1 Max-Planck-Institut für Hirnforschung, Abteilung Neurochemie,
Deutschordenstr. 46, 60528 Frankfurt/Main, Germany
2 CNRS UMR 8542, Département de Biologie, Ecole Normale
Supérieure, 46 rue d'Ulm, 75005 Paris, France
3 Department of Cell Biology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The
Netherlands
Author for correspondence (e-mail:
rohrer{at}mpih-frankfurt.mpg.de)
Accepted 20 July 2004
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SUMMARY |
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Key words: Autonomic, Ciliary, Cholinergic, Noradrenergic, Hand2 (dhand), Bmp, Phox2b, Gata2, Gata3, Chick, Mouse
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Introduction |
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Although Mash1 and Phox2b were shown to be essential and
sufficient to elicit noradrenergic neuron development from neural crest
precursor cells, they also function together in the generation of cholinergic
autonomic neurons and several other neuron subtypes. Thus, to generate
noradrenergic neurons, Mash1 and Phox2 genes must be
enforced by the action of additional autonomic and noradrenergic regulators
expressed in this lineage. The basic helix-loop-helix (bHLH) transcription
factor Hand2 (previously known as dHand) has recently been identified as a
noradrenergic co-determinant, due to its ability to elicit noradrenergic
differentiation in neural crest (Howard et
al., 2000) and parasympathetic precursors
(Müller and Rohrer,
2002
), and due to its expression in noradrenergic sympathetic but
not in parasympathetic ciliary neurons
(Müller and Rohrer,
2002
). The effects on the expression of the noradrenergic marker
gene dopamine-ß-hydroxylase (Dbh) can be explained by a direct
interaction with Phox2a to stimulate transcription from the Dbh promotor
(Xu et al., 2003
;
Rychlik et al., 2003
).
Finally, members of the Gata family of transcription factors have been
implicated in the control of noradrenergic differentiation
(Groves et al., 1995
;
Lim et al., 2000
).
The Gata transcription factors are key regulators of hematopoiesis
(Pevny et al., 1991;
Tsai et al., 1989
;
Maeno et al., 1996
;
Murphy and Reiner, 2002
),
cardiovascular and urogenital development
(Zhou et al., 1998
;
Molkentin et al., 1997
) and
nervous system development (Pandolfi et
al., 1995
; Nardelli et al.,
1999
; Pata et al.,
1999
; Dasen et al.,
1999
; van Doorninck et al.,
1999
; Craven et al.,
2004
; Karis et al.,
2001
). In hematopoiesis and developing heart and liver, Gata
transcription factors mediate the effects of Bmps
(Maeno et al., 1996
;
Schultheiss et al., 1997
;
Rossi et al., 2001
). Recent
evidence suggests that Gata factors maintain Bmp expression during cardiac
precursor maturation (Peterkin et al.,
2003
; Klinedinst and Bodmer,
2003
). Gata factors are characterized by two zinc finger domains
that mediate binding to a DNA motif centred around the nucleotide sequence
GATA (Yamamoto et al., 1990
;
Ko and Engel, 1993
;
Whyatt et al., 1993
). The Gata
family is composed of six vertebrate family members that are expressed in
distinct spatiotemporal patterns. However, of all family members, only Gata2
and Gata3 are present in the nervous system, where their expression overlaps
extensively (Kornhauser et al.,
1994
; Nardelli et al.,
1999
; Pata et al.,
1999
). The analysis of mice deficient for Gata2 or
Gata3 revealed that Gata2 controls Gata3 expression in many, but not
all expression domains (Nardelli et al.,
1999
; Pata et al.,
1999
). In the central nervous system, Gata2 is essential and
sufficient for spinal cord interneuron generation
(Zhou et al., 2000
;
Karunaratne et al., 2002
), for
the induction of ventral gonadotrope and thyrotrope fates in the pituitary
(Dasen et al., 1999
) and for
the generation of serotonergic neurons in rostral hindbrain
(Craven et al., 2004
).
Gata3 was shown to be involved in the development of serotonergic
neurons in the caudal raphe nuclei (van
Doorninck et al., 1999
; Pattyn
et al., 2004
), in ear formation
(Karis et al., 2001
;
Lawoko-Kerali et al., 2002
),
and in the expression of the noradrenergic marker genes Th and
Dbh in the peripheral nervous system
(Lim et al., 2000
).
Since in the sympathetic ganglia of Gata3-deficient mice
abrogation of Th and Dbh expression, but normal generic
neuronal differentiation, has been reported
(Lim et al., 2000), Gata3 was
considered to selectively control neuron subtype differentiation and to
represent a noradrenergic co-determinant for Phox2a/b and Mash1. Although the
Gata3 knockouts demonstrated the importance of this factor, its
position in the transcriptional network specifying sympathetic neurons was not
clear. Here, we have analysed the action of Gata transcription factors in
autonomic neuron development in the chick and re-investigated the sympathetic
neuron phenotype in Gata3-deficient mouse embryos. We demonstrate that
Gata2 but not Gata3 is expressed in the avian autonomic
nervous system. Gata2 expression in the chick starts after the
expression of Cash1, Phox2b, Phox2a and Hand2 and is induced
by overexpression of these transcription factors. Bmp-dependent expression
characterizes Gata2 as an additional member of the transcriptional network
acting in the sympathetic lineage downstream of Bmps. The elimination of Gata3
in the mouse and the knockdown of Gata2 in the chick result in a strong
decrease in both sympathetic ganglion size and Th expression. These results,
together with the effect of Gata2 overexpression demonstrate a function for
Gata2/3 in the type-specific, as well as generic, differentiation of
noradrenergic neurons, acting in the context of other autonomic
co-determinants.
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Materials and methods |
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Implantation of agarose beads loaded with noggin or BSA in chick embryos
The implantation technique used is described in detail by Schneider et al.
(Schneider et al., 1999).
Agarose beads (Affi-Gel blue beads; Biorad, Hercules, CA) were incubated for
at least 1 hour in a small volume of loading buffer containing either 1 mg/ml
noggin or bovine serum albumin (BSA). Two beads were implanted into the trunk
region of 2-day-old chick embryos, placed at the last somite and 2-3 somites
more rostral. The eggs were further incubated until stage 19, fixed, embedded
and sectioned. Cryosections of 12 µm were collected, including from the
implantation area, and analysed for expression of Sox10 and
Gata2 by in-situ hybridisation. The area of Sox10 and
Gata2 expression was quantified morphometrically and the areas were
expressed in µm2/section. The results are given as the mean area
per section±s.e.m. of at least six embryos analysed.
Expression of transgenes in vivo using retroviral replication-competent avian sarcoma (RCAS) vectors
Fertilized virus-free chicken eggs were obtained from Charles River
(Sulzfeld, Germany) and incubated for 2 days. Cell aggregates of DF1
fibroblasts infected with RCASBP(B)-Hand2
(Howard et al., 2000),
RCASBP(B)-Phox2b (Stanke et al.,
1999
), RCASBP(B)-CNS-Gata2, RCASBP(B)-CNS-dnGata2,
RCASBP(B)-CNS-engrailed and RCASBP(B)-CNS-VP16-Gata2 were implanted on the
right site of the embryos at brachial levels between the neural tube and the
last somite formed (Reissman et al., 1996). The eggs were further incubated
until E8. Embryos were killed by decapitation. The trunk and cervical region
of the embryos were fixed in 4% paraformaldehyde in 0.1 M sodium phosphate
buffer overnight, kept in 15% sucrose in 0.1 M sodium phosphate buffer
overnight, embedded in Tissue Tek (Sakura Finetek Europ BV, Zoeterwoude, The
Netherlands) and sectioned. Cryosections of 12 µm were collected and
analysed for expression of reverse trancriptase (RT), Th,
Scg10 and Gata2 (RCASBP(B)-Hand2 and RCASBP(B)-Phox2b
infections); RT, Th, Scg10, NF160, Phox2b and Cash1
(RCASBP(B)-CNS-Gata2 infections) and RT, Th, Dbh, Scg10, NF160 and
Phox2b (RCASBP(B)-CNS-dnGata2-, RCAS-BP(B)-CNS-engrailed and
RCASBP(B)-CNS-VP16-Gata2 infections) by in-situ hybridisation. For the
quantitative analysis, between 5 and 13 embryos were analysed for each of the
genes investigated.
Mouse breeding, genotyping and rescue
The generation and genotyping of Phox2b mutant mice and
Gata3 mutant mice have been reported
(Pattyn et al., 1999;
Pandolfi et al., 1995
;
Lim et al., 2000
). Homozygous
Gata3 mutants, which normally die at mid-gestation, were rescued
beyond E10 with noradrenergic agonists as described for Phox2b mutants
(Pattyn et al., 2000
).
In-situ hybridisation on sections
Non-radioactive in-situ hybridisation on cryosections and preparation of
digoxigenin- or fluoresceine-labelled probes for chick RT, Th, Scg10,
NF160, Gata2, Gata3, Phox2b, Phox2a, Hand2, Cash1 and Sox10 were
carried out as described previously
(Ernsberger et al., 1997;
Stanke et al., 1999
)
(Gata2 and Gata3 plasmids were generously provided by M.
Zenke). For double in-situ hybridisations Fast Red (Roche Diagnostics,
Mannheim, Germany) was used for staining the first probe. Sections were
photographed and the antibody was stripped off by washing twice for 10 minutes
with 1 ml 0.1 M glycine pH 1.8. After equilibration in MABT for 1 hour, the
second colour reaction with Nitroblue
Tetrazolium/S-bromo-
-chloro-3-indolyl phosphate (NBT/BCIP) was
carried out.
In-situ hybridisation using mouse Dbh, Gata2 (gift of J.
Nardelli), Gata3 (gift of D. Engel), Hand2 (gift of Y.-S.
Dai), Mash1 (gift of F. Guillemot), Ret, Sox10 (gift of K.
Kulbrodt) and Th antisense riboprobes, immunohistochemistry using
Phox2a, Phox2b, ß-galactosidase (Cappel) antisera or Tuj1 monoclonal
antibody (Covance), and combined in-situ hybridisation with
immunohistochemistry were performed as previously described
(Tiveron et al., 1996).
Double-immunofluorescence experiments using Phox2a and Tuj1 antibodies were
analysed on a Leica microscope. Pictures were superimposed in Photoshop.
Morphometric analysis
Chick
The area of Th, Dbh, Phox2b, NF160 and Scg10 expression
was quantified morphometrically using the Metamorph Imaging System (version
4.6, Universal Imaging Corporation) on all sections infected by the virus, as
indicated by expression of RT mRNA. Areas were expressed in
µm2/section. The results are given as the mean area per
section±s.e.m. of at least five embryos analysed. Student's
t-test was used for statistical analysis.
Mouse stellate ganglion
The surface of the stellate ganglion stained with Phox2b antibody
has been calculated on saggital sections of E13.5 embryos using the
Leica Qfluoro Program. Measurements of control ganglia were considered as
100%. For each genotype, four sections were counted on four chains
corresponding to two embryos.
Mouse thoracic chain
The number of Phox2b-positive cells were counted on saggital sections at
E13.5 at the thoracic level on a segment spanning three vertebrae, at the same
level in the control and the mutants. For each genotype, four sections were
counted on four chains corresponding to two embryos.
TUNEL analysis
TUNEL-positive cells were detected using the apoptag detection kit
(Appligene) following the manufacturer's instructions. The rostralmost part of
the sympathetic chain (anterior to the fusion of the dorsal aorta) was
delimited on transverse sections at E11.5 using a Sox10 in-situ
hybridisation signal on adjacent sections, and cells were counted within that
area. For each genotype, six sections were analysed on four chains
corresponding to two embryos.
Construction of plasmid transgenes
RCAS-BP(B)-CNS-Gata2
PCR technology was used to insert a Kozak sequence linked to a
ClaI site and a NotI site flanking the coding sequence of
ggGata2 (Yamamoto et al.,
1990). Primer: sense: 5'-AGT ATC GAT GAC CAC C
AT G GA GGT GGC CAC GGA TCA GC-3'; antisense: 5'-GAT
CGA GCG GCC GC T TA T CCC ATG GCT GTA ACC AT-3'.
(sense primer: bold, ClaI site; underlined, Kozak sequence;
bold+underlined, Start) (antisense primer: bold, NotI site;
bold+underlined, Stop)
The PCR product was then cloned directly into the pCRII-TOPO vector
(Invitrogen). After restriction analysing and sequencing, the insert was
cloned into the ClaI and NotI sites of the avian retroviral
vector RCAS-BP(B)-CNS. The RCAS-BP(B)-CNS is a modification of the RCAS-BP(B)
vector (Hughes and Kosik,
1984), inserting a unique NotI and SpeI site
directly behind ClaI(7029).
RCAS-BP(B)-CNS-dnGata2
PCR technology was used to insert a BamHI site and an
XbaI site flanking the two zinc finger domains of ggGata2 (ggGata2
S276-I379, equivalent to hsGata3
S258-I361) (Yamamoto
et al., 1990; Yang et al.,
1994
). Primer: sense: 5'-CTG GGA TCC TCA GAA GGC AGA
GAG TGT GTG AA-3'; anisense: 5'-TTC TCT AGA TTC GTT TTT CAT
GGT CAG AGG CC-3'. (sense primer: bold: BamHI site; antisense
primer: bold: XbaI site)
The BamHI-XbaI digested PCR fragment was ligated into the
pIEP vector (pIEP vector was generously provided by C. Goridis) after
eliminating the original BamHI-XbaI fragment (Phox2a DNA
binding site). The original pIEP vector contains the engrailed effector domain
from Drosophila melanogaster (AA1-AA298)
(Han and Manley, 1993)
upstream from the Phox2a homeodomain, that is flanked by a BamHI site
and an XbaI site. The pIEP vector also contains two myc-tags
downstream from the XbaI site. PCR technology was then used to insert
a Kozak sequence linked to a ClaI site and a SpeI site
flanking the dnGATA2 coding region. The PCR product was cloned into the
ClaI and NotI site of the RCAS-BP(B)-CNS vector. Primer:
sense: 5'-ACA ATC GAT GCC GCC A AT G GC CCT GGA
GGA TCG CTG CA-3'; antisense: 5'-TCT ACT AGT TCA
CAG GTC CTC CTC GCT GAT CAG-3'. (sense primer: bold, ClaI site;
underlined, Kozak sequence; bold+underlined, start) (antisense primer: bold,
SpeI site; bold+underlined, stop)
RCAS-BP(B)-CNS-VP16-Gata2
The engrailed domain of RCAS-BP(B)-CNS-dnGata2 was substituted by the VP16
domain (Ala481-Gly541 + spacer of 6 AA) (VP16 vector was
generously provided by C. Goridis).
RCAS-BP(B)-CNS-engrailed
The zinc finger domain of RCAS-BP(B)-CNS-dnGata2 was substituted by the
spacer Ala-Gly-Gly.
Semiquantitative RT-PCR analysis
Total RNA from chick sympathetic ganglia was isolated by using an RNeasy
kit (Qiagen). Relative levels of Gata2 and Gata3 expression
were determined by RT-PCR. Primer pairs were designed for amplification of
specific cDNA fragments.
Gata2 primers: sense: 5'-CAA CTA CAT GGA ACC AGC GC-3'; antisense: 5'-AGG CTG CTG CTG TAG TCA TG-3'; Gata3 primers: sense: 5'-CTC CGT ATT ACG GCA ACT CC-3'; antisense: 5'-GCT GCA GAC AGC CTT CTC TT-3'.
cDNA from total RNA was synthesised with oligo(dT) primers and Moloney
murine leukaemia virus reverse transcriptase (Superscript II; Life
Technologies) at 45°C for 1 hour. cDNA derived from 20-30 ng RNA was used
as template for PCR amplification in a 50 µl reaction volume containing
1x PCR buffer, 0.2 mM dNTPs and 0.1 µM each primer. Hot start was
performed by adding 1.5 units of AmpliTaq. The temperature profile consisted
of 25-36 cycles (95°C for 15 seconds, 65°C for 30 seconds and 72°C
for 30 seconds) and a final 5 minutes extension at 72°C. To achieve
accurate quantification, 10 µl aliquots were collected during the PCR run
at various cycle numbers. PCR products were separated by electrophoresis on 1%
agarose gel and stained with Ethidiumbromide. Their fluorescence intensities
were measured by using the Gel Doc 2000 (Bio Rad Laboratories). In all
experiments, amplification of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) cDNA fragments was run in parallel to normalize different
cDNA samples (Friedel et al.,
1997).
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Results |
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Essential role of Gata2 in chick sympathetic neuron generation
The strong effects of the Gata3 knockout on noradrenergic gene expression
in the mouse (Lim et al.,
2000) raised the question whether elimination of Gata2, which in
the chick sympathetic lineage appears to be functionally equivalent to mouse
Gata3, would also impair the development of the noradrenergic phenotype. To
interfere with the action of endogenous Gata2, a repressive form of Gata2, in
which the engrailed repressor domain is fused to Gata2 zinc finger domains,
was expressed in sympathetic neuron precursor cells. Previous studies have
demonstrated that the engrailed repressor does not function by titrating
promoter binding sites but rather interferes with transcription initiation
(Han and Manley, 1993
;
Jaynes and O'Farrell, 1991
).
Indeed, a similar engrailed-Gata2 fusion protein has very recently been
constructed and was shown to act as dominant-negative Gata2 (dnGata2)
(Craven et al., 2004
). DnGata2
was expressed unilaterally using RCAS retroviral vectors, so that the
sympathetic ganglia of the contralateral side could be used as internal
control. As additional controls, ganglia were infected with RCAS vectors
expressing only the engrailed repressor domain or a VP16-Gata2 variant, where
the transactivating N-terminal region of Gata2 is replaced by the VP16
activation domain. The expression of dnGata2 in sympathetic neuron precursors
resulted in a strong (50%) reduction of Th expression
(Fig. 6), and smaller effects
on Scg10 (Stmn2 Mouse Genome Informatics) (35%), Dbh
(41%) and Phox2b (31%) (Fig.
6). The expression of neurofilament (NF 160) was also
strongly reduced (not shown). All reductions were highly significant
(P<0.01) with respect to the control side. Th expression
was also significantly reduced compared with Phox2b
(P<0.05; n=11) and Scg10 (P<0.05;
n=11), whereas Dbh was not significantly more reduced than Phox2b and
SCG10. Control infections with the engrailed RCAS virus or RCAS-VP16-GATA2
affected neither Th nor Scg10 expression
(Fig. 6), excluding the
possibility that dnGata2 would act in an unspecific manner by titrating
transcription factors unrelated to endogenous Gata2. In conclusion, these
results demonstrate a dual effect of Gata2 knockdown: a reduction in
sympathetic ganglion size, reflected by a smaller area of Phox2b- and
SCG10-expressing cells, and an additional effect on Th expression in the
remaining cells. These effects suggest a more general role in sympathetic
neuron development than reported for murine Gata3
(Lim et al., 2000
).
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Gata2 expression in parasympathetic ganglia and the locus coeruleus
The importance of Gata2/3 in sympathetic neuron development raised
the issue whether Gata2/3 would play a similar role in other
autonomic ganglia and/or in central noradrenergic neurons. The parasympathetic
chick ciliary ganglion was found to completely lack Gata2 and
Gata3 expression at all stages analysed by in-situ hybridisation
between stage 20 (E3) and stage 35 (E8)
(Fig. 10). The result from the
in-situ hybridisation was confirmed by semiquantitative RT-PCR, which revealed
in E5 ciliary ganglia 1000-fold and 200-fold lower levels of Gata2
and Gata3 mRNA, respectively, compared with Gata2 mRNA in E7
sympathetic ganglia. Low-level signals for Gata2 and Gata3
by RT-PCR can be explained by Gata expression in cells of the
retro-orbital mesenchyme, contaminating to some extent the ganglion
preparation. Also the chick parasympathetic sphenopalatine ganglion was devoid
of Gata2 expression (Fig.
10). However, Gata2 was detectable in the submandibular
ganglion (Fig. 10), and strong
Gata2 expression was present in trunk parasympathetic ganglia, i.e.
cardiac ganglia (Fig. 10) and
the Remak ganglion (Groves et al.,
1995). From all chick ganglia investigated, Gata3 was
detected only in the cardiac ganglia (not shown). In the mouse, the
sphenopalatine, otic and submandibular ganglia were devoid of Gata3
expression at E13.5, with low, but detectable expression in cardiac ganglia
(not shown). Interestingly, low-level Th expression was present in
the chick parasympathetic sphenopalatine, submandibular, cardiac
(Fig. 10) and the Remak
ganglion (Cantino et al., 1982
;
Suzuki et al., 1994
), whereas
in the ciliary ganglion only very few Th-positive cells remained
(Müller and Rohrer, 2002
)
(Fig. 10). Dbh
expression paralleled Th expression, both with respect to ganglion
type and expression levels (not shown). The expression of a variable subset of
noradrenergic traits in cholinergic parasympathetic neurons has been described
previously in several species (Grzanna and
Coyle, 1978
; Landis et al.,
1987
; Leblanc and Landis,
1989
; Baluk and Gabella,
1990
; Hardebo et al.,
1992
).
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Discussion |
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Gata factors in relation to other Bmp-induced transcriptional determinants of the sympathetic lineage
The members of the Gata family of zinc finger transcription factors have
selective effects on various aspects of tissue and organ development. Of the
six family members, only Gata2 and Gata3 are expressed in
the vertebrate nervous system and were shown to control the development and
differentiation of specific neuronal subpopulations
(Yamamoto et al., 1990;
Ko and Engel, 1993
;
Whyatt et al., 1993
;
Zhou et al., 2000
;
Karunaratne et al., 2002
;
Dasen et al., 1999
;
van Doorninck et al., 1999
;
Craven et al., 2004
;
Lim et al., 2000
;
Karis et al., 2001
). In
general, Gata2 and Gata3 are coexpressed in the central
nervous system and Gata3 is downstream of Gata2
(Nardelli et al., 1999
;
Pata et al., 1999
). The
expression of these factors in the peripheral nervous system is much less
clear, as data are often available for only one factor in a single species and
tissue (Groves et al., 1995
;
Nardelli et al., 1999
;
George et al., 1994
). We
demonstrate here that Gata3 is not expressed in chick sympathetic
ganglia, whereas Gata2 is detectable throughout development. The
onset of Gata2 expression in the chick was found to occur after the
sequential expression of Mash1, Phox2b, Hand2 and
Phox2a.
The timing of Gata2 expression and its proposed role in
noradrenergic differentiation raised the question whether Gata2
expression is dependent on Bmps, which have been shown to control the
expression of Mash1, Phox2a/b and Hand2 in sympathetic
neuron precursors at the dorsal aorta
(Schneider et al., 1999;
Howard et al., 2000
) and which
control Gata factor expression in other developmental contexts
(Maeno et al., 1996
;
Schultheiss et al., 1997
;
Rossi et al., 2001
;
Patient and McGhee, 2002
).
Alternatively, Gata2 expression might be induced by additional,
independent signals. The present data firmly establish that Gata2
expression is prevented by the Bmp inhibitor noggin and is expressed in
Bmp-induced ectopic neurons. Thus, Gata2 represents an additional member of
the group of transcription factors induced by Bmps. The epistatic relationship
between Gata2 and the other factors has been addressed by
overexpression of Phox2b, Hand2 and Mash1. The induction of
Gata2 expression by each of these factors is in agreement with the
timing of expression and suggests that Gata2 may be directly or
indirectly controlled by these transcription factors. The notion that
Gata2 is expressed downstream of Phox2b is confirmed by the
lack of Gata2/3 expression in the Phox2b knockout mice and,
conversely, the initial presence of cells expressing Phox2b, Phox2a
and Hand2 in the absence of Gata3.
Generic and subtype-specific role of Gata factors in the sympathetic lineage
A major problem in the control of neurogenesis is how and when the
expression of neuron subtype-specific properties and generic neuronal
characteristics are coordinated. For sympathetic neuron development this issue
is still unclear. The loss of Mash1 and Phox2b does affect
both noradrenergic and pan-neuronal gene expression
(Guillemot et al., 1993;
Hirsch et al., 1998
;
Pattyn et al., 1999
) and in
gain-of-function experiments no selective effects were observed for
Phox2a, Phox2b and Hand2
(Stanke et al., 1999
;
Howard et al., 2000
). Under
certain in-vitro conditions, Mash1 was able to induce properties of
autonomic neurons, but not noradrenergic differentiation
(Lo et al., 1998
). Also in
vivo, Mash1 overexpression in peripheral nerve precursors results in
the preferential generation of non-adrenergic neurons, suggesting a major role
of Mash1 in the control of generic neuronal traits
(Stanke et al., 2004
). The
previous analysis of Gata3-deficient mice suggested that
Gata3 may selectively control the noradrenergic phenotype in this
lineage (Lim et al., 2000
). To
define the role of Gata2 in chick sympathetic neurons, a
dominant-negative variant of Gata2 was expressed in developing sympathetic
ganglia. The Gata2 knockdown resulted in reduced Th expression and in
a smaller size of sympathetic ganglia, identified by Phox2b and
Scg10. In agreement with this action of dnGATA2 on ganglion size, we
found a virtually complete loss of the superior cervical and stellate ganglia
and the strong atrophy of thoracic sympathetic chain ganglia in
Gata3/ mice, not reported in the first
description of these mutants (Lim et al.,
2000
). In the cells of the rudimentary sympathetic ganglia the
expression of Th is almost abolished and that of Dbh diminished.
In Gata3/ embryos, expression of
Phox2b, Mash1 and Dbh were initially normal, while
Th expression was already substantially reduced and that of
Gata2 virtually absent. The lack of Gata2 expression is
surprising, as Gata2 has been shown to be upstream of Gata3
in several systems. However, reciprocal crossregulations between
Gata2 and Gata3 have also been observed in the spinal cord
(Karunaratne et al., 2002) and
hindbrain (Craven et al.,
2004
). The strong reduction in Th expression at E10.5
suggests a role for Gata3 in the establishment of high-level
Th expression. Already at E11.5 the sympathetic ganglion size is
reduced and, in parallel, the number of apoptotic cells is strongly increased.
The continued cell death is thought to result in the rudimentary sympathetic
ganglia observed at E13.5.
These findings raise the question of how Gata2/3 functions in sympathetic precursor cells and why the cells die in the absence of Gata2/3. Gata2/3 factors could specifically control sympathetic neuron survival (but not differentiation) or, alternatively, control sympathetic neuron differentiation in a more general way. In the latter case, immature cells would be generated in Gata3/ ganglia that subsequently die since they are deficient in many properties, including survival signalling. We favour the latter possibility, since in gain-of-function experiments Gata2 acts as a differentiation factor rather than as a survival factor inducing the production of neurons in peripheral nerves, devoid of neurons during normal development.
The ectopic expression of Phox2a, Phox2b and Hand2 in
neural crest precursor cells elicits the generation of noradrenergic neurons
(Stanke et al., 1999;
Howard et al., 2000
). This is
explained by the strong crossregulations among these factors, resulting in the
induction of the complete network by each individual factor. Thus, it was
expected that Gata2 may be able to induce the corresponding set of
co-regulators required for noradrenergic and generic neuronal differentiation.
The present results do not support this possibility and reveal for Gata2 a
potential to control generic neuronal differentiation. This indicates that the
function of Gata2 is dependent on the interaction with co-regulators,
resulting in the induction of noradrenergic genes in the context of Mash1,
Phox2a/b and Hand2 in sympathetic precursors, while non-autonomic neurons are
generated in peripheral nerve precursors. Whereas overexpression of
Phox2a and Hand2 induce upstream members of the
transcriptional network involved in sympathetic neuron differentiation,
Gata2, perhaps as the most downstream factor, has only a very weak
crossregulating activity with respect to Phox2a, Hand2, Phox2b and
Cash1.
What is the reason for the generation of a small population of
noradrenergic, autonomic neurons and of some Th-positive cells devoid
of Scg10 in Gata2-infected nerves? The most likely
explanation is that peripheral nerve precursors represent a mixture of cells
at different stages of commitment and differentiation. Only in a minor
fraction of the cells Gata2 may be able to induce Phox2a and
additional upstream transcription factors that would elicit, together with
Gata2, noradrenergic neuron development. It should be noted that
peripheral nerve precursors are biased towards autonomic neuron
differentiation (White et al.,
2001). Th-positive, Scg10-negative cells might
be explained by the very low Cash1 expression in Gata2
overexpression experiments.
Gata function and the noradrenergic phenotype
In the autonomic nervous system, Mash1 and Phox2 transcription factors are
essential for the generation of both sympathetic and parasympathetic ganglion
neurons, i.e. functionally noradrenergic and cholinergic phenotypes (for the
most part). Therefore, additional regulators have to be hypothesized which
modify Phox2 and Mash1 action and are selectively expressed in noradrenergic
or cholinergic neurons. There is evidence that the bHLH transcription factor
Hand2 is such a factor: it is expressed selectively in sympathetic neurons and
capable, upon ectopic expression, of inducing adrenergic differentiation in
neural crest precursors and to maintain the normally transient Th
expression of parasympathetic ciliary neurons
(Müller and Rohrer,
2002). The present observations identify another such factor in
the form of Gata2/3, also absent from ciliary and sphenopalatine
parasympathetic ganglia, and which, in combination with Hand2 (and possibly in
direct interaction with it (Dai et al.,
2002
)) may contribute to the continued expression of Th
and Dbh in sympathetic neurons. However, investigation on a larger
scale shows that Gata2/3 function is not associated with
noradrenergic properties per se. Although Gata2 expression in the
parasympathetic cardiac, submandibular and Remak's ganglion
(Groves et al., 1995
)
correlates with the presence of noradrenergic gene expression, Gata2
and Gata3 are not expressed in the chick and mouse sphenopalatine
ganglion, also containing considerable numbers of neurons expressing Th and/or
Dbh (Fig. 10). It should be
mentioned in this context that variable aspects of noradrenergic traits are
expressed in cholinergic parasympathetic neurons, often transiently, and never
resulting in a functionally noradrenergic phenotype
(Grzanna and Coyle, 1978
;
Landis et al., 1987
;
Leblanc and Landis, 1989
;
Baluk and Gabella, 1990
;
Hardebo et al., 1992
).
Finally, Gata2/3 are absent in both chick and mouse from the major
noradrenergic centre of the brain, the locus coeruleus, and the development of
the locus coeruleus does not depend on Gata3. The selective function of
Gata2/3 in the development of noradrenergic sympathetic but not LC neurons
illustrates differences in the molecular control of the noradrenergic
phenotype in different lineages, after the initial, common dependence on Mash1
and Phox2a/b.
In conclusion, Gata2/3 have been identified as members of the group of Hand2-induced transcription factors that are essential for the generation and differentiation of sympathetic neurons. Among the sympathetic phenotypic traits that were tested to date, Gata2/Gata3 displays a preferential role in the expression of Th, a function that depends, however, on the presence of additional co-regulators present in the sympathetic neuronal lineage. It will be interesting to investigate whether Phox2a/b and Hand2 and/or unknown co-regulators are physically interacting with Gata2/3 and to identify the target genes controlled by Gata2/3 in the sympathetic lineage.
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ACKNOWLEDGMENTS |
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Footnotes |
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