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First published online 27 June 2007
doi: 10.1242/dev.000141
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1 Division of Developmental Neurobiology, MRC National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, UK.
2 Laboratory for Embryonic Induction, RIKEN Center for Developmental Biology,
2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe, Hyogo 650-0047, Japan.
3 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center
and University of Cincinnati College of Medicine, 3333 Burnet Avenue,
Cincinnati, OH 45229-3039, USA.
Author for correspondence (e-mail:
sang{at}nimr.mrc.ac.uk)
Accepted 24 May 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Forkhead genes, Cell fate specification, Differentiation, Dopaminergic, Midbrain, Gene dosage
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Barzilai and Melamed,
2003). In addition, mDA neurons regulate reward-based behaviours
and have been implicated in schizophrenia and attention-deficit disorders
(Tzschentke and Schmidt,
2000).
mDA neurons arise from neural progenitors in the ventral midline, probably
corresponding to the floor plate region. The generation of mDA neurons from a
neural progenitor cell can be divided into three distinct phases based on the
expression of molecular markers and the state of differentiation: regional and
neuronal specification (phase I), early (phase II) and late differentiation
(phase III) (reviewed by Smits et al.,
2006; Ang, 2006).
During phase I, sonic hedgehog (Shh), fibroblast growth factor 8 (Fgf8), Wnt1
and other extrinsic factors control regional and neuronal identity by
regulating the expression of Otx2 (Ye et
al., 1998; Puelles et al.,
2004; Vernay et al.,
2005; Prakash et al.,
2006), Lmx1a (Andersson et al.,
2006a), Lmx1b (Smidt et al.,
2000), Msx1 and Msx2 (Andersson
et al., 2006a), neurogenin 2 (Ngn2; also known as Neurog2 - Mouse
Genome Informatics) and Mash1 (also known as Ascl1) (Anderson et al., 2006b;
Kele et al., 2006).
Subsequently these progenitors undergo an early differentiation step to
generate immature mDA neurons expressing Ngn2, Nurr1 (also known as Nr4a2),
Lmx1a, Lmx1b, engrailed 1 and engrailed 2 (En1/2) and ßIII-tubulin (Tuj1;
also known as Tubb3) (reviewed by Wallen
and Perlmann, 2003; Prakash
and Wurst, 2006; Smits et al.,
2006; Ang, 2006).
Immature mDA neurons then further differentiate into mature DA neurons that
express tyrosine hydroxylase (TH) and aromatic L-amino acid
decarboxylase (AADC), in addition to the other markers mentioned above in
immature neurons. Loss-of-function studies in mice have revealed roles for
Nurr1 (Zetterstrom et al.,
1997), Lmx1b (Smidt et al.,
2000), Pitx3 (Hwang et al.,
2003; Nunes et al.,
2003; van den Munckhof et al.,
2003) and En1/2 (Simon et al.,
2001; Alberi et al.,
2004) in late differentiation and maintenance of these
neurons.
Less is known about the role of transcription factors in specification and
early differentiation of mDA progenitors. The homeodomain protein Otx2
regulates mDA specification by inhibiting the expression of Nkx2.2 homeodomain
protein (Puelles et al., 2004;
Prakash et al., 2006) and
regulating the expression of the proneural basic helix-loop-helix genes
Ngn2 and Mash1. Ngn2 has also been shown to be the key
proneural gene regulating neurogenesis of mDA progenitors in the midbrain;
Mash1 is partially able to compensate for Ngn2 function
(Andersson et al., 2006b;
Kele et al., 2006). Msx genes,
Msx1 and Msx2, are able to prematurely turn on Ngn2
expression and neurogenesis when expressed earlier in mice carrying a
Shh enhancer-Msx1 transgene
(Andersson et al., 2006a). In
addition, there is a reduction of mDA neurons in Msx1-/-
mutant embryos. The LIM homeodomain protein Lmx1a has been shown to be
necessary and sufficient for the generation of mDA neurons in the ventral
midbrain of chick embryos and can promote the generation of mDA neurons from
mouse ES cells treated with Fgf8 and Shh
(Andersson et al., 2006a).
However, Lmx1a is unable to generate mDA neurons in the absence of Shh or from
dorsal midbrain progenitors, indicating that other molecules that contribute
to the specification of mDA progenitors remain to be discovered.
In search of other factors regulating development of mDA neurons, we have
focused on Foxa1 and Foxa2 (Foxa1/2), members of the forkhead/winged helix
transcription factors, because Foxa1/2, but not Foxa3, are expressed in a wide
domain in ventral midbrain progenitors (Ang
et al., 1993; Monaghan et al.,
1993; Sasaki and Hogan,
1993). Foxa2-null mutants die at embryonic day (E) 9.5
owing to gastrulation defects (Ang and
Rossant, 1994; Weinstein et
al., 1994). These mutant embryos lack a node and notochord;
consequently, floor plate development and dorsal-ventral patterning of the
neural tube are also disrupted owing to loss of Shh signalling from the
notochord (Ang and Rossant,
1994). In addition, when Foxa2 is expressed ectopically in the
dorsal mid-hindbrain region of En1-Foxa2 transgenic mouse embryos,
ectopic floor plate-like regions develop in the dorsal hindbrain, suggesting a
role for Foxa2 in the development of the floor plate in mice
(Sasaki and Hogan, 1994);
however, in these studies it is unclear whether Foxa2 or Shh, a downstream
target, is responsible for this effect. A recent study on zebrafish
foxa2 (also known as monorail and axial), has also
demonstrated a role for Foxa2 in floor plate differentiation and specification
of oligodendrocytes, serotonergic raphe neurons and cranial motoneurons in
zebrafish embryos (Norton et al.,
2005). In contrast to the early function of Foxa2,
Foxa1-null embryos do not show early patterning defects, but Foxa1 is
required for the regulation of pancreatic islet genes essential for glucose
homeostasis (Kaestner et al.,
1999). Foxa1/2 also regulate metabolic responses during
gluconeogenesis (Zhang et al.,
2005), and are required for the development of endodermal organs
including liver (Lee et al.,
2005), pancreas (Lantz et al.,
2004), prostate (Gao et al.,
2003) and lung (Wan et al.,
2004).
In this study, we show that Foxa1/2 are continually required during multiple phases of mDA neuron development. Our data, from loss-of-function studies in mice, demonstrate that Foxa1/2 regulate the extent of neurogenesis in mDA progenitors by positively regulating Ngn2 expression. Subsequently, Foxa1/2 are required for the expression of Nurr1 and En1 in immature mDA neurons, and for the expression of TH and AADC in mature mDA neurons during early and late differentiation of mDA neurons. Genetic evidence indicates that these functions require different gene dosages of Foxa1/2. Altogether, these data demonstrate that Foxa1/2 regulate multiple phases of mDA neuron development in a dosage-dependent manner.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) with
animals homozygous for the Foxa2flox allele
(Hallonet et al., 2002).
Nestin-Cre/+;Foxa2flox/+ male animals were then
mated to Foxa2flox/flox females.
Nestin-Cre/+;Foxa2flox/+ males were also mated
with Foxa1lacZ/+ females
(Kaestner et al., 1999) to
generate
Nestin-Cre/+;Foxa2flox/+;Foxa1lacZ/+
male animals. Simultaneously, Foxa2flox/flox males were
mated with Foxa1lacZ/+ females to generate
Foxa2flox/+;Foxa1lacZ/+
female animals. Foxa2cko;Foxa1lacZ/lacZ mutants were
obtained by crossing
Nestin-Cre/+;Foxa2flox/+;Foxa1lacZ/+
males with Foxa2flox/flox;Foxa1lacZ/+ females.
The Foxa2flox and Foxa1lacZ alleles
were detected by PCR (Hallonet et al.,
2002; Kaestner et al.,
1999), and the Cre transgene was detected using a pair of
primers and PCR conditions as described by Indra et al.
(Indra et al., 1999).
Shhflox/flox (Lewis et
al., 2001) animals were purchased from Jackson Laboratories. At
all times, animals were handled according to the Society of Neuroscience
Policy on the Use of Animals in Neuroscience Research, as well as the European
Communities Council Directive.
Immunohistochemistry of brain sections
Embryos or dissected brains were fixed overnight at 4°C in 4%
paraformaldehyde in 0.1 M PBS and cryoprotected with 30% sucrose in PBS,
embedded in OCT compound (VWR International, Poole, UK) and sectioned on a
cryostat (CM3050S; Leica, Nussloch, Germany). A minimum of three control and
three mutant embryos were analysed, except where indicated. For
immunohistochemistry, sections were incubated overnight at 4°C with the
appropriate primary antibody diluted in 1% BSA in PBS. Sections were then
extensively washed in PBS containing 0.1% BSA and incubated 1 hour at room
temperature with a secondary antibody conjugated with a fluorochrome
(Molecular Probes). Sections were then washed and mounted in Vectashield
H-1000 (Vector Laboratories, Burlingame, CA). The following primary antibodies
were used: rabbit anti-Foxa2 (1:1000)
(Filosa et al., 1997), goat
anti-Foxa2 (1:100; sc-6554, Santa Cruz), guinea-pig anti-Foxa1 (1:500)
(Wan et al., 2004), rabbit
anti-Lmx1a (1:1000; gift of M. German, University of California San Francisco
Diabetes Center, San Francisco, CA), guinea-pig anti-Lmx1b (1:20,000; gift of
T. Müller and C. Birchmeier, Max-Delbruck-Center for Molecular Medicine,
Berlin, Germany), mouse anti-Nkx2.2 [1:5; 74.5A5, Developmental Studies
Hybridoma Bank (DSHB)], rabbit anti-Nkx6.1 (1:1500; gift of Martyn Goulding,
Salk Institute, La Jolla, CA), rabbit anti-TH (1:200; AB152, Chemicon), mouse
anti-En1 (1:40; 4G11, DSHB), rabbit anti-AADC (1:200; ab3905), mouse anti-Ngn2
(1:5; gift of D. J. Anderson, California Institute of Technology, Pasadena,
CA), rabbit anti-Shh (1:100; sc-9024, Santa Cruz), mouse anti-Brn3a (1:100;
sc-8429, Santa Cruz), mouse anti-Islet 1 (1:20; 40.2D6, DSHB), rabbit
anti-Nurr1 (1:200; sc-990, Santa Cruz), and mouse anti-Tuj1+
(1:1000; SDL.3D10, Sigma). In some cases, staining of nuclei with Toto-3
iodide (1:1000, Molecular Probes) was performed. All images were collected on
a Leica TCS SP2 confocal microscope and processed with Adobe Photoshop 7.0
software (Adobe Systems, San Jose, CA). Quantitative immunocytochemical data
represent mean±s.d. for cell counts of half the ventral midbrain in
consecutive sections through the entire midbrain, every 60 µm. The
percentage of Nkx2.2+ cells in Lmx1a+ mDA progenitor
domain=average percentage of midbrain Lmx1a+ Nkx2.2+
cells/number of midbrain Lmx1a+ cells of mutant embryo 1 and mutant
embryo 2=(24/1398x100+32/1871x100)/2.
Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick-end labelling (TUNEL)
Cryostat sections were washed once for 5 minutes in PBS containing 0.1%
Triton X-100, permeabilised in ice-cold 0.01 M citrate buffer and 0.1% Triton
X-100 for 2 minutes, and washed again in PBS containing 0.1% Triton X-100. The
enzymatic reaction was then performed at 37°C according to the
manufacturer's protocol (1 684 795; Roche Diagnostics, Mannheim, Germany).
BrdU labelling
Pregnant females were injected intraperitoneally with a solution of BrdU
(B-5002, Sigma; at 10 mg/ml in physiological serum) at 100 µg per g of body
weight and sacrificed 1 hour later. Proliferating cells were revealed by
immunohistochemistry on frozen sections.
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), Foxa1/2 were found to be expressed in ventricular zone
progenitors in the ventral midbrain of mouse embryos at E10.5 and E12.5
(Fig. 1A-F). Foxa1/2 were
expressed in all midbrain basal and floor plate progenitors at these stages,
except for a few cells around the alar/basal boundary that only expressed
Foxa2 (see Fig. S1 in the supplementary material). Co-expression of Foxa1/2
with Lmx1a confirmed that both Foxa proteins are expressed in mDA progenitors
at E10.5 (Fig. 1A,B). Foxa1/2
were also expressed in Nurr1+
(Fig. 1C,D) immature and
TH+ mature mDA neurons (Fig.
1E,F) and Brn3a+ (also known as Pou4f1 - Mouse Genome
Informatics) red nuclei neurons, but not in Islet 1+ (Isl1)
oculomotor (see Fig. S1 in the supplementary material) neurons in the ventral
midbrain at E12.5. In summary, Foxa1/2 proteins are expressed in all ventral
midbrain progenitors and some postmitotic neurons, including all stages of mDA
cells, suggesting possible roles in regulating their specification and
differentiation. A summary of the expression of Foxa1/2 proteins and other
markers of mDA cells used in this study is provided in
Fig. 1G.
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), was used to inactivate Foxa2 in CNS progenitors from E10.5
onwards. Analysis of Foxa2 expression by immunohistochemistry revealed that
loss of Foxa2 protein in Nestin-Cre/+;Foxa2flox/flox
(referred to henceforth as Foxa2cko) embryos began at E10.5. In
Foxa2cko embryos, Foxa2 was only detected in some cells within the
floor plate in the midbrain at E10.5, and by E12.5 all Foxa2 expression was
missing from the midbrain (Fig.
1H-H''',I-I'''). This timing of deletion of Foxa2 by
Nestin-Cre correlates well with the timing of activation of
ß-galactosidase expression in Nestin-Cre/+;R26Rflox/+
embryos (Vernay et al., 2005).
Hence, Foxa2cko embryos allow us to study the functions of Foxa2 in
mDA progenitors during neurogenesis, which occurs mainly between E10.5 and
E13.5.
Foxa1/2 positively regulate the expression of Ngn2 in mDA progenitors
mDA progenitors are distinguishable from other progenitors in the ventral
midbrain by their co-expression of Lmx1a/b at E10.5. Lmx1a and Lmx1b were
found to be similarly expressed in mDA progenitors at E10.5 and E12.5,
indicating that these markers are properly specified in these progenitors in
Foxa2flox/flox (control), Foxa2cko,
Foxa1lacZ/lacZ (henceforth referred to as
Foxa1-/-) and Foxa1-/-;Foxa2cko
double-mutant embryos (Fig.
2A-D,A'-D'). Msx1, a homeodomain protein, is also
normally expressed in mDA progenitors in wild-type and all mutant embryos
(Andersson et al., 2006a) (data
not shown).
Nkx homeodomain proteins, Nkx2.2 and Nkx6.1 (also known as Nkx2-2 and
Nkx6-1, respectively - Mouse Genome Informatics), were found to be expressed
in more-dorsal midbrain progenitors, and their expression patterns were not
modified in Foxa1/2 single mutants
(Fig. 2E-G,E'-G')
at E10.5 and E12.5. However, Foxa1-/-;Foxa2cko double
mutants showed a ventral expansion of Nkx2.2 at E10.5. This expansion extended
further into the Lmx1a+ mDA progenitor domain
(Fig. 2L), as 1.7% of
Lmx1a+ mDA progenitors expressed Nkx2.2 in the ventral midbrain of
Foxa1-/-;Foxa2cko double-mutant embryos at E12.5 (see
Materials and methods). By contrast, Lmx1a+ Nkx2.2+
cells were not found in control and single Foxa1/2 mutant embryos at
E12.5 (Fig. 2I-K). Nkx6.1
expression in midbrain basal plate progenitors immediately adjacent to mDA
progenitors was also reduced in Foxa1-/-;Foxa2cko double
mutants at both stages (Fig.
2H,H'). Since Foxa2 has previously been shown to regulate
the expression of Shh and Shh positively regulates Nkx2.2 expression
(Pabst et al., 2000), we next
determined whether Nkx2.2 expression is still dependent on Shh at E10.5. We
therefore examined the expression of Nkx6.1 and Nkx2.2 in Nestin-
Cre/+;Shhflox/flox (referred to as Shhcko) mutant
embryos that lacked Shh from E10.5 onwards
(Fig. 3A,E and data not shown).
Normal expression of Nkx2.2 and Nkx6.1 was observed in Shhcko
(Fig. 3G) compared with control
embryos (Fig. 3C). In addition,
Foxa1/2, Nurr1 and TH were similarly expressed in mDA cells in Shhcko
(Fig. 3F,H) and control
(Fig. 3B,D) embryos, suggesting
normal development of mDA progenitors and neurons in Shhcko embryos
at E12.5. Altogether, these results suggest that Foxa1/2 act cooperatively to
regulate the ventral limit of Nkx2.2 expression in the midbrain of mouse
embryos from E10.5 onwards. This role of Foxa1/2 is independent of Shh
signalling.
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;
Kele et al., 2006). We
therefore examined the expression of Ngn2 in Foxa1 and Foxa2
single- and double-mutant embryos at E12.5. Similar expression of Ngn2 was
observed in Foxa1-/-, Foxa2cko and control embryos (data
not shown). By contrast, a 57% reduction in the number of cells expressing
Ngn2 (mutant, 114±4; control, 264±9; n=3) was observed
in the Lmx1a+ mDA progenitor domain in
Foxa1-/-;Foxa2cko double mutant compared with control
embryos (Fig. 4A-D). These
results indicate that Ngn2 expression is dramatically reduced only when both
Foxa1 and Foxa2 are removed, suggesting that
Foxa1-/-;Foxa2cko double mutants may also show defects in
neuronal differentiation. Altogether, these results demonstrate that whereas
the majority of mDA progenitors acquire regional identity based on their
expression of Lmx1a, Lmx1b and Msx1, neuronal specification is compromised
because there is a 57% decrease in the number of mDA progenitors expressing
Ngn2.
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In summary, analyses of Foxa1/2 single and double-mutant embryos revealed that Foxa1/2 proteins are required during multiple phases of mDA neuronal development. In single mutants, there is a block in the late differentiation phase, with a reduction in the number of mature mDA neurons. Foxa1-/-;Foxa2cko double-mutant embryos exhibited a more severe phenotype than Foxa1/2 single mutants because neuronal specification and early differentiation of mDA progenitors were affected, leading to an almost complete loss of mDA neurons.
Dosage-dependent functions of Foxa1/2 in mDA cells
To better characterise similar functions of Foxa1/2 in mDA cells,
we compared the expression of Nurr1 and TH by double-antibody labelling
experiments in the ventral midbrain of mutant embryos carrying 0-4 copies of
Foxa1/2 genes in their genome at E12.5, E15.5 and E18.5. Reducing the
dose of Foxa1/2 resulted in a proportionate decrease in the number of
Nurr1+ TH+ mature mDA neurons in mouse embryos at E12.5
(Fig. 6A and data not shown),
indicating that Foxa1/2 regulate differentiation of these neurons in
a dose-dependent manner. Interestingly, a progressive rescue in the number of
TH+ mature mDA neurons was observed in all
Foxa1/2 single and double mutants except in
Foxa1-/-;Foxa2cko double mutants at E15.5 and E18.5,
indicating that a single copy of Foxa1 or Foxa2 is required
for this recovery (Fig. 6B,C
and data not shown). It is noteworthy that Foxa2cko,
Foxa1+/-;Foxa2flox/flox and
Foxa1-/-;Foxa2flox/+ embryos only showed
partial rescue of Nurr1+ TH+ mDA neurons even at E18.5
(Fig. 6C and data not shown).
As these mutant embryos do not survive beyond E18.5, the status of mDA neurons
cannot be determined at later stages. Importantly,
Foxa1+/-;Foxa2flox/+ double-heterozygous
embryos showed a similar reduction in the number of mature mDA neurons as
Foxa1-/- and Foxa2cko single mutants at E12.5
(Fig. 6). This reduction was
fully recovered in the double-heterozygous mutant embryos at E18.5
(Fig. 6). Altogether, these
data indicate that Foxa1 and Foxa2 are functionally capable of compensating
for each other in regulating the development of mDA neurons.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Foxa1/2 regulate Ngn2 expression and consequently neurogenesis in mDA progenitors
Since Ngn2 has previously been shown to regulate neuronal differentiation
of mDA progenitors, the partial inhibition of neuronal differentiation is
likely to be due to reduced Ngn2 expression in Foxa1/2
double-homozygous mutant embryos. How Foxa1/2 regulates Ngn2 expression at the
molecular level remains to be determined. The partial effects on Ngn2
expression could be due to the perdurance of Foxa2 protein in progenitors in
mutant embryos at the beginning of mDA neurogenesis around E10.5 and/or
alternative redundant mechanisms for regulating these genes in these
progenitors. Support for the latter hypothesis comes from the findings that
Lmx1a and Otx2 are positive regulators of Ngn2 expression
(Vernay et al., 2005;
Andersson et al., 2006b), and
Otx2 is also required for the repression of Nkx2.2 expression in mDA
progenitors (Puelles et al.,
2004; Vernay et al.,
2005; Prakash et al.,
2006).
Our studies also showed that there is abnormal expression of Nkx2.2 in a
small percentage of Lmx1a+ mDA progenitors, suggesting that Foxa1/2
might be required to prevent Nkx2.2 expression in these cells. However, the
consequence of this abnormal expression in mDA progenitors remains to be
determined. Previous studies have suggested that abnormal expression of Nkx2.2
in mDA progenitors may result in these progenitors acquiring a serotonergic
fate (Puelles et al., 2004;
Prakash et al., 2006). Since
Foxa2 is also required for the generation of serotonergic neurons in the
hindbrain (J. Jacob, A.L.M.F., C. Milton, F. Prin, P. Pia, W.L., A. Gavalas,
S.-L.A. and J. Briscoe, unpublished) it is unlikely that serotonergic neurons
are generated in the midbrain of Foxa1-/-;Foxa2cko
double-mutant embryos.
Foxa1/2 regulate the acquisition of dopaminergic properties in immature and mature midbrain neurons
Analyses of Foxa1/2 double and single mutants also revealed
essential roles for Foxa1/2 in early and late differentiation phases of mDA
neurons, respectively. Specifically, Foxa1/2 are required for the expression
of Nurr1 and En1 in immature mDA neurons and for the expression of AADC and TH
in mature mDA neurons. Altogether, these results indicate that Foxa1/2
regulate distinct genes at different phases of mDA neuron development. It is
noteworthy that the neuron promoter of human AADC contains a FOXA2-binding
site that overlaps with a consensus binding site for POU-domain transcription
factors, including BRN2 (also known as POU3F2)
(Raynal et al., 1998). Brn2 is
widely expressed in midbrain progenitors and neurons at E12.5 (our unpublished
results) and hence is available to cooperate with Foxa1/2 proteins. Whether
AADC is a direct target of the combined activities of Foxa2 and a POU-domain
transcription factor requires further studies.
Foxa1/2 regulate mDA neuron development in a dosage-dependent manner
Analyses of embryos with different copy numbers of Foxa1/2
indicated a dose-dependent requirement for these genes during the
differentiation of mDA neurons. Foxa1/2 are required for
specification and early and late differentiation steps of mDA cells; however,
Foxa1/2 appear to be required at progressively higher doses as mDA
cells differentiate. Loss of two copies of Foxa1/2 resulted in a late
differentiation defect, whereas an early differentiation defect was only
observed when three or four copies of Foxa1/2 genes were removed.
Moreover, a specification defect was only observed in embryos lacking all four
copies of Foxa1/2. These results suggest that higher doses of
Foxa1/2 are required at progressively later differentiation phases
and raise the intriguing possibility that the timing and duration of mDA
neuron differentiation might be regulated by the concentration of Foxa1/2 in
these cells.
One mechanism that could explain the requirement for higher doses of
Foxa1/2 for late rather than early differentiation targets is
differences in affinity of binding sites. For example, if late differentiation
target genes of Foxa1/2 have lower affinity binding sites, they might require
a higher dose of Foxa1/2 transcription factors for their activation. Such a
mechanism has already been shown to operate in the regulation of early and
late target genes of PHA-4, the C. elegans homologue of Foxa, during
pharyngeal development (Gaudet and Mango,
2002). Sequences that bind PHA-4 with high affinity in vitro are
typically found in promoters of genes expressed early during pharyngeal
development, whereas low-affinity sites are restricted to late promoters in
C. elegans. By analogy with PHA-4, the binding site affinity for
Foxa1/2 proteins might thus be a crucial determinant of gene expression in mDA
neurons. However, it is also likely that additional factors function in
combination with Foxa1/2 for temporal control of gene expression at different
stages. Future work will concentrate on identifying molecular targets for
Foxa1/2 in order to provide further understanding of molecular mechanisms of
gene regulation by Foxa1/2 within the CNS. These experiments will also
determine whether Foxa1/2 have identical or overlapping molecular targets.
Given their important regulatory roles in mDA neuron development, further
understanding of the molecular mechanisms of action of Foxa1/2 is likely to
facilitate attempts to differentiate mDA neurons from stem cells.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/15/2761/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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