathetic ganglia and nerves never
reached the high BMP levels required to elicit TH/DBH expression,
because of differential expression of BMP inhibitors. However, there are
several factors that do not substantiate this explanation. (i) Assuming that
TH/DBH expression requires high BMP concentrations, the presence of
TH/DBH-positive cells would imply that high BMP levels are present
and can be reached in the ciliary ganglion environment. (ii) The generation of
ectopic neurons by BMP overexpression demonstrates that significant increases
in BMP levels were reached in vivo in both cases. (iii) The lack of
dHand expression in TH/DBH ciliary neurons generated in
response to BMPs argues for a difference between ciliary and sympathetic
neuron precursors. In conclusion, the present results are more compatible with
the notion of differences between autonomic precursor cells in the head region
where the ciliary ganglion forms and autonomic precursor cells in sympathetic
ganglia and trunk peripheral nerve. It will be interesting to investigate to
what extent the present findings can be generalized to cranial and trunk
parasympathetic ganglia.
What may cause a difference between sympathetic and ciliary precursors?
These differences may reflect different anteroposterior positional values
(Jessell and Lumsden, 1998;
Rubinstein and Shimamura,
1998
) since ciliary and sympathetic precursors are derived from
different axial levels (Le Douarin and
Kalcheim, 1999
), or local signals in the environment of the
forming ganglia. Heterotopic neural crest transplantation experiments
demonstrated that the developmental capacities of the neural crest cells are
qualitatively equivalent at all axial levels (Le Dourin et al., 1975;
Le Douarin and Kalcheim,
1999
), which strongly suggest that local signals specify the fate
of neural crest cells following their migration. The finding that autonomic
precursor cells are present in the ciliary ganglion at E4.5 and later that can
differentiate to noradrenergic neurons upon heterotopic transplantation
(Le Douarin et al., 1978
;
Dupin, 1984
) seems to
contradict our inability to elicit noradrenergic differentiation by BMP4
overexpression. It should, however, be noted that the potential of these cells
is repressed during normal development, apparently by local signals, and is
revealed and realized only after ganglia disassemble during
backtransplantation (Le Douarin et al.,
1978
; Dupin, 1984
;
Schweizer et al., 1983
). Thus,
we propose that the local environmental, signals that define parasympathetic
neuron identity in the ciliary ganglion primordium e.g. the repression of
dHand expression, cannot be overcome by increased expression of BMPs
and downstream signaling in vivo, in the ciliary ganglion environment. This
may be possible in vitro or after transplantation of these cells into a
different environment in vivo. Interestingly, not only neural crest precursors
from the ciliary ganglion, but also ciliary neurons are able to acquire a
noradrenergic phenotype upon transplantation and migration into the trunk
(Coulombe and Bronner-Fraser,
1986
). The finding that noradrenergic differentiation of immature
ciliary neurons does not occur upon implantation into the head mesenchyme of
young embryos (Sechrist et al.,
1998
) supports our conclusion that signals from the head
mesenchyme environment prevents full noradrenergic differentiation.
What is the reason for a low number of neurons maintaining TH and
DBH in the ciliary ganglion in the absence of dHand? The
compensation by the closely related factor eHand
(Srivastava et al., 1995;
Hollenberg et al., 1995
;
Howard et al., 1999
) is
excluded as eHand expression was not detected in the ciliary ganglion
(data not shown). We propose that dHand function may be required only
during a relatively early developmental period and that its function can be
replaced later by other signaling pathways. For dopaminergic neurons in
cranial sensory ganglia, in particular in the petrosal ganglion, there is
evidence for an early, transient TH expression, followed later by a
second, sustained TH expression, elicited by signals from the target
(Katz and Erb, 1990
) and/or
maintained by electrical activity
(Brosenitsch and Katz, 2001
). A
similar scenario would account for early and late expression of TH
and DBH in mammalian parasympathetic ganglia
(Hirsch et al., 1998
;
Landis et al., 1987
;
Leblanc and Landis, 1989
). In
view of the very low number of TH/DBH-positive cells that remain in
chick ciliary ganglia, this issue was not addressed in our study.
Whereas the present study revealed mechanisms involved in the control of
noradrenergic differentiation in the ciliary parasympathetic ganglion versus
sympathetic ganglia, the extrinsic signals and the transcription factors
controlling the early, target-independent expression of cholinergic marker
genes are not known (Ernsberger and
Rohrer, 1999) and it remains a matter of speculation whether
similar factors are involved in sympathetic and parasympathetic ganglia.
Candidate cholinergic differentiation factors are ligands for the c-ret
receptor, since c-ret is selectively expressed in cholinergic sympathetic
neurons (Ernsberger et al.,
2000
) and ciliary neurons
(Hashino et al., 2001
) and
since c-ret ligands are able to induce cholinergic marker genes in vitro
(Brodski et al., 2002
).
Neuropoietic cytokines acting through gp130/LIFRß receptors are involved
in the expression of VIP but not of ChAT and VAChT in chick sympathetic neuron
development in vivo (Geissen et al.,
1998
; Duong et al.,
2002
).
In conclusion, our results provide evidence that BMPs are essential for the generation of both parasympathetic and sympathetic neurons and suggest that BMPs act on neural crest precursor cells that display specific location-dependent differences that in turn determine the response to BMP signaling and eventually the autonomic neuron subtype. The transcription factor dHand is not only an indicator for this difference but may be responsible for the differential expression of the noradrenergic marker genes TH and DBH in sympathetic and parasympathetic ciliary neurons.
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ACKNOWLEDGMENTS |
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