1 Department of Biomedical Science, University of Sheffield, Firth Court,
Western Bank, Sheffield S10 2TN, UK
2 National Cancer Institute, National Institute of Health, MD, 20892, USA
* Authors for correspondence (e-mail: k.ohyama{at}sheffield.ac.uk and m.placzek{at}sheffield.ac.uk)
Accepted 19 September 2005
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SUMMARY |
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Key words: Shh, Bmp7, Chick, Dopamine, Hypothalamus
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Introduction |
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The link between inductive signalling, transcription factor expression and
neuronal fate is particularly well understood in the developing spinal cord
(Edlund and Jessell, 1999;
Briscoe and Ericson, 2001
;
Marquardt and Pfaff, 2001
;
Jessell, 2002
). By contrast,
far less is known of these events within the hypothalamus, a major component
of the ventral diencephalon. Thus, despite their key role in mediating
homeostasis in the adult, little is known about the differentiation of
hypothalamic neurons. Studies in zebrafish embryos have suggested that ambient
levels of Wnt activity determine early hypothalamic character
(Kapsimali et al., 2004
).
However, the source, nature and mechanism of action of signals required to
induce and specify neuronal identities within the hypothalamic anlage remains
largely unclear. The tuberal (mid) hypothalamus is generally accepted to be
induced by underlying prechordal mesoderm, a structure that is likely to
initiate hypothalamic neuronal induction
(Muenke and Beachy, 2000
;
Kiecker and Niehrs, 2001
;
Wilson and Houart, 2004
); but
to date, there has been little systematic analysis of hypothalamic neuronal
differentiation in response to prechordal mesoderm-derived signals.
Sonic hedgehog (Shh) and bone morphogenetic proteins (Bmps) are expressed
within prechordal mesoderm (Patten and
Placzek, 2000) and interact to control development of ventral-most
cells within the tuberal hypothalamus (Dale
et al., 1997
; Dale et al.,
1999
), cells that initially share an origin with the anterior
floor plate (Patten et al.,
2003
; Placzek and Briscoe,
2005
). Whether Shh and Bmps similarly act to establish
hypothalamic neuronal identity is unclear. Hedgehog signalling is required
cell-autonomously for the differentiation of some characteristics of
hypothalamic neurons (Mathieu et al.,
2002
; Wilson and Houart,
2004
), but is not sufficient to promote all aspects of
hypothalamic neuronal character. Overexpression of Hedgehog proteins does not,
for example, lead to the ectopic expression of the homeodomain (HD)
transcription factor Nkx2.1 (Rohr et al.,
2001
; Wilson and Houart,
2004
). As yet, no study has examined whether Bmp signalling
contributes to the differentiation of hypothalamic neurons, nor established
whether, and how, Shh and Bmps may cooperate to direct hypothalamic neuronal
fate.
Here, we perform experiments in chick embryos to analyse the differentiation of tuberal hypothalamic neurons. Our studies identify a set of progenitor cells that give rise to hypothalamic dopaminergic (DA) neurons that transiently co-express tyrosine hydroxylase (Th) and the hypothalamic regional markers Nkx2.1 and Msx1/2. We show that sequential Shh and Bmp signals from the prechordal mesoderm control the neurotransmitter identity and regional characteristics of these cells, the two signals required in a temporally distinct sequence. The induction of DA identity is initiated by Shh signalling; it occurs independently of, and precedes, induction of the hypothalamic regional markers Nkx2.1 and Msx1/2. Bmp7 acts on cells that are ventralised by Shh to induce these regional hypothalamic markers, and can elicit such hypothalamic characteristics in late-differentiating or postmitotic cells. In ovo electroporation studies reveal that Bmp7 and Shh cooperate to specify hypothalamic DA neuronal identity in a manner that depends on the transcriptional repressor Six3. Finally, we demonstrate that the combined action of Shh and Bmp7 can direct mouse embryonic stem (ES)-derived neural progenitor cells to a hypothalamic DA fate.
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Materials and methods |
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In situ hybridisation
Embryos and explants were processed for in situ hybridisation as described
previously (Dale et al., 1999).
The following template DNAs were used to generate digoxigenin-labelled
antisense RNA probes: pcvhh encoding chick Shh (linearised with SalI
and transcribed with SP6 RNA polymerase); pBH2 encoding chick Bmp7 (linearised
with XhoI and transcribed with T3 RNA polymerase); and psix3 encoding
chick Six3 (linearised with XhoI and transcribed with T3 RNA
polymerase). As controls, sense RNA probes were used.
Tissue dissection and explant culture
All embryos were staged according to Hamburger-Hamilton
(Hamburger-Hamilton, 1951) and
dissected in cold L15 medium (Gibco-BRL). Explant cultures were performed in
collagen gels according to published techniques
(Patten et al., 2003
). Based
upon fate-mapping analyses (Dale et al.,
1999
; Patten et al.,
2003
), prospective hypothalamic tissue was dissected out after
Dispase treatment (1 mg/ml in L15 medium at room temperature, 5-15 minutes).
For co-culture of hypothalamus and prechordal mesoderm, both dissected tissues
were recombined in vitro. Explants were cultured for 2-3 days for the analysis
of progenitor cells, 5-7 days for DA neurons and 13 days for the analysis of
DßH expression (onset of culture designated as day 0). In BrdU labelling
experiments, explants were incubated with 10 µM BrdU on day 1, 2, 3, 4, 5
or 6.
Real-time PCR analysis
Real-time quantitative RT-PCR analysis was performed using ABI Prism 7700
sequence detection system (Applied Biosystems). RNA in each sample was
standardised using ß-actin amplification as an internal control. Primers
used were: Shh, forward primer 5'-CGGCTTCGACTGGGTCTACT-3'
and reverse primer 5'-CGCTGCCACTGAGTTTTCTG-3'; the Taqman probe,
5'-CGAGTCCAAGGCGCACATCCACR-3' (labelled with the reporter dye FAM
on the 5' nucleotide and the quenching dye TAMRA on the 3'
nucleotide); ß-actin forward primer,
5'-GGTCATCACCATTGGCAATG-3' and reverse primer,
5'-CCCAAGAAAGATGGCTGGAA-3'; the Taqman fluorogenic probe,
5'-TTCAGGTGCCCCGAGGCCCT-3' labelled with the reporter dye VIC on
the 5' nucleotide and the quenching dye TAMRA on the 3'
nucleotide. Relative quantification of Shh mRNA was calculated using the
comparative Ct method.
DiI labelling
Chick embryos were incubated to stage 15. The lipophilic carbocyanine dye,
1, 1-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine
perchlorate (DiI) (Molecular Probes) was injected into the germinal zone of
the lateral tuberal hypothalamus, targeting Shh+ cells. Embryos were either
fixed immediately, or cultured to E6 and fixed in 4% paraformaldehyde (PFA),
then analysed by immunolabelling for Shh and Th.
Proteins
Transient transfection of 293T cells were performed to obtain supernatants
containing Shh, Bmp7 and chordin. The following plasmids were used: SHH-N IRES
GFP, encoding the N terminus of Shh and green fluorescent protein (GFP);
pdMb7, encoding a chimaera of dorsalin (pre-pro region) and chick Bmp7, with a
Myc epitope inserted at the junction (gift of T. Jessell); and
pMT11HA.1-chordin, encoding chick chordin with an HA epitope. Shh activity was
evaluated by examining Nkx2.2 and Pax6 expression after exposure of chick
lateral neural plate (LNP) explants. Shh-containing culture supernatant
(1x) showed an activity equivalent to 3 nM Shh. In blocking experiments,
anti-Shh IgG (20 µg/ml) was used as previously described
(Ericson et al., 1996).
Bead implantation
Affigel beads (Pharmacia Biotech) were incubated with transfected culture
supernatant, prepared as described above, at 4°C overnight. After a brief
PBS wash, beads were implanted and embryos developed further.
Electroporation
Electroporation of HH stage 10-11 chick embryos was performed in ovo.
Expression plasmids [either the repressor or the activator form of Six 3
(RDSix3, ADSix3) (Kobayashi et al.,
2002)] were electroporated into the lateral mesencephalon.
Efficiency of gene introduction was confirmed in separate experiments by
co-electroporating a red fluorescent protein (RFP) construct. After
electroporation, mesencephalic lateral neural tube was dissected and cultured,
with factors, in collagen gels.
Differentiation of hypothalamic DA neurons using mouse ES cells-derived neural progenitor cells
Maintenance of undifferentiated ES cells (CCE), embryoid body (EB)
formation, selection of nestin-positive cells, and neuronal differentiation
were performed as described (Okabe et al.,
1996; Lee et al.,
2000
). Nestin-positive cells were cultured in the presence of Shh
and Bmp7, Shh alone or Bmp7 alone for 8 days. In control cultures, supernatant
produced by 293T cells transfected with control plasmid DNA, or no DNA, was
added to the culture medium. Differentiation was initiated by withdrawal of
basic Fgf and the signalling molecules, and supported through addition of
ascorbic acid (200 µM, Sigma). Medium was changed every 2 days. After a
further 8 days in culture, cells were fixed with 4% PFA and processed for
immunolabelling.
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Results |
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We examined whether a defined class of neurons arise from progenitor cells
within the lateral tuberal hypothalamus. In rodents, A12 tubero-infundibular
dopaminergic (DA) neurons arise from the ventral lobe of the tuberal
hypothalamus (Altman and Bayer
1978; Daikoku et al.,
1986
). Similarly, we find that tyrosine hydroxylase
(Th)-immunoreactive neurons appear in the lateral tuberal hypothalamus at
embryonic day 5 (E5) (Fig.
2A,A',B,B' and data not shown). These neurons do not
co-express dopamine ß hydroxylase (DßH), defining them as
dopaminergic, not noradrenergic. To test whether DA neurons arise from
progenitor cells within the Nkx2.1+/Shh+ domain, we fate-mapped the
Nkx2.1+/Shh+ germinal zone of the lateral tuberal hypothalamus. At E6, cells
that had been DiI-labelled at stage 15
(Fig. 2C,D) continued to reside
within the Shh+ territory (Fig.
2E). Furthermore, many Th+ cells were labelled with DiI
(Fig. 2F, arrows). Thus, many
tuberal DA neurons originate in the lateral tuberal hypothalamus and
differentiate in situ.
|
Prechordal mesoderm triggers the induction of hypothalamic DA neurons
Previously, we have found that prechordal mesoderm governs ventral tuberal
hypothalamic cell identity (Dale et al.,
1997), but no study has yet examined whether prechordal mesoderm
is sufficient to trigger the differentiation of hypothalamic neurons. To test
this, we first examined whether prechordal mesoderm could induce Nkx2.1+/Shh+
cells. In stage 4-5 lateral neural plate (LNP) explants (blue square in
Fig. 4) cultured alone for 2
days, no cells expressed Nkx2.1 or Shh
(Fig. 3A), although Lim1/2,
Pax6 and Pax7 were detected (Fig.
3B-D). By contrast, co-culture of prechordal mesoderm with LNP
explants induced Nkx2.1+/Shh+ cells in the neural explant
(Fig. 3E; data not shown), many
of which co-expressed Lim1 (Fig.
3F). Nkx2.1+/Shh+ and Nkx2.1+/Lim1+ cells were induced some
distance from the prechordal mesoderm, whereas cells expressing Nkx2.1 alone
were induced immediately adjacently (brackets in
Fig. 3E,F). These latter cells
co-expressed Foxa2 and Bmp7, which transiently mark ventral tuberal
hypothalamic cells (Dale et al.,
1997
; Dale et al.,
1999
) (Fig.
3G,H,O). This shows that prechordal mesoderm triggers the
induction of ventral tuberal hypothalamic cells in immediately adjacent
tissue, and the induction of Nkx2.1+/Shh+ cells more distantly. To address
whether signals from prechordal mesoderm can directly trigger the induction of
Nkx2.1+/Shh+ cells, LNP explants were cultured at a distance from prechordal
mesoderm. In this contact-independent context
(Tanabe et al., 1995
),
Nkx2.1+/Shh+ cells that co-expressed Lim1 were induced
(Fig. 3I,J) but Foxa2+/Bmp7+
ventral tuberal hypothalamic cells were not detected
(Fig. 3K,L).
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Progressive specification of hypothalamic characteristics
To address when different aspects of hypothalamic cell identity are
specified, we analysed cell differentiation in pHyp explants isolated at
progressive stages of development (Fig.
4A,F,K). Stage 4 pHyp explant cultures express Pax6 and Lim1
(Fig. 4B,C) but not Nkx2.1,
Nkx2.2 or Shh (Fig. 4B-E).
Stage 5 pHyp explants express Pax6, Lim1, Shh and Nkx2.2, but not Nkx2.1
(Fig. 4G-J). Stage 6 pHyp
explants express Nkx2.1, and as in vivo, many Nkx2.1+ cells co-express Shh and
Lim1 (Fig. 4L-O). In addition,
an upregulation of Msx is initiated in Nkx2.1+ cells
(Fig. 2L;
Fig. 4O inset). Together, these
analyses show a progressive specification of lateral tuberal hypothalamic
cells; first Shh+/Lim1+ cells are specified; subsequently, Nkx2.1/Msx
expression is upregulated in Shh+/Lim1+ progenitors.
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Shh is required, but not sufficient, to induce Nkx2.1+/Shh+ cells and hypothalamic DA neurons
The prechordal mesoderm expresses Shh at stage 4-5
(Marti et al., 1995;
Dale et al., 1999
;
Patten et al., 2003
). To
examine the role of Shh in hypothalamic neuronal differentiation, we exposed
LNP explants to Shh. After 2 days, Shh+ cells that co-expressed Lim1/2 and
Six3 were detected, but none co-expressed Nkx2.1, suggesting that Shh
signalling leads to a partial, but incomplete, specification of lateral
tuberal hypothalamic cells. Similarly, after 6 days, Th+ neurons were
generated, but they did not co-express Nkx2.1 or Msx
(Fig. 5C, blue arrowheads; data
not shown). To exclude the possibility that prechordal mesoderm-mediated
induction of Nkx2.1+/Msx+ hypothalamic DA neurons is mediated by particularly
high levels of Shh that are not achieved in our in vitro experiments, we
measured levels of Shh mRNA in prechordal mesoderm by real-time
quantitative RT-PCR. No significant increase in Shh was detected
within prechordal mesoderm at stage 5, when Nkx2.1 is not specified, and stage
6-7, when Nkx2.1 is specified (Fig.
5D).
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Cooperative action of Shh and Bmp7 in the differentiation of hypothalamic DA neurons in vitro
Previous analyses have shown that Bmp7 is expressed by prechordal mesoderm
from stage 5-6 (Dale et al.,
1999; Vesque et al.,
2000
), and have suggested that it is required for the induction of
Nkx2.1 in ventral tuberal hypothalamic cells
(Dale et al., 1997
). These
findings, together with the correlation between Bmp7 signalling and Msx
expression observed in other tissues (Lee
and Jessell, 1999
), prompted us to ask whether Bmp7 plays a key
role in hypothalamic neuronal identity, acting with Shh to induce Nkx2.1+/Msx+
hypothalamic DA neurons. Incubation of LNP explants for 2 days in the presence
of both Shh and Bmp7 induced Nkx2.1+/Shh+ cells
(Fig. 5G), many of which
co-expressed Lim1 (Fig. 5H).
After 5-7 days, Nkx2.1+/Msx+ DA neurons were generated
(Fig. 5I and inset). Neither
cell type was generated in LNP explants exposed to Bmp7 alone (data not
shown). These experiments show that Bmp7 can cooperate with Shh to induce
Nkx2.1+/Shh+ cells and Nkx2.1+/Msx+ hypothalamic DA neurons. However, the LNP
shows a restricted competence to differentiate with hypothalamic character:
Shh and Bmp7 could induce hypothalamic cells in LNP taken anterior to Hensen's
node, but not in LNP from more posterior regions (purple square in
Fig. 4F; data not shown).
To test the requirement for Bmp7 in hypothalamic DA neuronal specification,
conjugate explants of prechordal mesoderm-pHyp were incubated with chordin, a
Bmp inhibitor (Piccolo et al.,
1996). In 2-day cultures exposed to chordin, Shh+ cells were
detected, but there was a dramatic decrease in the number of Nkx2.1+/Shh+
cells (compare Fig. 5E with
5J). Likewise, in 6-day cultures, Th+ cells were detected, but no
Nkx2.1+ hypothalamic DA neurons differentiated
(Fig. 5L; compare with
Fig. 3N). These cells do not
default to A13 thalamic, to A8-A10 mesencephalic DA or to hindbrain
noradrenergic fates (not shown), suggesting they are immature hypothalamic
neurons that have initiated an incomplete differentiation programme.
Bmp7 can induce hypothalamic regional markers in late-differentiating/postmitotic cells that are ventralised by Shh signalling
A remaining issue is the mechanism of co-operation of Shh and Bmp7. Shh and
Bmp7 signalling could induce distinct characteristics of progenitors that,
together, specify hypothalamic identity. Alternatively, Bmp7 could induce
hypothalamic character in cells that are ventralised by Shh signalling. To
distinguish these possibilities, we asked whether there is a temporally
distinct requirement for the two signalling molecules in hypothalamic DA
neuronal specification by exposing explants only transiently to either Shh or
Bmp7, and altering the order of their addition.
We first asked how transient exposure of explants to Shh or Bmp7 alone affects cell fate. In anterior LNP (aLNP) explants (Fig. 4F, blue square) exposed transiently to Shh, a downregulation of Pax6 and Pax7, and a concomitant upregulation of Shh was observed (Fig. 5M: compare with Fig. 3A; data not shown). However, no expression of Nkx2.1 was detected (Fig. 5M, inset). In aLNP explants exposed to Bmp7, Pax6, Pax7 and Shh were unaffected (data not shown), Msx was upregulated (Fig. 5N), but Nkx2.1 was not (data not shown). Thus, a transient addition of Shh and Bmp7 is sufficient to alter cell fate within aLNP, but neither alone can upregulate Nkx2.1, nor induce Nkx2.1+/Msx+ hypothalamic cells.
aLNP explants were next transiently exposed to Bmp7 under conditions in which Msx was upregulated. Subsequently, Shh was added to the cultures. Under these conditions, Pax6 and Pax7 were downregulated, and Shh was upregulated; transiently, Shh+/Msx+ cells were detected (not shown). However, although after 7 days, Th+ neurons were detected, Nkx2.1+/Msx+/Th+ neurons were not (Fig. 5O; data not shown). Thus, Bmp7 and Shh do not appear to operate in independent parallel pathways, mediating distinct characteristics that together induce hypothalamic identity.
By contrast, in explants exposed transiently to Shh and then to Bmp7, Nkx2.1+/Msx+/Th+ neurons were detected (Fig. 5P; data not shown), suggesting that Bmp7 can induce hypothalamic characteristics in cells that are ventralised by Shh signalling. Both sustained and transient exposure of explants to Bmp7 resulted in the appearance of hypothalamic DA neurons (Fig. 5P,Q). Remarkably, addition of Bmp7 on days 1-2 or 5-6 of culture resulted in the differentiation of equivalent numbers of Nkx2.1+/Th+ neurons (Fig. 5P-R). Only when Bmp7 was added on day 6 were no Nkx2.1+/Th+ neurons detected (Fig. 5S).
The ability of Bmp7 to induce Nkx2.1 when added to day 5 cultures led us to examine whether Bmp7 signalling can act directly on differentiating or postmitotic DA neurons to induce Nkx2.1 in the neurons themselves. To address this, we examined when Th+ neurons become postmitotic in pHyp explants. Stage 5 or stage 7 pHyp explants were cultured and BrdU added to parallel cultures on successive days. When explants were incubated with BrdU on days 2 or 3, BrdU+/Th+ neurons were detected on day 7 (Fig. 5T, inset; not shown). By contrast, when explants were incubated with BrdU on day 5, Th+ neurons were detected, but none of these co-labelled with BrdU (Fig. 5T). Despite the fact that Th+ neurons are differentiating or postmitotic by day 5, addition of Bmp7 to stage 5 pHyp explants on day 5 resulted in the appearance of Nkx2.1+/Th+ neurons on day 7 (Fig. 5U). Together, these data indicates that Bmp7 can act on differentiating or postmitotic DA neurons to induce hypothalamic regional identity.
Bmp7 induces Nkx2.1+/Shh+ cells in vivo in a Six3-dependent manner
We tested the requirement in vivo for Bmp7 for the induction of
Nkx2.1+/Shh+ cells. Beads soaked with chordin were implanted adjacent to the
prospective ventral tuberal hypothalamus of stage 5 embryos, and embryos
developed until stage 15 (Fig.
6). In embryos exposed unilaterally to chordin, Nkx2.1+ expression
was abolished from the neural tube ipsilateral to the bead
(Fig. 6A,B), showing that Bmp7
is necessary for the induction of Nkx2.1+/Shh+ cells in vivo.
To determine whether Shh and Bmp7 can induce ectopic Nkx2.1+/Shh+ cells in vivo, beads soaked in Shh and Bmp7 were placed adjacent to the lateral anterior neural plate of stage 5 embryos, and embryos developed until stage 15. In control embryos, Nkx2.1+/Shh+ cells are restricted to the anterior ventral, and the lateral tuberal, hypothalamus, but are not found elsewhere (Fig. 1A, Fig. 6C,D). By contrast, in embryos exposed to Shh and Bmp7, ectopic Nkx2.1+/Shh+ cells are detected (Fig. 6D,D'). Beads soaked in either Bmp7 alone or Shh alone did not induce Nkx2.1+/Shh+ cells (data not shown).
|
To test directly whether Six3 acts as a competence factor for Shh and Bmp7 to induce hypothalamic cells, we electroporated a Six3 repressor expression construct, RD Six3, into the mesencephalon in ovo. Electroporated lateral neural tube cells were immediately explanted, and a subset examined to establish that exogenously introduced Six3 could be detected (Fig. 6E,F). The remaining explants were exposed in vitro to Shh and Bmp7. After a 7-day culture, we detected many Nkx2.1+/Th+ DA neurons in RD Six3-electroporated tissue (Fig. 6G). By contrast, in tissue electroporated either with a control vector, or with an activator form of Six3 (AD Six3), and then exposed to Shh and Bmp7, Th+ cells appeared, but none co-expressed Nkx2.1 (Fig. 6H,I). These neurons co-expressed En1 and Lmx, suggestive of mesencephalic DA character (Fig. 6J,K). Together, these data suggest that Shh and Bmp7 induce hypothalamic fate in a Six3-dependent manner, and show that repressor activity is required for Six3 function.
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Discussion |
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At early stages, Shh ventralises prospective lateral tuberal hypothalamic
progenitors, upregulating expression of Shh itself in Lim1+ progenitors and
specifying DA neuronal generation. Incubation of prechordal mesoderm/pHyp
explants with anti-Shh IgG results in a failure to specify Shh or Th;
conversely, Shh is sufficient to downregulate Pax6 and Pax7, and to specify
Shh+/Lim1+ progenitor cells and Th+ neurons in LNP explants. A number of lines
of evidence suggest that induction of Shh and Th occurs through the Shh
signalling pathway. Shh signalling components, including Gli1 and Gli2 are
expressed in lateral tuberal hypothalamic progenitor cells (M.P.,
unpublished), and Gli2-null mice lack Shh expression in the lateral
tuberal hypothalamus (Matise et al.,
1998). Similarly, the Th promoter region has binding sites for
Gli1/Gli2 (Schimmel et al.,
1999
) and Th+ DA neurons fail to differentiate in
Gli2-null mice (Matise et al.,
1998
).
By contrast, late aspects of hypothalamic regional identity, evidenced by the upregulation of Nkx2.1 and Msx in cells that are specified as DA neurons, is imposed by Bmp signalling. Neither Shh nor Bmp7 is sufficient to induce Nkx2.1 in aLNP explants but addition of Shh and Bmp7 results in the robust induction of Nkx2.1+/Msx+ hypothalamic DA neurons. Conversely, addition of chordin to prechordal mesoderm/pHyp recombinates prevents prechordal mesoderm from inducing hypothalamic DA neurons.
A number of lines of evidence show that Bmp7 imposes hypothalamic identity
on cells that are ventralised by Shh signalling. The transient exposure of
progenitor cells first to Bmp7 and then to Shh results in the induction of Msx
and Shh, with some progenitor cells co-expressing Msx/Shh. However, these
cells do not differentiate into hypothalamic Nkx2.1+/Msx+ DA neurons. This
argues against a model in which Bmp7 and Shh signalling function in parallel
to induce distinct characteristics that, together, specify hypothalamic
identity. Instead our data indicate that Bmp7 acts on cells that are
ventralised by Shh to promote the hypothalamic regional identity of DA
neurons. First, Nkx2.2 and Shh+/Lim1+ progenitor cells that require Shh
signalling alone are specified before Nkx2.1+/Shh+/Lim1+ cells that require
both Shh and Bmp7. Second, Bmp7 can induce the expression of Nkx2.1/Msx in
either cells that are about to exit the cell cycle, or in postmitotic neurons
that are committed to a DA neuronal fate. Although our experiments do not
indicate whether the action of Bmp7 occurs exclusively at this stage, these
results add to the growing body of evidence that signalling molecules that act
early in embryogenesis to govern the developmental potential of neural
progenitor cells can elicit regional changes in neural identity
postmitotically (Livet et al.,
2002; William et al.,
2003
; Sockanathan et al.,
2003
). Finally, in vivo, the onset of expression of Bmp7 in
prechordal mesoderm occurs some hours after that of Shh
(Dale et al., 1999
;
Vesque et al., 2000
;
Patten et al., 2003
).
Our studies show that Shh signalling plays a dual role in the
differentiation of hypothalamic DA neurons, controlling neurotransmitter
phenotype and acting with Bmp7 to control regional character. This is distinct
from the role of Shh signalling in the differentiation of midbrain DA neurons:
analysis of Lmx1b-deficient mice reveals that two independent signalling
pathways govern midbrain DA neuronal differentiation, a Shh-dependent pathway
that controls neurotransmitter phenotype and a Shh-independent region-specific
pathway (Sakurada et al.,
1999; Smidt et al.,
2000
). Together, these results suggest that Shh controls
pan-dopaminergic character, but that region-specific character is controlled
through complex pathways that either cooperate with Shh, as shown here, or
operate independently of Shh.
Co-operative signalling by Bmp7 and Shh
In the posterior CNS, Bmps at the dorsal epidermal ectoderm and roof plate
oppose and constrain the expression and actions of Shh emanating from ventral
tissues (Lee and Jessell,
1999; Liem et al.,
2000
; Patten and Placzek,
2002
). By contrast, in this study we show that Msx/2, a read-out
of active Bmp signalling, is expressed in the Shh-expressing lateral tuberal
hypothalamus. This unusual expression of Msx within a Shh-expressing region
suggests the existence of a molecular mechanism to prevent Shh downregulating
in the presence of active Bmp signalling. How this mechanism operates remains
unproven, but a likely transcriptional candidate is Nkx2.1. Our studies show
that Nkx2.1 is induced by Bmp7 signalling, and show a correlation in Nkx2.1
expression and the maintenance of Shh expression. In support of this
interpretation, Nkx2.1-null embryos lack Shh expression in the
hypothalamus (Sussel et al.,
1999
). As our data shows that Shh expression in hypothalamic
explants precedes that of Nkx2.1, these results together support a
cell-autonomous role for Nkx2.1 in the maintenance of Shh expression. Further
support for a role for HD factors in regulating Shh expression derives through
a recent dissection of the Shh enhancer element, SFPE2, which reveals that a
HD site is required for Shh transcription in the posterior spinal cord
(Jeong and Epstein, 2003
).
Thus, our studies suggest that Bmp signalling may play a dual role in the
hypothalamus, inducing regional identity and maintaining Shh expression.
Shh and Bmp7 act with Six3 to establish hypothalamic identity
Our data suggest that Shh and Bmp7 can suffice to promote hypothalamic DA
neurons, but only in tissue that has acquired an earlier competence. Blockade
of Bmp7 signalling abolishes the ability of prechordal mesoderm to induce
Nkx2.1+/Th+ neurons in pHyp explants. Under these conditions, the prechordal
mesoderm induces Th+ neurons, but these do not adopt characteristics of DA
neurons from other regions. Such Th+ neurons do not default to A13 thalamic,
A8-A10 mesencephalic DA fates or to a hindbrain noradrenergic fate. Instead,
our data suggest that these display the characteristics of immature
hypothalamic neurons that have initiated an incomplete differentiation
programme, as evidenced through their continued expression of Lim1 and
Six3.
Previous studies in mice have suggested that Six3 not only marks
hypothalamic cells (Bovolenta et al.,
1998), but is required for hypothalamic cell differentiation:
Nkx2.1 expression is absent in the hypothalamus of Six3-null mice
(Lagutin et al., 2003
). Our
studies support and extend these findings, showing that Shh and Bmp7 induce
hypothalamic neuronal identity only in the presence of Six3 repressor
activity. First, the ability of Shh and Bmp7 to induce Nkx2.1+/Shh+ cells and
Nkx2.1+/Th+ neurons correlates, in vivo and in vitro, with Six3 expression.
Second, ES cell-derived neural cells that express Shh and that can respond to
Bmp7 by upregulating Nkx2.1 in Th+ neurons also express Six3. Finally,
exposure to Shh and Bmp7 of lateral mesencephalic tissue electroporated with a
Six3-repressor, but not a Six3-activator, construct results in the induction
of Nkx2.1+ DA neurons.
Six3 directly binds to the Wnt1 promoter to repress its expression
(Lagutin et al., 2003),
suggesting that a key function of Six3 is to abolish expression of Wnt1.
Intriguingly, recent studies in zebrafish have provided evidence that levels
of Wnt activity are crucial to the acquisition of hypothalamic identity,
suggesting that lower levels of Wnt activity are required to promote
hypothalamic fates versus more posterior fates
(Kapsimali et al., 2004
).
Together, these analyses suggest that Six3 represses Wnt, and that ambient
levels of Wnt activity promote an early hypothalamic competence/fate that
dictates the ability of Shh and Bmp7 to govern later aspects of hypothalamic
identity (Fig. 8).
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ACKNOWLEDGMENTS |
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