MRC Centre for Developmental Neurobiology, King's College London, 4th Floor New Hunt's House, Guy's Campus, London SE1 1UL, UK
Author for correspondence (e-mail:
frank.schubert{at}port.ac.uk)
Accepted 1 February 2005
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SUMMARY |
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Key words: Sax1, Emx2, Six3, Pax6, Mesencephalon, Tegmentum, Medial longitudinal fascicle, Posterior commissure, Early axon scaffold
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Introduction |
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The specification of neurons, however, has been studied in great detail in
the spinal cord, where the initial dorsoventral patterning of the neural tube,
resulting from the antagonistic action of ventralising and dorsalising signals
is translated into spatially restricted expression of homeobox genes. The
longitudinal, dorsoventrally restricted expression domains of the homeobox
genes prefigure the longitudinal columns of neuronal subtypes in the spinal
cord, as differentiating neurons adopt their distinct identities as a result
of expressing a specific combination of homeodomain transcription factors
(reviewed by Goulding and Lamar,
2000).
The organisation of the brain is more complex, and it harbours a greater
diversity of neurons than does the spinal cord. Neurons are either organised
into different layers, as in the tectum or the cerebral cortex, or into
nuclei, as in the ventral midbrain. Nuclei occupy distinct positions along the
rostrocaudal and dorsoventral axes, where their specification is likely to be
controlled by coordinate patterning of the neural tube. Interestingly,
parallels exist between the dorsoventral patterning of spinal cord and
midbrain at the molecular level. As in the spinal cord, ventral patterning in
the midbrain is governed by floor plate-derived Shh signalling: overexpression
of sonic hedgehog (Shh) throughout the midbrain leads to an expansion
of the basal plate-derived tegmentum territory at the expense of the dorsal
tectum (Watanabe and Nakamura,
2000), while local misexpression induces the expression of
ventrally expressed homeobox genes in a dose-dependent pattern
(Agarwala et al., 2001
).
Several homeobox genes are normally expressed in the ventral midbrain in
longitudinal domains so-called arcs in a similar arrangement
to the homeobox gene expression domains in the spinal cord
(Sanders et al., 2002
). Up to
five arcs, defined by the differential expression of homeobox genes, are
established in response to a presumed gradient of Shh emanating from the floor
plate. Ventral signalling, with resulting expression of homeobox genes, is
crucial for correct development of the ventral midbrain. A direct requirement
for Shh has been demonstrated for two groups of neurons in the
tegmentum, the somatic motoneurones of the oculomotor nucleus
(Chiang et al., 1996
), and the
dopaminergic neurons of substantia nigra and ventral tegmental area
(Hynes et al., 1995
).
Consistent with an instructive function of homeobox genes in the specification
of ventral mesencephalic neurons, Isl1 and Phox2a are
essential for the formation of the oculomotor nucleus
(Nakano et al., 2001
;
Pfaff et al., 1996
),
Emx2 is required for proper development of the red nucleus
(Agarwala and Ragsdale, 2002
),
and several homeobox genes including Pitx3 and Lmx1 are
involved in the specification of dopaminergic neurons (reviewed by
Smidt et al., 2003
).
We are interested in whether homeobox genes similarly play a role in the
formation of the early axon scaffold. The dominating longitudinal tract in the
early scaffold is the medial longitudinal fascicle (mlf). Very little is known
about the molecular mechanisms that underlie the formation of the mlf and its
contributing nucleus the interstitial nucleus of Cajal (INC). Tight
genetic regulation seems particularly important for distinguishing the fate of
mlf neurons from those forming the posterior commissure (pc), as neurons for
both tracts are located principally in the same ventral cluster at the
midbrain-forebrain border (MFB) (Tallafuss
et al., 2003). Could homeobox genes play a role in specifying
these early neuronal subtypes, and especially the mlf and pc cells? To answer
this question, we first analysed the expression patterns of the homeobox genes
Sax1, Six3, Emx2 and Pax6 in the ventral midbrain of chick
embryos between HH15 and HH25, when the early axon scaffold is formed. These
genes have previously been described to be expressed at the ventral MFB (e.g.
Agarwala and Ragsdale, 2002
;
Bovolenta et al., 1998
;
Schubert et al., 1995
), but
their precise temporal and spatial patterns of expression have not been
determined. Out of the expression analysis, Sax1 emerged as a prime
candidate for neuronal specification at the ventral MFB, as it is expressed
predominantly in the ventral neuronal cluster, coincident with the INC, from
the time the first neurons appear. Sax1 is a member of the NK1 class
of homeobox genes, which in vertebrates is usually represented by two closely
related genes, Sax1 and Sax2
(Bae et al., 2004
;
Bober et al., 1994
;
Schubert et al., 1995
;
Simon and Lufkin, 2003
;
Spann et al., 1994
), while
additional, more divergent members have recently been described in
Xenopus (Kurata and Ueno,
2003
) and zebrafish (Bae et
al., 2003
). We have employed electroporation in the chick to study
the function of Sax1 in the specification of neurons at the ventral
MFB. Misexpression of Sax1 leads to an increase in the size of the
mlf, and affects the expression of other ventral homeobox genes, suggesting
that a homeobox gene code underlies the formation of the early axon scaffold,
and that Sax1 in particular regulates the formation of the mlf.
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Materials and methods |
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Expression analysis
We employed RNA probes for chick Sax1
(Spann et al., 1994),
Emx2 (Bell et al.,
2001
), Isl1 (Tsuchida
et al., 1994
), Pax6
(Goulding et al., 1993
),
Phox2a (Groves et al.,
1995
) and Six3
(Chapman et al., 2002
) for our
analysis. For details of the whole-mount in situ hybridisation protocol, see
Dietrich et al. (Dietrich et al.,
1997
). In double labelling experiments, digoxigenin- and
fluorescein-labelled probes were consecutively detected with alkaline
phosphatase-conjugated antibodies (Roche), using NBT/BCIP (Roche) and Vector
Red (Vector Labs) as blue and red substrates.
To analyse the expression of Sax1 in relation to mlf neurons, we
retrograde labelled the mlf from the rostral hindbrain using
fluorescein-labelled dextran (Molecular Probes). Embryos at HH23 were
dissected in PBS, the ventral hindbrain was cut at the level of rhombomere 2,
and a crystal of the dye applied. Embryos were incubated at 37°C in L15
medium (GibcoBRL) for 3 hours, and then fixed. Following in situ hybridisation
for Sax1, the fluorescein label was detected with an alkaline
phosphatase-conjugated anti-fluorescein antibody (Roche), as in
double-labelling in situ hybridisation, using Vector Red as substrate (see
also Agarwala and Ragsdale,
2002).
Retrograde labelling of mlf and pc
Specific axon tracts in the embryonic brain were retrograde labelled with
lipophilic dyes. The mesenchyme was removed from day 5 chick embryos, and
isolated, hemisected brains were fixed flat on black nitrocellulose membrane
(Schleicher and Schuell). Crystalline DiI was applied on to the ventral part
of rhombomere 2 to label the mlf, while DiO was used to label the pc from the
roof plate of the caudal pretectum.
Immunohistochemistry
Neurons (cell bodies and axons) were detected with an antibody against
Neurofilament-M (Zymed RMO270), visualised by a peroxidase-conjugated
anti-mouse antibody (Jackson Laboratories) using Diaminobenzidine (Vector
Labs) as substrate. When combined with in situ hybridisation, the primary
antibody for immunohistochemical detection of neurofilament protein was
applied after completing the colour reaction of the whole-mount in situ
hybridisation procedure.
Electroporation
Two different expression constructs were used, both based on
pCAß-LINK-IRESeGFPm5-ClaI (Fig.
3I) (J. Gilthorpe, A. Hunter and A.L., unpublished), an expression
vector in which a hybrid CMV/chick ß-actin promoter
(Miyazaki et al., 1989) drives
the transcription of a polycistronic message encoding the gene of interest and
linked by an IRES element enhanced green fluorescent protein
(eGFP). pCAß-Sax1-IRES-GFP was constructed by inserting the full
coding region of the murine Sax1 gene
(Schubert et al., 1995
) into
the expression vector (Fig.
3L). For the assembly of pCAß-VP16Sax1-IRES-GFP,
first the eh1-like domain of the murine Sax1 gene
(Smith and Jaynes, 1996
) was
removed, and the transactivation domain of Herpes simplex VP16
(Triezenberg et al., 1988
) was
introduced in its place (Fig.
3L). The coding region for the hybrid VP16Sax1 protein
was again cloned into the base expression vector. The integrity of the
expression constructs was confirmed by sequencing. The
pCAß-LINK-IRESeGFPm5-ClaI vector itself was used as control for
non-specific effects of the electroporation.
|
Sectioning and photography
Where required, embryos were sectioned after the whole-mount in situ
hybridisation or immunohistochemistry procedures. Sections of 30 µm were
cut on a Leica vibratome and mounted in 80% glycerol.
Photographs of whole embryos, dissected brain tissue or sections were taken on a Zeiss Axiophot microscope with differential interference contrast, using a Zeiss Axiocam digital camera. Subsequent processing and assembly of the images was carried out with Adobe Photoshop.
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Results |
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Using whole-mount RNA in situ hybridisation, we detected the first expression of a homeobox gene in the ventral midbrain at HH15, when Sax1 signals appeared at the ventral MFB. These were followed shortly by Six3 and Emx2 signals in the same region (not shown). In contrast, Pax6 is not expressed in the midbrain until HH19. By HH20, distinct differences in the expression patterns of the different homeobox genes are evident (Fig. 1A-E). Signals for Six3 and Emx2 are split into ventral and dorsal stripes in the midbrain, separated by the emerging Pax6 expression domain, and ventrally delimited by the Isl1-positive oculomotor nucleus (Fig. 1C-E). The Sax1 signal spans the whole dorsoventral extent of the Six3/Emx2 domains, including also the intervening Pax6-positive stripe (Fig. 1B). Interestingly, although all four genes are expressed in the mantle layer, the mRNA for Sax1 is found exclusively in the outer margin of the mantle layer, aligning the marginal zone, while the Six3 and Emx2 signals are located further medially (Fig. 1B-D, the asterisk marks the medial limit of the Sax1 signals).
|
|
To identify which neurons might express Sax1, we stained chick embryonic brains at different stages simultaneously for neurofilament protein and Sax1 mRNA (Fig. 2E-H). At HH17, neurofilament staining detects the mlf as a bundle of longitudinal axons just dorsal to the floor plate. These can be traced to cell bodies at the ventral MFB, located within the Sax1 expression domain at the MFB (Fig. 2E). The location of these neurons at the ventral MFB, immediately rostral and dorsal to the oculomotor nucleus, and their caudal ipsilateral projection identify them as mlf neurons. While at HH17 Sax1 is expressed just in a small domain, the Sax1 signals at HH21 are much stronger and mask the neurofilament staining. Still, the axons of the mlf are visible as they extend from the Sax1 domain (Fig. 2F).
To confirm the conclusion that the mlf neurons express Sax1, we sectioned double-labelled embryos horizontally. Sections through the ventral mesencephalon show the Sax1 expression domain located just rostral to the oculomotor nucleus (Fig. 2G). Higher magnifications revealed Sax1-expressing neurons projecting into the mlf (Fig. 2H). Furthermore, we combined retrograde labelling of the mlf with in situ hybridisation for Sax1 mRNA. At HH23, cell bodies of mlf neurons were concentrated in two clusters around the MFB, a caudodorsal patch and a rostroventral area (see also Fig. 3J). Both areas overlap with the Sax1 expression domains in ventral mesencephalon and pretectum (Fig. 2D). Our analysis shows that Sax1 is expressed in the INC, the nucleus of the mlf.
Ectopic expression of Sax1 disrupts the early axon scaffold
The close association of Sax1 expression and mlf neurons raised
the possibility that Sax1 could be involved in establishing this
early tract. To test this hypothesis, we used a gain-of-function approach
where we expressed Sax1 ectopically to study the effect on the
morphology of the mlf (Fig. 3).
We employed two different expression constructs, based on the
pCAß-LINK-IRESeGFPm5-ClaI vector (J. Gilthorpe, A. Hunter and A.L.,
unpublished) (Fig. 3L).
pCAß-Sax1-IRES-GFP contains the coding sequence for the mouse
Sax1 gene (Fig. 3O).
Assuming that Sax1 normally acts as a transrepressor, mediated
through the binding of Groucho co-factors to its eh-1 like domain
(Smith and Jaynes, 1996), this
construct would repress the expression of Sax1 target genes. We also
designed a modified version, VP16Sax1, in which the transactivation
domain of Herpes Simplex VP16 (Triezenberg
et al., 1988
) replaced the eh1-like transrepression domain
(Fig. 3O). The
pCAß-VP16Sax1-IRES-GFP construct therefore encodes a protein
that would transactivate Sax1 target genes, thus acting as a
dominant-negative regulator of Sax1 function.
When we introduced the Sax1 expression constructs at HH10-13, we observed changes in the morphology of the early axon scaffold just 1 day after electroporation. While the mlf axons normally run close to the floor plate in a compact bundle (Fig. 3A), the fibres in the Sax1-expressing embryos stretch further dorsally, and their course is less regular (Fig. 3B). This phenotype appears even more pronounced after 2 days of ectopic Sax1 expression, when immunohistochemical staining for neurofilament protein shows the irregular pattern of the longitudinal axon tract in the ventral midbrain (Fig. 3D-I). While in embryos expressing the control construct the axon scaffold appeared normal (Fig. 3D,G), following electroporation of pCAß-Sax1-IRES-GFP the mlf expanded dorsally, occupying a larger region of the tegmentum (Fig. 3E,H). By contrast, the pc, although prominently stained in control embryos (Fig. 3D), was barely visible in the Sax1-expressing embryos (Fig. 3E). Ectopic expression of VP16Sax1 did not result in such a strong phenotype, probably owing to lower levels of expression consistently achieved with the pCAß-VP16Sax1-IRES-GFP construct. Still, VP16Sax1 seems to have the opposite effect on the mlf, as the tract appeared less prominent than in control embryos (compare Fig. 3C,F,I with Fig. 3A,D,G).
The results of the immunohistochemical analysis are mirrored by retrograde labelling of the ventral longitudinal tract from the ventral hindbrain (Fig. 3J,K). Again, the mlf was enlarged in the Sax1-expressing embryos (Fig. 3K) compared with embryos just expressing the control construct (Fig. 3J). In addition, while in the control embryo cell bodies were organised into two subclusters, located caudodorsally and rostroventrally (Fig. 3J, arrowheads), mlf neurons in the Sax1-expressing embryos were scattered throughout the ventral MFB, following no apparent pattern (Fig. 3K).
The expansion of the mlf after ectopic Sax1 expression could be the result of increased proliferation of mlf precursors, or of mis-specification of neurons normally destined for a different fate. Using an antibody against the mitosis marker phospho-Histone H3 (PH3), we analysed the electroporated embryos for differences in cell proliferation in the tegmentum. We found no obvious differences in the PH3 staining between embryos electroporated with either pCAß-LINK-IRESeGFPm5-ClaI (Fig. 3M) or pCAß-Sax1-IRES-GFP (Fig. 3N) that could explain the expansion of the mlf, suggesting that Sax1 misexpression leads to the mis-specification of neurons at the ventral MFB towards mlf neuron fate.
Ectopic expression of Sax1 disrupts the homeobox gene code in the tegmentum
In the spinal cord, homeobox genes regulate their expression by mutual
cross repression (Muhr et al.,
2001). This mechanism ensures that sharp expression boundaries are
formed, translating into distinct neuronal fate decisions. If similar
mechanisms act in the midbrain, Sax1 misexpression should affect the
expression of other homeobox genes in the ventral midbrain. To test this
hypothesis, we studied the expression patterns of Six3, Emx2, Pax6
and Phox2a in electroporated embryos
(Fig. 4). Normally,
Six3 and Emx2 are expressed in a subdomain of the
Sax1-expressing region, albeit in cells located more medially
(Fig. 1). This pattern is
unchanged in embryos expressing the GFP expression construct
(Fig. 4A,D). By contrast, 1 day
after electroporation of pCAß-Sax1-IRES-GFP, signals for both
genes are reduced or lost, depending on the level of ectopic Sax1
expression (Fig. 4B,E).
Misexpression of VP16Sax1 has a profound effect on Emx2
expression, leading to the upregulation of Emx2 ventrally, and even
to ectopic transcription of Emx2 in the dorsal midbrain
(Fig. 4C). Six3
expression, by contrast, is not altered by VP16Sax1
(Fig. 4F).
|
These results show that like the homeobox genes in the spinal cord the homeobox genes in the ventral midbrain can apparently crossregulate each other. Sax1 in particular has a profound effect on the expression of Emx2 and Six3.
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Discussion |
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Expression domains for homeobox genes subdivide the ventral midbrain and pretectum
We have found that a number of homeobox genes are expressed in distinct
domains in the ventral midbrain and pretectum during early brain development
in the chick. Among these, Emx2 and Pax6 have been described
previously as part of the arcuate plan that suggests the organisation of the
ventral midbrain into longitudinal domains, called arcs
(Agarwala and Ragsdale, 2002;
Sanders et al., 2002
). The
arcs can be visualised by the expression of homeobox genes such as
Phox2a for arc 1 or Pax6 dividing arcs 2 and 3
(Sanders et al., 2002
). In our
analysis, we have included two further genes labelling arcs 2 and 3,
Six3 (Bovolenta et al.,
1998
) and Emx2 (Bell
et al., 2001
). The expression patterns of both genes in the
ventral midbrain are largely overlapping. However, only the Emx2
signal also extends ventrally into the rostral part of arc 1, where it labels
the prospective red nucleus (Agarwala and
Ragsdale, 2002
). All of these genes are expressed throughout most
of the midbrain, stretching from the MFB almost to the isthmus. This suggests
that they may form part of a general patterning machinery for the whole
ventral midbrain. By contrast, Sax1 is expressed predominantly around
the MFB, abutting the oculomotor nucleus dorsally and rostrally. This
Sax1 expression pattern in the chick is similar to its orthologue
Sax1 (Schubert et al.,
1995
) and its paralogue Sax2
(Simon and Lufkin, 2003
) in
mouse. Likewise, the zebrafish sax2 gene is expressed in the
ventrocaudal cluster at the MFB (Bae et
al., 2004
). In double labelling experiments for Sax1 mRNA
and either neurofilament protein or retrograde labelling to visualise the mlf,
we have demonstrated that the Sax1 expression domain overlays the
INC, hinting at a specific function of Sax1 in the specification of
neurons at the MFB, particularly those forming the mlf.
Sax1 regulates the formation of the mlf
What is the role of Sax1 in the formation of the mlf? Our
misexpression experiments demonstrate that the expression of Sax1 has
to be tightly regulated to ensure the normal development of the mlf: ectopic
expression of Sax1 interferes with the patterning at the ventral MFB,
and leads to an expansion of the mlf. This result suggests that Sax1
is involved in the formation of the mlf, possibly by specifying mlf fate in
differentiating neurons. However, in the converse experiment,
VP16Sax1 expression reduces the size of the mlf, but does not
completely abolish its formation. This might be explained by incomplete
penetrance of the constitutively activating construct against the background
of endogenous Sax1 and Sax2 expression. Sax2
expression in the mouse midbrain overlaps the Sax1 expression domain,
and mice lacking Sax2 do not show an apparent midbrain phenotype
(Simon and Lufkin, 2003),
arguing for possible compensation by its paralogue.
The same cluster of neurons that includes the INC also harbours the ventral
part of the nucleus of the pc. The pc is formed well after the mlf, with the
first axons extending dorsally visible at HH17. In embryos expressing
Sax1 ectopically, the number of pc neurons is reduced so that the pc
is barely visible. It is unclear how neurons in the ventral cluster are
specified to mlf or pc fate, but ectopic expression of Sax1 seems to
interfere with this process. There is a possibility that fate specification is
influenced by the birth date of individual neurons, as in the case of
oculomotor and red nucleus neurons developing successively from arc 1
(Agarwala and Ragsdale, 2002).
In such a scenario, early birth would supports mlf fate, while later birth
would favour pc neurons. Sax1 would be thus linked to the timing of
neurogenesis. Indeed, in the early embryo Sax1 is transiently
expressed alongside Cash4 in the caudal neural plate, preceding
neurogenesis (e.g. Henrique et al.,
1997
). Although the role of Sax1 in the caudal neural
plate is unknown, it may be involved in neurogenesis in the caudal CNS, a role
also recently assigned to the distantly related NK1-class homeobox gene
Pnx (Bae et al.,
2003
). In the brain, Sax1 is normally only expressed in
postmitotic neurons, while with the electroporation method we introduce
Sax1 into neural progenitors, which may influence the time point when
neural cells leave the cell cycle.
Alternatively, the specification of mlf and pc could be the result of
intrinsic differences, possibly the differential expression of homeobox genes.
Again, our results are consistent with this mechanism. The mlf enlargement
after ectopic expression of Sax1 is not linked to any apparent change
in cell proliferation. Although formally it is also possible that ectopic
Sax1 expression changes axon guidance cues to misroute pc neurons
onto a caudal path, our findings argue for a change of cell fate in the
affected cells as the most likely cause of the observed phenotype. In
addition, the repression of Emx2 and Six3 indicates an
altered spatial patterning of the ventral MFB. In zebrafish, Six3
labels pc neurons as well as the INC
(Tallafuss et al., 2003).
Possibly, the specification of pc or mlf neurons depends on the balance of
homeobox gene expression at the ventral MFB. Loss of Six3 and
Emx2 expression together with increased or ectopic
Sax1 expression might shift this balance in favour of mlf
specification.
It is possible that both mechanisms, temporal and molecular difference, work hand in hand, as Sax1 is expressed closer to the marginal surface of the neural tube than Six3 and Emx2. This not only explains how the latter escape transcriptional repression by Sax1, but may also reflect a link between the time of neuronal differentiation and the expression of specific homeobox genes, thus adding a temporal dimension to the spatial pattern of differential gene expression.
A genetic network governing nuclei formation at the ventral MFB
Recently, several studies have described the molecular patterning of the
ventral midbrain, and together with the data presented in our study
we can begin to assemble the genetic network that controls the
formation of ventral midbrain nuclei. Patterning of the ventral midbrain along
the two main axes occurs under the influence of neighbouring tissues: Fgf8
from the isthmus sets up the caudorostral polarity of the midbrain (e.g.
Crossley et al., 1996), and
Shh derived from notochord and floor plate constitutes the ventralising signal
(Watanabe and Nakamura, 2000
).
In response to both signals, homeobox genes are expressed in distinct patterns
in the ventral midbrain (Agarwala et al.,
2001
; Sanders et al.,
2002
). The midbrain arcs largely subdivide the tegmentum into
distinct domains along the dorsoventral axis, but they also display distinct
rostrocaudal features. Thus, Emx2 is expressed in arcs 2 and 3 in the
entire midbrain, but only rostrally extends into arc 1. Sax1
expression in arcs 2 and 3 and the intervening region is restricted to the
rostral midbrain, close to the MFB.
Several studies have now implicated homeobox genes with the formation of
particular nuclei in the ventral midbrain, indicating the importance of proper
patterning for the correct development of tegmental neurons. Our study
demonstrates that the homeobox `code' is already crucial for the specification
of neurons that form the early axon scaffold. We also show for the first time
that a homeobox gene expressed around the ventral MFB can directly or
indirectly regulate the expression of other homeobox genes, providing a
possible patterning mechanism. The regulatory activity of Sax1 appears to be
highly specific, as ectopic expression of Sax1 abolishes the
expression of Emx2 and Six3, but not Pax6 in the
ventral midbrain. However, Pax6 expression in the adjacent ventral
pretectum, where Pax6 and Sax1 are expressed exclusively, is
lost after ectopic expression of Sax1. At the same time, the
Isl1 expression domain extends rostrally into the area where normally
Emx2 would be expressed. Quite possibly, the rostral extension of the
Isl1 domain (and the oculomotor nucleus) is an indirect effect of
Sax1, reflecting the loss of Emx2 expression. Likewise, the
loss of the oculomotor nucleus following expression of VP16Sax1 could
be an indirect effect of the ectopic Emx2 expression induced by the
dominant-negative variant of Sax1. In this scenario, Emx2 would repress the
expression Isl1 in the rostral arc 1. Although such an effect has yet
to be investigated, it is a conceivable mechanism by which Emx2 may
specify differentiating neurons towards red nucleus rather than oculomotor
nucleus fate (Agarwala and Ragsdale,
2002). This would resemble the mechanism suggested by our own
data, with Sax1 crucially influencing the fate decision between mlf
and pc.
These lines of evidence point to a possible recurring theme for
nucleogenesis in the ventral midbrain: differentiating neurons at a given
position face binary decisions of cell fate, and their choice is influenced by
their relative birth date and is controlled by the differential expression of
homeobox genes. An important characteristic of this possible mechanism is the
mutual repression of the fate-determining transcription factors to avoid
ambiguity in the cell fate, a strategy also employed in other examples of cell
fate selection from a common precursor pool such as the specification of
neuronal fate in the vertebrate spinal cord
(Briscoe et al., 2000) and of
muscle cell identity in the Drosophila embryo
(Jagla et al., 2002
).
Interestingly, NK1 class genes are involved in both processes: the fly
homologue of Sax1, slouch, is a muscle identity gene
(Knirr et al., 1999
); and
Sax1 itself is expressed in a subset of interneurones in the spinal
cord (Schubert et al.,
1995
).
Conclusion
We propose that, as a result of broad rostrocaudal regionalisation mediated
by the isthmic organiser, by local interactions at the MFB, and by
dorsoventral patterning by floor plate and roof plate, homeobox genes are
expressed in distinct domains in the ventral midbrain and pretectum. Their
expression domains may become sharpened by reciprocal repressive interaction
between the homeobox genes. We show that perturbing this intricate pattern by
overexpressing the ventrally expressed homeobox gene Sax1 ectopically
leads to disturbed dorsoventral patterning of the midbrain and affects the
organisation of the early axon scaffold. We conclude that the (combinatorial)
expression of specific homeodomain transcription factors determines neuronal
cell fate in the tegmentum of midbrain and pretectum.
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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Agarwala, S. and Ragsdale, C. W. (2002). A role for midbrain arcs in nucleogenesis. Development 129,5779 -5788.[CrossRef][Medline]
Agarwala, S., Sanders, T. A. and Ragsdale, C. W.
(2001). Sonic hedgehog control of size and shape in midbrain
pattern formation. Science
291,2147
-2150.
Bae, Y. K., Shimizu, T., Yabe, T., Kim, C. H., Hirata, T.,
Nojima, H., Muraoka, O., Hirano, T. and Hibi, M. (2003). A
homeobox gene, pnx, is involved in the formation of posterior neurons in
zebrafish. Development
130,1853
-1865.
Bae, Y. K., Shimizu, T., Muraoka, O., Yabe, T., Hirata, T., Nojima, H., Hirano, T. and Hibi, M. (2004). Expression of sax1/nkx1.2 and sax2/nkx1.1 in zebrafish. Gene Expr. Patt. 4,481 -486.[CrossRef]
Bell, E., Ensini, M., Gulisano, M. and Lumsden, A. (2001). Dynamic domains of gene expression in the early avian forebrain. Dev. Biol. 236, 76-88.[CrossRef][Medline]
Bober, E., Baum, C., Braun, T. and Arnold, H. H. (1994). A novel NK-related mouse homeobox gene: expression in central and peripheral nervous structures during embryonic development. Dev. Biol. 162,288 -303.[CrossRef][Medline]
Bovolenta, P., Mallamaci, A., Puelles, L. and Boncinelli, E. (1998). Expression pattern of cSix3, a member of the Six/sine oculis family of transcription factors. Mech. Dev. 70,201 -203.[CrossRef][Medline]
Briscoe, J., Pierani, A., Jessell, T. M. and Ericson, J. (2000). A homeodomain protein code specifies progenitor cell identity and neuronal fate in the ventral neural tube. Cell 101,435 -445.[CrossRef][Medline]
Chapman, S. C., Schubert, F. R., Schoenwolf, G. C. and Lumsden, A. (2002). Analysis of spatial and temporal gene expression patterns in blastula and gastrula stage chick embryos. Dev. Biol. 245,187 -199.[CrossRef][Medline]
Chedotal, A., Pourquie, O. and Sotelo, C. (1995). Initial tract formation in the brain of the chick embryo: selective expression of the BEN/SC1/DM-GRASP cell adhesion molecule. Eur. J. Neurosci. 7,198 -212.[Medline]
Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H. and Beachy, P. A. (1996). Cyclopia and defective axial patterning in mice lacking Sonic hedgehog gene function. Nature 383,407 -413.[CrossRef][Medline]
Chitnis, A. B. and Kuwada, J. Y. (1990). Axonogenesis in the brain of zebrafish embryos. J. Neurosci. 10,1892 -1905.[Abstract]
Crossley, P. H., Martinez, S. and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380,66 -68.[CrossRef][Medline]
Dietrich, S., Schubert, F. R. and Lumsden, A.
(1997). Control of dorsoventral pattern in the chick paraxial
mesoderm. Development
124,3895
-3908.
Easter, S. S., Ross, L. S. and Frankfurter, A. (1993). Initial tract formation in the mouse brain. J. Neurosci. 13,285 -299.[Abstract]
Goulding, M. and Lamar, E. (2000). Neuronal patterning: Making stripes in the spinal cord. Curr. Biol. 10,565 -568.[CrossRef]
Goulding, M. D., Lumsden, A. and Gruss, P.
(1993). Signals from the notochord and floor plate regulate the
region-specific expression of two Pax genes in the developing spinal cord.
Development 117,1001
-1016.
Groves, A. K., George, K. M., Tissier-Seta, J. P., Engel, J. D.,
Brunet, J. F. and Anderson, D. J. (1995). Differential
regulation of transcription factor gene expression and phenotypic markers in
developing sympathetic neurons. Development
121,887
-901.
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.[CrossRef]
Henrique, D., Tyler, D., Kintner, C., Heath, J. K., Lewis, J. H., Ish-Horowicz, D. and Storey, K. G. (1997). cash4, a novel achaete-scute homolog induced by Hensen's node during generation of the posterior nervous system. Genes Dev. 11,603 -615.[Abstract]
Hynes, M., Poulsen, K., Tessier-Lavigne, M. and Rosenthal, A. (1995). Control of neuronal diversity by the floor plate: contact-mediated induction of midbrain dopaminergic neurons. Cell 80,95 -101.[CrossRef][Medline]
Jagla, T., Bidet, Y., da Ponte, J. P., Dastugue, B. and Jagla, K. (2002). Cross-repressive interactions of identity genes are essential for proper specification of cardiac and muscular fates in Drosophila. Development 129,1037 -1047.[Medline]
Knirr, S., Azpiazu, N. and Frasch, M. (1999).
The role of the NK-homeobox gene slouch (S59) in somatic muscle patterning.
Development 126,4525
-4535.
Kurata, T. and Ueno, N. (2003). Xenopus Nbx, a novel NK-1 related gene essential for neural crest formation. Dev. Biol. 257,30 -40.[CrossRef][Medline]
Miyazaki, J., Takaki, S., Araki, K., Tashiro, F., Tominaga, A., Takatsu, K. and Yamamura, K. (1989). Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5. Gene 79,269 -277.[CrossRef][Medline]
Muhr, J., Andersson, E., Persson, M., Jessell, T. M. and Ericson, J. (2001). Groucho-mediated transcriptional repression establishes progenitor cell pattern and neuronal fate in the ventral neural tube. Cell 104,861 -873.[CrossRef][Medline]
Nakano, M., Yamada, K., Fain, J., Sener, E. C., Selleck, C. J., Awad, A. H., Zwaan, J., Mullaney, P. B., Bosley, T. M. and Engle, E. C. (2001). Homozygous mutations in ARIX(PHOX2A) result in congenital fibrosis of the extraocular muscles type 2. Nat. Genet. 29,315 -320.[CrossRef][Medline]
Pfaff, S. L., Mendelsohn, M., Stewart, C. L., Edlund, T. and Jessell, T. M. (1996). Requirement for LIM homeobox gene Isl1 in motor neuron generation reveals a motor neuron-dependent step in interneuron differentiation. Cell 84,309 -320.[CrossRef][Medline]
Sanders, T. A., Lumsden, A. and Ragsdale, C. W.
(2002). Arcuate plan of chick midbrain development. J.
Neurosci. 22,10742
-10750.
Schubert, F. R., Fainsod, A., Gruenbaum, Y. and Gruss, P. (1995). Expression of the novel murine homeobox gene Sax-1 in the developing nervous system. Mech. Dev. 51, 99-114.[CrossRef][Medline]
Simon, R. and Lufkin, T. (2003). Postnatal
lethality in mice lacking the Sax2 homeobox gene homologous to Drosophila
S59/slouch: evidence for positive and negative autoregulation. Mol.
Cell. Biol. 23,9046
-9060.
Smidt, M. P., Smits, S. M. and Burbach, J. P. (2003). Molecular mechanisms underlying midbrain dopamine neuron development and function. Eur. J. Pharmacol. 480, 75-88.[CrossRef][Medline]
Smith, S. T. and Jaynes, J. B. (1996). A
conserved region of engrailed, shared among all en-, gsc-, Nk1-, Nk2- and
msh-class homeoproteins, mediates active transcriptional repression in vivo.
Development 122,3141
-3150.
Spann, P., Ginsburg, M., Rangini, Z., Fainsod, A., Eyal-Giladi,
H. and Gruenbaum, Y. (1994). The spatial and temporal
dynamics of Sax1 (CHox3) homeobox gene expression in the chick's spinal cord.
Development 120,1817
-1828.
Tallafuss, A., Adolf, B. and Bally-Cuif, L. (2003). Selective control of neuronal cluster size at the forebrain/midbrain boundary by signaling from the prechordal plate. Dev. Dyn. 227,524 -535.[CrossRef][Medline]
Triezenberg, S. J., Kingsbury, R. C. and McKnight, S. L. (1988). Functional dissection of VP16, the trans-activator of herpes simplex virus immediate early gene expression. Genes Dev. 2,718 -729.[Abstract]
Tsuchida, T., Ensini, M., Morton, S. B., Baldassare, M., Edlund, T., Jessell, T. M. and Pfaff, S. L. (1994). Topographic organization of embryonic motor neurons defined by expression of LIM homeobox genes. Cell 79,957 -970.[CrossRef][Medline]
Watanabe, Y. and Nakamura, H. (2000). Control
of chick tectum territory along dorsoventral axis by Sonic hedgehog.
Development 127,1131
-1140.
Wilson, S. W., Ross, L. S., Parrett, T. and Easter, S. S. (1990). The development of a simple scaffold of axon tracts in the brain of the embryonic zebrafish, Brachydanio rerio. Development 108,121 -145.[Abstract]
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