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First published online 30 August 2006
doi: 10.1242/dev.02553
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1 Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba 305-8575,
Japan.
2 Department of Cell and Developmental Biology, University of Michigan, Ann
Arbor, Michigan 48109-0616, USA.
3 Department of Biochemistry, University of Calgary, Canada.
4 Department of Pathology and Laboratory Medicine, Children's Memorial Hospital,
Feinberg School of Medicine, Northwestern University, Chicago, IL 60614-3394,
USA.
5 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba
305-8575, Japan.
6 Laboratory Animal Resource Center, University of Tsukuba, Tsukuba 305-8575,
Japan.
* Author for correspondence (e-mail: engel{at}umich.edu)
Accepted 27 July 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Gata3, Chromaffin cells, Sympathetic neurons, Tyrosine hydroxylase, Dopamine ß-hydroxylase
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Howard, 2005). Those
progenitors subsequently migrate again to their final destinations: the
sympathetic ganglia (SG), the adrenal medulla (AM) and the extra-adrenal
chromaffin tissue. Transcription factors that mediate BMP signaling in
specifying the SA lineage have been identified and include the zinc-finger
protein, Gata3, the bHLH proteins Mash1 (Ascl1 - Mouse Genome Informatics) and
Hand2, as well as the closely related homeodomain transcription factors Phox2a
and Phox2b.
Gata3 encodes a transcription factor containing two steroid
hormone receptor-like zinc fingers that serve as its DNA-binding domain
(Yamamoto et al., 1990) that
binds most avidly to the consensus motif AGATCTTA
(Ko and Engel, 1993) and is
highly conserved among all six members (Gata1 to Gata6) of this multigene
family (Patient and McGhee,
2002). Gata3 is prominently expressed in the primary sympathetic
chain and persists during the development of sympathetic neurons, adrenal
chromaffin cells and para-aortic chromaffin cells [the organ of Zuckerkandl,
OZ, which degenerates in adult animals
(George et al., 1994;
Lakshmanan et al., 1999;
Lim et al., 2000)].
Gata3 homozygous mutants die at around E11
(Pandolfi et al., 1995) but
can be rescued by feeding heterozygous intercrossed dams with catecholamine
intermediates, demonstrating a central role for Gata3 in catecholamine
biosynthesis (Lim et al.,
2000).
In the SG of Gata3 mutants, normal expression of Phox2 and other
neuronal markers, but not Th or Dbh, was detected, suggesting that
Gata3 acts genetically downstream of Phox2 and is required
only for the expression of terminal noradrenergic traits. Loss- and
gain-of-function experiments examining Mash1, Hand2, Phox2a and
Phox2b indicated that they act before Gata3 in the SA regulatory
cascade (Goridis and Rohrer,
2002; Lim et al.,
2000). More-recent studies have implied that Gata3 may also be
essential for aspects of early sympathetic neuronal development
(Tsarovina et al., 2004).
However, the effect of Gata3 loss-of-function during adrenal
medullary development is largely undetermined.
Here we have examined the consequences of Gata3 deficiency on sympathetic
neuron and adrenal medullary chromaffin cell development, anticipating
possible revelation of new insights into Gata3 neuroendocrine function(s). We
show that Gata3 is essential not only for Th and Dbh
activation, but also for cell survival and differentiation of sympathetic
neurons and adrenal chromaffin cells. A human DBH (hDBH)
gene promoter was used to direct Gata3 expression specifically to the murine
SA system (Mercer et al.,
1991), and this significantly restores Th and Dbh deficiency in
Gata3 mutant mice, thereby overcoming the Gata3
mutation-induced embryonic lethality. In contrast to drug-rescued
Gata3 mutants, SA tissue-specific Gata3 complementation restored
nearly normal development of sympathetic neurons and neuroendocrine chromaffin
cells as well as of Mash1, Hand2 and Phox2b expression. A host of previous
studies suggested that Mash1, Hand2 and Phox2b function both genetically and
biochemically earlier than Gata3 in SA differentiation
(Tsarovina et al., 2004);
however, the synthesis of previous observations with these new data indicates
the more likely possibility that these SA lineage-restricted transcription
factors collaborate in a mutually interdependent cell-autonomous manner to
regulate the interactive genetic circuitry governing SA differentiation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) or eGFP gene
was inserted 3' to the 5.8 kb hDBH promoter
(Kapur et al., 1991;
Mercer et al., 1991). Both
expression constructs were co-microinjected into BDF1
(C57BL/6xDBA/2)-fertilized ova, and two independent transgenic lines
were recovered. Each line was mated with either heterozygotes of
Gata3 germline knockout (Gata3+/-)
(Pandolfi et al., 1995) or
nlslacZ knock-in (Gata3Z/+)
(van Doorninck et al., 1999)
to generate Gata3 heterozygotes that were hemizygous for the
transgene. Pharmacological rescue of Gata3 mutant embryos has been
described (Lim et al., 2000).
Animals were genotyped using PCR and/or Southern blotting. Primers used to
detect the hDBH-Gata3 transgene are shown in
Table 1.
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200 cells/embryo) using ISOGEN (Nippon Gene). First-strand
cDNA was synthesized from 0.5 µg of total RNA in a 20 µl reaction using
Superscript II (Invitrogen). Q-PCR was performed using 2xSYBR Green PCR
master mix (PE-Applied Biosystems), reverse-transcribed cDNA and gene-specific
primers according to the manufacturer's protocol in an ABI PRISM 7000 sequence
detector system (PE-Applied Biosystems). To quantify the amount of target mRNA
in the samples, a standard curve was prepared using a cDNA containing the
target transcript as the control. This procedure enabled standardization of
the initial mRNA content of cells relative to the amount of GAPDH mRNA. The
sequences of gene-specific primers used are listed in
Table 1. The data were recorded
as means±s.e.m. The statistical significance of differences among means
of several groups was determined by Student's t-test.
TUNEL and immunofluorescent analyses
Embryos (E10.5, E12.5, E13.5, E15.5, E18.5) or neonates were fixed
overnight in 4% PFA at 4°C and then processed for immunostaining as frozen
or paraffin-embedded sections. For immunofluorescence analysis, rabbit
anti-tyrosine hydroxylase (Pel-Freez Biological), rabbit anti-Phox2b (gift
from C. Goridis), rabbit anti-chromogranin A (Santa Cruz Biochemicals), rabbit
anti-SF1 (gift from G. Hammer), and rabbit anti-GFP (Molecular Probes)
antibodies were used. For co-staining of Th and GFP, monoclonal anti-Th
antibody (Sigma) was used. Co-localization of ß-galactosidase and GFP was
performed using a Cy3-conjugated rabbit anti-ß-galactosidase antibody
(K.C.L., unpublished) and rabbit anti-GFP antibody (Molecular Probes).
Fluorescence was visualized using a Leica DM inverted microscope. Separate
images were taken and merged using OpenLab software. Whole-mount X-gal
staining of embryos was performed as previously described
(Lakshmanan et al., 1999). For
detection of apoptosis, TUNEL assays were performed on 10 µm tissue
cryosections using an In Situ Cell Death Detection Kit (Roche Diagnostics)
according to the manufacturer's instructions.
Morphometric analysis
Meta Imaging Series 6.1 (Molecular Device) was used to quantify
morphometrically the surface area morphometrically and the anti-ß-gal,
anti-Th and anti-Phox2b immunoreactive areas, as well as the TUNEL-positive
cells in SA tissues. Measurements from at least four sections of bilateral
sympathetic ganglia or adrenal glands from at least two different wild-type
embryos of each genotype were arbitrarily set at 100%.
Electron microscopy
For electron microscopy, E18.5 adrenal glands were fixed overnight in 2.5%
gluteraldehyde in 0.1 M Sorenson's buffer (pH 7.4), washed with 0.1 M
Sorenson's buffer and then post fixed in 1.0% OsO4. After more
rinsing with 0.1 M Sorenson's buffer, tissues were stained `en-block' using 3%
uranyl acetate and processed for embedding in Epon. Ultrathin sections (75 nm)
were examined using a Philips CM-100 transmission electron microscope.
Flow cytometry
E18.5 adrenal glands were treated with 0.25% collagenase type B (Roche
Diagnostics) for 1 h at 37°C, and cells were then dissociated using
finetipped pipettes. After the cells had been filtered through a 35 µm
nylon mesh, they were resuspended in PBS containing 4% FCS. Determination of
ß-gal activity using FDG and subsequent incubations with mAb have been
described previously (Hendriks et al.,
1999). FACS analysis and sorting was carried out using FACS
VantageSE and CellQuest software (Becton Dickinson, San Jose, CA).
Phycoerythrin (PE)-conjugated CD45 antibody was purchased from BD pharmingen
(San Diego, CA). The isolated cells were resuspended in ISOGEN (Nippon Gene)
for RT-PCR analysis.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mercer et al.,
1991). In order to generate SA lineage-specific forced
co-expression of Gata3 and eGFP in transgenic animals, the hDBH
promoter was cloned upstream of both mouse Gata3 and eGFP DNAs
(Fig. 1A). Both constructs were
co-microinjected into fertilized ova to generate double transgenic animals.
The hDBH/Gata3 and hDBH/eGFP transgenes cosegregated through
multiple generations in two independent TghDBH-G3 lines,
demonstrating co-integration of the two expression constructs.
Immunofluorescent studies examining tissues from E18.5
Gata3Z/+:TghDBH-G3 embryos confirmed that
transgenic eGFP (Fig. 1D,G,J)
and lacZ expression [from a Gata3/lacZ knock-in allele
(Gata3 KI) (van Doorninck et al.,
1999); Fig. 1C,F,I]
were completely coincident (Fig.
1E,H,K) in the AM, the SG and the OZ. These data demonstrate that
the hDBH-directed transgenes accurately recapitulate endogenous
Gata3 SA expression.
To determine whether TghDBH-G3 could rescue the embryonic
lethality conferred by the Gata3 germline mutation,
Gata3Z/+:TghDBH-G3 and
Gata3+/- animals were intercrossed
(Fig. 1B). Only 15.3% (21/254)
of the Gata3Z/-:TghDBH-G3 mutants survived to
E18.5 and both transgenic lines did not differ in their ability to rescue
Gata3 mutation-induced midgestational lethality
(Fig. 1B and
Table 2), so only one line was
used for all subsequent experiments. We conclude that transgenic expression of
Gata3, when placed under the control of the hDBH promoter, rescued
Gata3-deficient mice from mid-embryonic demise. As with the
drug-rescued mutants (Lim et al.,
2000), Tg-rescued mutant embryos were similar in size to their
wild-type littermates, but additionally exhibited hypoplastic mandibles and
glandular aplasia (the parathyroid glands and thymuses) as well as inner ear
and kidney deficiencies (Fig.
2A-H; data not shown). These phenotypes are representative of many
of the same affected tissues and organs in individuals with GATA3
haploinsufficient HDR (hypoparathyroidism, sensorineural deafness and renal
dysplasia) syndrome (Van Esch et al.,
2000), underscoring the central role Gata3 plays in the
development of each of these organ systems.
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; Thomas et al.,
1995; Zhou and Palmiter,
1995). The cardiac wall thickness of the Tg-rescued Gata3
mutants was fully restored, suggesting that SA system-specific Gata3
complementation reconstituted the noradrenalin biosynthesis pathway, which in
turn leads to fully restored myocardial compaction in the mutants
(Fig. 2I,J).
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). In E10.5 Gata3 mutant embryos, Th expression is
significantly weaker in both the SG and NT
(Fig. 3C)
(Lim et al., 2000), suggesting
that both SG- and NT-specific Th expression may be under the regulatory
influence of Gata3. The hDBH promoter directs transgenic eGFP
expression in the SG and in the NT in E10.5 embryos
(Fig. 3D)
(Kapur et al., 1991;
Mercer et al., 1991). As
anticipated, Th immunoreactivity was significantly restored both in the SG and
NT of E10.5 Tg-rescued Gata3 null mutants
(Fig. 3E).
By stark contrast, Th expression was observed only in the SG of E12.5
wild-type embryos, and not in the NT (Fig.
3F) (Son et al.,
1996), whereas Th was fully suppressed in the E12.5 Gata3
mutant SG (Fig. 3G). Transgenic
eGFP expression was still observed weakly in the NT at E12.5
(Fig. 3H), although that also
gradually extinguished and was undetectable by E18.5 (data not shown)
(Kapur et al., 1991;
Mercer et al., 1991). As
anticipated, Th expression was restored in the SG, but not in the NT, of the
E12.5 Tg-rescued Gata3 mutant
(Fig. 3I). These results
demonstrate that SG-specific stable and NT-specific transient Gata3
complementation restored Th expression in both tissues, thus leading to
restoration of the catecholamine deficiency in the E10.5 Gata3
mutants. From E12.5 onwards, SA lineage-restricted restoration allows survival
of Gata3 mutant embryos to birth.
To evaluate SA development in older embryos, we examined whole-mount X-gal-stained E18.5 Gata3 knock-in embryos using the lacZ gene as a marker for the SA lineage. We observed that E18.5 drug-rescued Gata3Z/- mutants had significantly smaller thoracic paravertebral SG in comparison with Gata3Z/+ littermates (Fig. 4A,C). This conclusion was further substantiated when Hematoxylin and Eosin (HE)-stained histological sections of E18.5 embryos were examined. In drug-rescued Gata3 mutants, the thoracic paravertebral SG were reduced by 65% in area, with few visible neurons, when compared with that of wild-type littermates (Fig. 4D,F).
Phox2b is a paired/homeodomain transcription factor that has been shown to
play a key regulatory role in early SA differentiation in a common genetic
pathway upstream of Gata3 (Goridis and
Rohrer, 2002; Huber et al.,
2005; Pattyn et al.,
1999; Tsarovina et al.,
2004). As anticipated, in Gata3-deficient embryos, Phox2b staining
was evident in only a few surviving sympathetic neurons, and the
Phox2b-immunoreactive area was significantly reduced (by over 80%) in
comparison with that in wild-type littermates
(Fig. 4G,I,M). At the same
time, Th expression was virtually extinguished in the Gata3 mutant
sympathetic neurons (Fig.
4J,L,M).
In Tg-rescued Gata3 embryos, the size as well as anti-Phox2b and
anti-Th immunoreactivities of the paravertebral SG were concomitantly restored
to 70-80% of wild-type levels by E18.5
(Fig. 4B,E,H,K,M,N). These data
underscore the previous observations concluding that Gata3 is indispensable
for the differentiation of sympathetic neurons
(Tsarovina et al., 2004) and
that Th and Phox2b gene expression is suppressed in the (few) remaining viable
sympathetic neurons after Gata3 ablation. Restoration of Gata3
function in the mutants by transgenic complementation results in restoration
of reasonably normal differentiation of sympathetic neurons accompanied by the
recovery of Phox2b and Th expression.
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In the AM of Tg-rescued embryos, GFP and Th immunostaining were almost
completely coincident, indicating that Th immunoreactivity was restored by
transgenic Gata3 complementation in a cell-autonomous manner
(Fig. 5P,Q,R). Chromogranin A
(ChrA), a major soluble protein occupying the core of peptide hormone and
neurotransmitter secretory vesicles
(Mahata et al., 2003), was
markedly deficient in Gata3Z/- adrenal glands, indicating
that the few remaining adrenal chromaffin cells are bereft of functional
secretory vesicles (Fig. 5M,O).
As an independent measure of chromaffin cell differentiation, ChrA expression
was, like Phox2b and Th, largely restored in Tg-rescued Gata3 mutants
(Fig. 5N).
Chromaffin cells have distinctive ultrastructural features, most notably
large chromaffin granules that allow their unique identification as distinct
from sympathetic neurons (Coupland and
Tomlinson, 1989). Fig.
6A shows typical chromaffin cells, filled with numerous large
secretory granules (core diameter >100 nm), from an E18.5 wild-type adrenal
gland. By contrast, the adrenal gland of an E18.5 Gata3-/-
embryo harbors few cells that can be readily identified as chromaffin cells,
based on the presence of similar granules
(Fig. 6A,C). As anticipated, SA
tissue-specific transgenic Gata3 rescue restored chromaffin cell
ultrastructural characteristics (Fig.
6B). Taken together, these observations suggest that Gata3 is
indispensable for conferring the functional properties attributed to fully
differentiated chromaffin cells. Despite the fact that Gata3 mutant
embryos lack a compact AM, the size of the adrenal cortex (as measured by
examining Sf1 expression, a transcription factor exclusively expressed in
steroidogenic cells) (Luo et al.,
1994), is unaltered in the Gata3 mutants
(Fig. 6F). Sf1-negative
chromaffin cells were abundant in wild-type and Tg-rescued Gata3
mutants, whereas a clearly defined AM was not apparent in drug-rescued
Gata3 mutant embryos (Fig.
6D-F).
Reduced number of adrenal chromaffin progenitors in Gata3 mutant mice
We next asked whether Gata3-deficient chromaffin cells are able to
home to the adrenal gland anlagen during early embryogenesis. This was
addressed by following lacZ expression from the knock-in mutant
allele as a marker for the SA lineage cells. At E13.5, fewer
ß-gal+ cells were detected in the adrenal gland primordium of
homozygous mutant embryos in comparison with heterozygotes
(Fig. 7A,C).
ß-Gal+ cells clustered in the center of the AM in
Gata3 heterozygous mutants by E15.5, while Gata3-deficient
chromaffin cell number was clearly diminished and failed to aggregate normally
in the medullary region (Fig.
7D,F). Quantification of ß-gal+ cells in serial
sections showed that the chromaffin cell population in homozygous mutants
amounted to only 30% of heterozygous controls
(Fig. 7G). In Tg-rescued
Gata3 mutants, the chromaffin cell number was partially restored at
E13.5 and E15.5 of embryogenesis (Fig.
7B,E,G).
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Quantitative analysis supports a co-regulatory relationship among SA lineage control genes
To address the consequences of Gata3 deficiency on other SA cell-specific
transcription factors, we enriched lacZ+ E18.5 chromaffin
cells by flow cytometry using the Gata3 knock-in allele as a
chromaffin cell-specific marker. Hematopoietic or dead cells were eliminated
by gating out CD45+ and/or propidium-iodide (PI)+ cells.
The lacZ+CD45- cells constituted 4.06±1.14%
(Gata3Z/-; n=5), 5.43±0.005%
(Gata3Z/-:TgDBH-G3; n=5) or
8.02±1.48% (Gata3Z/+; n=5) of the E18.5
adrenal gland single cell suspension (Fig.
9A). The percent changes of fractionally recovered
lacZ+ cells is consistent with their fractional
representation observed in the X-gal-stained histological analyses of embryos
of the same genotypes (Fig.
7).
The purity of the flow-sorted chromaffin cells was assessed by monitoring
the expression of Sf1 mRNA, encoding an adrenal cortical
cell-specific nuclear receptor (Luo et
al., 1994). The lacZ+CD45- fraction was
largely devoid of Sf1, whereas the lacZ-CD45-
population contained abundant Sf1+ cells, indicating that adrenal
chromaffin cells were highly enriched in the
lacZ+CD45- fraction
(Fig. 9B).
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), is
more depleted than Phox2b, demonstrating an even stronger influence of Gata3
on Hand2 expression in Gata3-deficient chromaffin cells. Unexpectedly, we
found that Mash1 expression is elevated (approximately twofold) in purified
Gata3-deficient chromaffin cells, demonstrating that Gata3 (either
directly or indirectly) negatively regulates Mash1, the SA regulator
that is proposed to act earliest in this cascade.
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50% of haploid levels
(Fig. 9C), which might generate
either a hypomorphic or complete response if Gata3 indeed regulates those
other transcription factor genes. As anticipated, restoring even 25% of
wild-type Gata3 by breeding the TghDBH-G3 into the mutant
background created a strong hypomorphic allele, and significantly restored Th,
Dbh, Hand2 and Phox2b expression in those purified chromaffin cells, while
Mash1 levels reverted to almost the same level as in
Gata3Z/+ animals (Fig.
9C). These data clearly demonstrate that Th and Dbh expression as
well as the expression of putative regulators of Gata3 (Mash1, Phox2b
and Hand2) are under pronounced (direct or indirect) influence of Gata3 in
chromaffin cells. Importantly, Gata3 complementation in the mutant
neuroendocrine cells largely restores those transcription factor expression
levels. Taken together, these quantitative analyses are inconsistent with a
simple linear hierarchical model in which Mash1 activates
Phox2a and Hand2, while Phox2b activates
Phox2a, Hand2 and Gata3, which in concert activate
Th and Dbh, but instead indicate that Gata3 levels
significantly affect the expression of those `upstream' genes
(Goridis and Rohrer, 2002).
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Pata et al.,
1999). Gata3 has previously been shown to be involved in the
development of serotonergic neurons in the caudal raphe nuclei
(Pattyn et al., 2004;
van Doorninck et al., 1999),
in sensory otic neurons (Karis et al.,
2001; van der Wees et al.,
2004) and in the expression of the noradrenergic genes Th and Dbh
(Lim et al., 2000;
Tsarovina et al., 2004). Here,
we show that Gata3 is indispensable for the differentiation of adrenal
chromaffin cells as well as sympathetic neurons during murine embryogenesis.
Moreover, SA lineage-specific Gata3-expressing transgenic lines were able to
rescue developmental deficiencies of the sympathoadrenal system that are
attributable to Gata3 loss-of-function mutation.
Gata3 is thought to act as a `downstream' regulator in the molecular
pathway leading to SA differentiation
(Goridis and Rohrer, 2002). In
Gata3 mutants, sympathetic ganglia are formed and express Phox2 mRNA
and protein, but they exhibit dramatically reduced levels of Th and Dbh
(Lim et al., 2000).
Conversely, Phox2b-dependent expression of Gata3 has previously been
demonstrated in the sympathetic chain of Phox2b-null mutant mice
(Tsarovina et al., 2004).
Furthermore, chicken Gata2, which appears to be the functional counterpart of
murine Gata3 in the primary sympathetic chain, is expressed later than Mash1,
Phox2b, Hand2 and Phox2a during chick embryogenesis
(Tsarovina et al., 2004).
In contrast to our expectations, the present observations revealed that Gata3 plays a more complex role in SA differentiation than previous studies had indicated. The Gata3 homozygous mutants developed significantly smaller embryonic SG and AM throughout embryogenesis (E13.5 to E18.5), demonstrating that SA development was not simply delayed but permanently altered. There was increased apoptotic activity in Gata3 homozygous mutant adrenal glands from E13.5 onwards. This temporally and spatially restricted progressive cell death in SA tissues resulted in the accumulation of substantially fewer chromaffin cells by E18.5, demonstrating that Gata3 is essential for survival of adrenal chromaffin cells.
As previously reported, Th immunoreactivity was almost completely abolished
in Gata3-/- sympathetic ganglia and adrenal glands.
Meanwhile, only a fraction of viable sympathetic neurons and chromaffin cells
expressed (weak) Phox2b immunoreactivity in E18.5 Gata3-/-
mutants, whereas Phox2b is detected intensely in all sympathetic neurons and
adrenal chromaffin cells of control animals. This qualitative observation was
verified by Q-PCR as Gata3-/- chromaffin cells exhibited
modestly lower Phox2b (halved) and significantly diminished Hand2, Th and Dbh
mRNA accumulation. Consistent with these observations, previous studies
reported suppressed Hand2 expression in E10.5 Gata3 mutant
sympathetic ganglia (Tsarovina et al.,
2004), suggesting that the same pathways regulate noradrenalin
synthesis in all SA cells. Regulation of Hand2 by Gata factors has previously
been reported in various tissues. For example, Hand2 is prominently expressed
in the first branchial arch, which gives rise to lower jaw structures, and is
significantly reduced in the branchial arches of Gata3 mutants
(Ruest et al., 2004). In the
developing cardiac primordium, Hand2 is regulated by Gata4
(McFadden et al., 2000),
implying a potential regulatory function for Gata factors on Hand2 expression
during cardiogenesis.
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; Tsarovina et al.,
2004), and the observed de-repression of Mash1
transcription could be the causal event leading to differentiation failure in
Gata3-deficient chromaffin cells. The results presented here demonstrate that
Mash1, Phox2b and Hand2, all of which are putative upstream regulators of
Gata3 in early SA development, are under the control of Gata3 regulatory
influence in chromaffin cells.
Previous reports documented the transient expression of Th and Dbh in the
ventrolateral neural tube (NT) of mid-gestational embryos (E10.5-E13.5),
indicative of the transient noradrenergic cell lineage in this region
(Son et al., 1996;
Teitelman et al., 1981). It
was also reported that an hDBH promoter transgene faithfully
recapitulated the transient NT expression at this stage
(Kapur et al., 1991). As
anticipated, hDBH promoter-driven transgenic eGFP expression was
observed transiently in the NT of E10.5 and E12.5 transgenic rescued embryos.
Of potential significance here, Gata3 is also transiently expressed in the
ventral NT between E10.5 and E13, including expression in a subpopulation of
V2 interneurons (Smith et al.,
2002). Consistently, our observations reflected a significant
suppression of Th expression both in the SG and NT of E10.5 Gata3
mutant embryos and the restoration in both regions in the Tg-rescued
Gata3 mutants, suggesting that Gata3 directly or indirectly regulates
transient Th expression in the ventrolateral neural tube.
Several lines of evidence support the notion that sympathetic ganglia and
adrenal chromaffin cells share a common developmental origin
(Anderson, 1993;
Anderson et al., 1991;
Unsicker, 1993). Adrenal
chromaffin cells arise from a population of neural crest cells that initially
localize in the primary sympathetic chain of the embryo. When the primary
sympathoblasts re-migrate dorsolaterally to form the definitive sympathetic
ganglia, other progenitors migrate ventrally and then penetrate the medial
cranial end of the developing adrenal gland to become adrenal chromaffin cells
(Yamamoto et al., 2004). When
these neural crest derivatives colonize the adrenal anlagen, they become
associated with mesodermal cells that are destined to form the adrenal cortex.
In the environment of the adrenal gland, the ultimate development of these
precursor cells is thought to benefit from growth factors provided by the
developing cortex (e.g. IGF-1, FGF and glucocorticoids), and then be converted
into mature adrenalin-producing chromaffin cells
(Unsicker, 1993). The
experiments reported here show that the expression of Phox2b, Th and Dbh genes
and the sympathoadrenal development in the Gata3 mutants is
progressively suppressed until around the time of birth (E18.5). These
observations, when taken together with the observation that Gata3 is expressed
throughout SA cell development, suggest that Gata3 may play some role in the
initial specification of the common sympathoadrenal progenitor cell in the
primary sympathetic chain, but unquestionably demonstrate that Gata3 is
indispensable for successful execution of sympathetic neuronal and adrenal
chromaffin cell differentiation.
Previous reports have shown that Phox2b mutant animals are also
severely compromised in the number of adrenal chromaffin cells
(Huber et al., 2005;
Pattyn et al., 1999). This
observation suggests a common requirement for Phox2b and Gata3 in the survival
of sympathoadrenal progenitors in the adrenal medulla. Reduced Phox2b
expression in Gata3 mutant SA cells and the reported observation of
Phox2b-dependent Gata3 expression in Phox2b mutant raises the
possibility that there exists a reciprocal and mutually reinforcing
crossregulation by these transcription factors. Explicitly, these data are
inconsistent with the hypothesis that a simple genetic hierarchy exists
between these genes in the cascade of sympathoadrenal developmental events
(Goridis and Rohrer, 2002).
Unfortunately, the current literature sheds no additional light on whether
Gata3 directly or indirectly regulates the Hand2, Mash1 or
Phox2b genes, as SA lineage-specific transcriptional regulation of
these loci has not yet been reported. A neuroepithelial-specific proximal
promoter that regulates Phox2b transcription has been characterized,
but without specific reference to SA-restricted activity
(Samad et al., 2004).
Similarly, two in vitro studies have characterized the transcriptional
behavior of the Phox2a promoter in tissue culture cells
(Flora et al., 2001;
Hong et al., 2001) without
evidence that the regulation is SA lineage specific. Thus, the transcriptional
data characterizing the Phox2a and Phox2b (to date)
expression provide an inadequate foundation on which to base further
regulatory analysis.
Real-time quantitative RT-PCR demonstrated that transgenic Gata3 mRNA was
expressed at lower levels than the endogenous Gata3 mRNA in E10.5 embryo
caudal halves and in the E18.5 mutant adrenal gland; both transgenic lines
expressed comparable levels of transgene-derived Gata3 mRNA (data not shown).
Presumably, high level Gata3 expression in the SA system could result in
embryonic lethality in wild-type embryos as a consequence of overstimulation
of catecholamine synthesis, possibly preventing the establishment of highly
expressing transgenic lines. Alternatively, the nature of the hDBH
gene promoter may be intrinsically limited in its ability to express foreign
DNAs. The human Dbh gene is postulated to lie downstream of Gata3 in
the hierarchy of SA development as the onset of hDBH-directed
transgene expression occurs later than endogenous Gata3 expression
(George et al., 1994) and is
suppressed in the Gata3-null background (T.M., data not shown). A SA
system-specific enhancer of the Gata3 gene, which would completely
recapitulate endogenous Gata3 spatiotemporal expression, could be a more
appropriate element for directing full genetic complementation.
Taken together, the observations reported here show that Gata3 is essential for the development and survival of both sympathetic ganglia and adrenal chromaffin cells. These experiments are incompatible with a simple hierarchical regulatory cascade, but rather are more consistent with the hypothesis that Mash1, Phox2a/b, Hand2 proteins and Gata3 are individually required for mutually reinforcing activation or repression of one another. Whether these cell-autonomous, mutually reinforcing activities are direct or are mediated by intermediate effectors will be resolved only when the cis elements controlling the SA system-specific activity for each of these regulatory genes is defined. Finally, the hDBH-rescued Gata3 mutants should prove to be a useful tool to investigate currently cryptic Gata3 functions in many tissues (e.g. otic neurons, the thymus, parathyroid gland and kidneys) that display dysmorphic/amorphic phenotypes in the late gestational hDBH-Gata3-rescued animals.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Anderson, D. J. (1993). Molecular control of cell fate in the neural crest: the sympathoadrenal lineage. Annu. Rev. Neurosci. 16,129 -158.[Medline]
Anderson, D. J., Carnahan, J. F., Michelsohn, A. and Patterson, P. H. (1991). Antibody markers identify a common progenitor to sympathetic neurons and chromaffin cells in vivo and reveal the timing of commitment to neuronal differentiation in the sympathoadrenal lineage. J. Neurosci. 11,3507 -3519.[Abstract]
Coupland, R. E. and Tomlinson, A. (1989). The development and maturation of adrenal medullary chromaffin cells of the rat in vivo: a descriptive and quantitative study. Int. J. Dev. Neurosci. 7,419 -438.[CrossRef][Medline]
Flora, A., Lucchetti, H., Benfante, R., Goridis, C., Clementi,
F. and Fornasari, D. (2001). Sp proteins and Phox2b regulate
the expression of the human Phox2a gene. J. Neurosci.
21,7037
-7045.
George, K. M., Leonard, M. W., Roth, M. E., Lieuw, K. H.,
Kioussis, D., Grosveld, F. and Engel, J. D. (1994). Embryonic
expression and cloning of the murine GATA-3 gene.
Development 120,2673
-2686.
Goridis, C. and Rohrer, H. (2002). Specification of catecholaminergic and serotonergic neurons. Nat. Rev. Neurosci. 3,531 -541.[CrossRef][Medline]
Hendriks, R. W., Nawijn, M. C., Engel, J. D., van Doorninck, H., Grosveld, F. and Karis, A. (1999). Expression of the transcription factor GATA-3 is required for the development of the earliest T-cell progenitors and correlates with stages of cellular proliferation in the thymus. Eur. J. Immunol. 29,1912 -1918.[CrossRef][Medline]
Hong, S. J., Kim, C. H. and Kim, K. S. (2001). Structural and functional characterization of the 5' upstream promoter of the human Phox2a gene: possible direct transactivation by transcription factor Phox2b. J. Neurochem. 79,1225 -1236.[CrossRef][Medline]
Howard, M. J. (2005). Mechanisms and perspectives on differentiation of autonomic neurons. Dev. Biol. 277,271 -286.[CrossRef][Medline]
Huber, K., Bruhl, B., Guillemot, F., Olson, E. N., Ernsberger, U. and Unsicker, K. (2002). Development of chromaffin cells depends on MASH1 function. Development 129,4729 -4738.
Huber, K., Karch, N., Ernsberger, U., Goridis, C. and Unsicker, K. (2005). The role of Phox2B in chromaffin cell development. Dev. Biol. 279,501 -508.[CrossRef][Medline]
Kapur, R. P., Hoyle, G. W., Mercer, E. H., Brinster, R. L. and Palmiter, R. D. (1991). Some neuronal cell populations express human dopamine betahydroxylase-lacZ transgenes transiently during embryonic development. Neuron 7, 717-727.[CrossRef][Medline]
Karis, A., Pata, I., van Doorninck, J. H., Grosveld, F., de Zeeuw, C. I., de Caprona, D. and Fritzsch, B. (2001). Transcription factor GATA-3 alters pathway selection of olivocochlear neurons and affects morphogenesis of the ear. J. Comp. Neurol. 429,615 -630.[CrossRef][Medline]
Ko, L. J. and Engel, J. D. (1993). DNA-binding
specificities of the GATA transcription factor family. Mol. Cell.
Biol. 13,4011
-4022.
Ko, L. J., Yamamoto, M., Leonard, M. W., George, K. M., Ting, P.
and Engel, J. D. (1991). Murine and human T-lymphocyte GATA-3
factors mediate transcription through a cis-regulatory element within the
human T-cell receptor delta gene enhancer. Mol. Cell.
Biol. 11,2778
-2784.
Lakshmanan, G., Lieuw, K. H., Lim, K. C., Gu, Y., Grosveld, F.,
Engel, J. D. and Karis, A. (1999). Localization of distant
urogenital system-, central nervous system-, and endocardium-specific
transcriptional regulatory elements in the GATA-3 locus. Mol. Cell.
Biol. 19,1558
-1568.
Lim, K. C., Lakshmanan, G., Crawford, S. E., Gu, Y., Grosveld, F. and Engel, J. D. (2000). Gata3 loss leads to embryonic lethality due to noradrenaline deficiency of the sympathetic nervous system. Nat. Genet. 25,209 -212.[CrossRef][Medline]
Luo, X., Ikeda, Y. and Parker, K. L. (1994). A cell-specific nuclear receptor is essential for adrenal and gonadal development and sexual differentiation. Cell 77,481 -490.[CrossRef][Medline]
Mahata, S. K., Mahapatra, N. R., Mahata, M., Wang, T. C.,
Kennedy, B. P., Ziegler, M. G. and O'Connor, D. T. (2003).
Catecholamine secretory vesicle stimulus-transcription coupling in vivo.
Demonstration by a novel transgenic promoter/photoprotein reporter and
inhibition of secretion and transcription by the chromogranin A fragment
catestatin. J. Biol. Chem.
278,32058
-32067.
McFadden, D. G., Charite, J., Richardson, J. A., Srivastava, D., Firulli, A. B. and Olson, E. N. (2000). A GATA-dependent right ventricular enhancer controls dHAND transcription in the developing heart. Development 127,5331 -5341.[Abstract]
Mercer, E. H., Hoyle, G. W., Kapur, R. P., Brinster, R. L. and Palmiter, R. D. (1991). The dopamine beta-hydroxylase gene promoter directs expression of E. coli lacZ to sympathetic and other neurons in adult transgenic mice. Neuron 7, 703-716.[CrossRef][Medline]
Nardelli, J., Thiesson, D., Fujiwara, Y., Tsai, F. Y. and Orkin, S. H. (1999). Expression and genetic interaction of transcription factors GATA-2 and GATA-3 during development of the mouse central nervous system. Dev. Biol. 210,305 -321.[CrossRef][Medline]
Pandolfi, P. P., Roth, M. E., Karis, A., Leonard, M. W., Dzierzak, E., Grosveld, F. G., Engel, J. D. and Lindenbaum, M. H. (1995). Targeted disruption of the GATA3 gene causes severe abnormalities in the nervous system and in fetal liver haematopoiesis. Nat. Genet. 11,40 -44.[CrossRef][Medline]
Pata, I., Studer, M., van Doorninck, J. H., Briscoe, J., Kuuse, S., Engel, J. D., Grosveld, F. and Karis, A. (1999). The transcription factor GATA3 is a downstream effector of Hoxb1 specification in rhombomere 4. Development 126,5523 -5531.[Abstract]
Patient, R. K. and McGhee, J. D. (2002). The GATA family (vertebrates and invertebrates). Curr. Opin. Genet. Dev. 12,416 -422.[CrossRef][Medline]
Pattyn, A., Morin, X., Cremer, H., Goridis, C. and Brunet, J. F. (1999). The homeobox gene Phox2b is essential for the development of autonomic neural crest derivatives. Nature 399,366 -370.[CrossRef][Medline]
Pattyn, A., Simplicio, N., van Doorninck, J. H., Goridis, C., Guillemot, F. and Brunet, J. F. (2004). Ascl1/Mash1 is required for the development of central serotonergic neurons. Nat. Neurosci. 7,589 -595.[CrossRef][Medline]
Ruest, L. B., Xiang, X., Lim, K. C., Levi, G. and Clouthier, D.
E. (2004). Endothelin-A receptor-dependent and -independent
signaling pathways in establishing mandibular identity.
Development 131,4413
-4423.
Samad, O. A., Geisen, M. J., Caronia, G., Varlet, I.,
Zappavigna, V., Ericson, J., Goridis, C. and Rijli, F. M.
(2004). Integration of anteroposterior and dorsoventral
regulation of Phox2b transcription in cranial motoneuron progenitors by
homeodomain proteins. Development
131,4071
-4083.
Smith, E., Hargrave, M., Yamada, T., Begley, C. G. and Little, M. H. (2002). Coexpression of SCL and GATA3 in the V2 interneurons of the developing mouse spinal cord. Dev. Dyn. 224,231 -237.[CrossRef][Medline]
Son, J. H., Min, N. and Joh, T. H. (1996). Early ontogeny of catecholaminergic cell lineage in brain and peripheral neurons monitored by tyrosine hydroxylase-lacZ transgene. Brain Res. Mol. Brain Res. 36,300 -308.[Medline]
Teitelman, G., Gershon, M. D., Rothman, T. P., Joh, T. H. and Reis, D. J. (1981). Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats. Dev. Biol. 86,348 -355.[CrossRef][Medline]
Thomas, S. A., Matsumoto, A. M. and Palmiter, R. D. (1995). Noradrenaline is essential for mouse fetal development. Nature 374,643 -646.[CrossRef][Medline]
Tsarovina, K., Pattyn, A., Stubbusch, J., Muller, F., van der
Wees, J., Schneider, C., Brunet, J. F. and Rohrer, H. (2004).
Essential role of Gata transcription factors in sympathetic neuron
development. Development
131,4775
-4786.
Unsicker, K. (1993). The chromaffin cell: paradigm in cell, developmental and growth factor biology. J. Anat. 183,207 -221.
van der Wees, J., van Looij, M. A., de Ruiter, M. M., Elias, H., van der Burg, H., Liem, S. S., Kurek, D., Engel, J. D., Karis, A., van Zanten, B. G. et al. (2004). Hearing loss following Gata3 haploinsufficiency is caused by cochlear disorder. Neurobiol. Dis. 16,169 -178.[CrossRef][Medline]
van Doorninck, J. H., van Der Wees, J., Karis, A., Goedknegt,
E., Engel, J. D., Coesmans, M., Rutteman, M., Grosveld, F. and De Zeeuw, C.
I. (1999). GATA-3 is involved in the development of
serotonergic neurons in the caudal raphe nuclei. J.
Neurosci. 19,RC12
.
Van Esch, H., Groenen, P., Nesbit, M. A., Schuffenhauer, S., Lichtner, P., Vanderlinden, G., Harding, B., Beetz, R., Bilous, R. W., Holdaway, I. et al. (2000). GATA3 haplo-insufficiency causes human HDR syndrome. Nature 406,419 -422.[CrossRef][Medline]
Yamamoto, M., Ko, L. J., Leonard, M. W., Beug, H., Orkin, S. H.
and Engel, J. D. (1990). Activity and tissue-specific
expression of the transcription factor NF-E1 [GATA] multigene family.
Genes Dev. 4,1650
-1662.
Yamamoto, M., Yanai, R. and Arishima, K. (2004). Study of migration of neural crest cells to adrenal medulla by three-dimensional reconstruction. J. Vet. Med. Sci. 66,635 -641.[CrossRef][Medline]
Zhou, Q. Y. and Palmiter, R. D. (1995). Dopamine-deficient mice are severely hypoactive, adipsic, and aphagic. Cell 83,1197 -1209.[CrossRef][Medline]
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