Department of Neurobiology, Pharmacology and Physiology, The University of Chicago, Chicago, IL 60637, USA
* Author for correspondence (e-mail: cliff{at}drugs.bsd.uchicago.edu)
Accepted 12 September 2002
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SUMMARY |
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Key words: Bain nuclei, Oculomotor neurons, Red nucleus, Homeobox genes, Emx2, Sonic hedgehog, FGF8, Isthmus, Chick, Mouse
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INTRODUCTION |
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The ventral midbrain provides an elegant model system for addressing the
mechanisms of brain nucleogenesis. The adult midbrain tegmentum exhibits a
complex structural architecture featuring spherical, ovate and plate-shaped
nuclei. During embryogenesis, this nuclear organization is preceded by a more
regular organization into a reiterated series of arcuate territories called
midbrain arcs (Agarwala et al.,
2001; Sanders et al.,
2002
). Arcs are known to be molecularly distinct, differing, for
example, in their expression of homeobox genes
(Agarwala et al., 2001
).
Whether the arcs simply reflect a mechanism for regulating the numbers of
distinct midbrain cell-types or serve a more specific role in allocating
neurons to particular nuclear fates is unknown.
The possibility that developmental periodicities might provide a patterning
substrate for nucleogenesis has been addressed in connectional and
fate-mapping studies of hindbrain rhombomeres. The striking finding is that
most brainstem nuclei are generated from serially adjoining sets of
rhombomeres with few nuclei developing from single rhombomeres
(Cramer et al., 2000;
Diaz et al., 1998
;
Lumsden and Keynes, 1989
;
Marin and Puelles, 1995
).
Thus, the extent to which anteroposterior periodicities are crucial to
hindbrain nuclear specification remains uncertain
(Wingate and Lumsden, 1996
).
Indeed, the generation of hindbrain nuclei, many of which form longitudinal
columns, may be more tightly regulated in the mediolateral dimension
(Clarke et al., 1998
;
Marin and Puelles, 1995
). This
observation is of particular interest because midbrain arcs, unlike hindbrain
rhombomeres, are arrayed along the mediolateral axis parallel to the ventral
midline.
In these studies, we explored the relationship between arc pattern
formation and the generation of midbrain nuclei by focusing on the most medial
arc as a prototypical arc. Using diagnostic connectional and molecular
criteria we identified the anlagen for at least two midbrain nuclei within the
medial arc: the oculomotor complex (OMC) and the red nucleus (RN). Both of
these nuclei are part of the motor system, but their functions and connections
are so dissimilar that a shared origin in the same arc was unexpected. The OMC
contains motoneurons that control eye movements and the parasympathetic
regulation of accommodation and pupil contraction
(Evinger, 1988). The RN, by
contrast, contains no motoneurons but is a cerebellar-related nucleus
mediating motor cortex and cerebellar outflow to spinal cord in the control of
limb movements (Holstege and Tan,
1988
; Keifer and Houk,
1994
; ten Donkelaar,
1988
). The two medial arc pronuclei are also molecularly distinct,
differing in their expression of and dependence on homeodomain transcription
factors. In this report, we show that the prospective RN is distinguished by
expression of the POU domain gene BRN3A and the homeobox gene
EMX2, and that Emx2 function is required for RN development.
Within the medial arc, the RN and OMC anlagen occupy distinct spatial
positions in three dimensions. We reasoned that if the formation of these
midbrain nuclei is in fact governed by arc patterning, then any perturbation
of the medial arc should result in strictly coordinate perturbations of its
constituent pronuclei. The medial arc is flanked by two signaling centers, the
Sonic Hedgehog (SHH)-rich rostral floor plate and the FGF8- and WNT1-rich
midbrain-hindbrain junction (Agarwala et
al., 2001; Joyner et al.,
2000
). Manipulations of SHH and FGF8 signals are known to result
in perturbations of the midbrain arcs along the mediolateral axis (SHH)
(Agarwala et al., 2001
) and of
midbrain generally along the anteroposterior axis (FGF8)
(Crossley et al., 1996
). To
test the relationship between arc pattern formation and the generation of
midbrain nuclei, we misexpressed SHH and FGF8 with in vivo
electroporation technology. Results from these experiments show that ectopic
SHH expression anywhere within the midbrain is sufficient to induce
both medial arc pronuclei. Furthermore, SHH manipulations that distort the
midbrain arcs elicit completely parallel distortions of the OMC and RN
primordia, and FGF8 manipulations that shift the arcs forward coordinately
shift both medial arc pronuclei rostrally. These results suggest that the
midbrain arcs represent a patterning template for allocating midbrain
progenitor cells to their correct nuclear fates.
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MATERIALS AND METHODS |
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In ovo electroporation
Fertilized chicken eggs were incubated in a humidified forced-draft chamber
until the embryos reached stage 8-15
(Hamburger and Hamilton,
1951). Fifty to 250 nl of endotoxin-free plasmid DNA solution (1
µg/µl in 10 mM Tris, 1 mM EDTA, 0.02% Fast Green) was injected through a
glass capillary needle into the midbrain vesicles of chick embryos displayed
by windowing. For conventional macroelectroporations
(Muramatsu et al., 1998
;
Sakamoto et al., 1998
), two
platinum electrodes
2 mm apart were positioned to straddle the midbrain,
and a series of eight electric pulses (50 mseconds, 9V) were delivered with a
square wave electroporator (BTX 820, Genetronics, CA). Microelectroporation
techniques (Momose et al.,
1999
) were employed to elicit more restricted midbrain
transfections. A 500 µm tungsten wire was etched in 10% KOH to a tip
diameter of 40 µm. The tungsten electrode was inserted into the lumen of
the midbrain after plasmid injection and served as the negative electrode. The
positive terminal was a platinum electrode placed outside the midbrain above
the eye. The electrodes were held parallel at a distant of 1 mm and three
electric pulses (25 mseconds, 7V) were delivered. Electroporated embryos were
returned to the incubator for 3-4 days and on harvesting were submerged in 4%
paraformaldehyde in a phosphate-buffered saline (PBS) solution. Embryos that
showed some sign of injury from electroporation were excluded from further
study. Our analyses are based on whole-mount and section in situ hybridization
of 164 embryos successfully electroporated with the SHH
(n=54) or Fgf8 (n=110) constructs.
Axon tracing
Neural connections were studied in embryonic day (E) 5 and E6 chick brains
(n=94) with fluorescein-conjugated lysinated dextrans
(fluoro-emerald, 10 kDa, Molecular Probes). To label the OMC, the brain
neurectoderm and the third cranial nerve were dissected away from adhering
mesenchymal tissues in chilled Tyrode's solution and pinned to a SYLGARD dish.
Tracer resuspended in water was dried to a slurry, picked up on a pair of fine
forceps and applied to the dissected end of the nerve. For RN labeling, the
tracer was dried on a minuten pin and stuck into dissected brainstem. Labeled
embryo tissue was incubated for 30 minutes in chilled Neurobasal Medium (Life
Technologies) with 5% fetal calf serum and 30 mM glucose, and placed for 2-4
hours in a 6% CO2 incubator at 37°C. The tissue was then
immersion-fixed overnight in 4% paraformaldehyde-PBS. The fluoresceinated
dextrans were detected with anti-fluorescein Fab fragments conjugated to
alkaline phosphatase in modified in situ hybridization protocols.
Midbrain explants
Chick embryos were collected at stage 12-15 and dissected in cold Tyrode's
saline. Ventral midbrain explants with little or no adjoining dorsal midbrain
tissue were kept briefly at 4°C in L-15 medium containing 10%
heat-inactivated fetal bovine serum. The explants were then placed on a
Millipore filter in a drop of Neurobasal medium containing 20 mM glucose,
antibiotics and 10% fetal bovine serum, and were incubated in a 6%
CO2 incubator at 37°C for 2-3 days. Cultures were fixed in 4%
paraformaldehyde-PBS for analysis by in situ hybridization.
Mice
All mice were cared for according to animal protocols approved by the IACUC
of the University of Chicago. Outbred CD-1 timed pregnant mice were obtained
from the University of Chicago Cancer Research Center Transgenic Facility.
Emx2 heterozygote mice provided by P. Gruss
(Pellegrini et al., 1996) were
bred and genotyped by PCR. Noon of the day of vaginal plug discovery was
considered E0.5. Homozygous Emx2 mutant embryos (n=25)
between E12.5-E18.5 were recovered for gene expression analyses and compared
with equal or greater numbers of wild-type and heterozygote littermate mice
(n=27).
In situ hybridization
Two-color whole-mount in situ hybridization was carried out on chick brain
with digoxigenin- and fluorescein-labeled riboprobes synthesized from chick
cDNA plasmids for ACHE, BRN3A/POU4F1, EMX2, EVX1, FGF8, FOXA2, GBX2, ISL1,
OTX2, PAX3, PAX6, PHOX2A, SHH, TH and WNT1. Whole-mount and
section in situ hybridization was carried out on E12.5-E18.5 mouse
neurectoderm with riboprobes for mouse class III ß-tubulin/Tubb6,
Emx2 and Fgf8, and rat Brn3a, Isl1 and Th.
Riboprobe-mRNA duplexes and fluoresceinated dextrans were detected with
antibody-phosphatase conjugates (Roche Molecular Biochemicals) and
demonstrated by phosphatase histochemistry employing the distinguishable
tetrazolium chromagens NBT and TNBT.
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RESULTS |
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OMC neurons are not the sole constituents of the medial arc. Situated deep
to the OMC, near the pial surface of the midbrain, is a second group of cells,
identified as dopaminergic (DA) neurons by tyrosine hydroxylase (TH)
gene expression (Fig. 1C,D). In
the mouse, midbrain DA neurons have been reported to be migratory
(Marchand and Poirier, 1983),
raising the possibility that chick medial arc TH+ cells have migrated
in from the adjoining ventral midline. This observation, combined with the
late onset of TH gene expression in the chick and the lack of
availability of early markers of chick DA neurons
(Hynes et al., 1995
;
Smidt et al., 1997
;
Zetterstrom et al., 1997
),
leads to uncertainty about the origin of midbrain DA cells. We therefore
confined our analyses of the medial arc to the development of its other
constituents.
Overlapping the rostral end of the OMC along the anteroposterior axis and completely segregated from the OMC neurons along the ventricular-pial axis, is a third population of medial arc cells. This third territory is selectively enriched in mRNA for the homeodomain transcription factor EMX2 and for the POU domain transcription factor BRN3A (Fig. 1E-H; data not shown).
Connectional criteria establish that this EMX2+ and
BRN3A+ territory of the medial arc contains neurons of the
prospective red nucleus (pRN), a major brainstem nucleus involved in the
control of limb movement. In tetrapod vertebrates, the only exclusively
contralateral projection from midbrain tegmentum to lower brainstem and spinal
cord originates in the RN (ten Donkelaar,
1988). Thus, this projection is diagnostic of the RN. Small tracer
deposits of fluoresceinated dextrans centered in the medial arc at E6 elicit
anterograde labeling of multiple fiber-tracts descending into the hindbrain,
one of which crosses the midline (Fig.
2C). In reciprocal experiments, tracer injections placed in E5 or
E6 hindbrain produce retrograde labeling of a single population of exclusively
contralaterally projecting cells (Fig.
2D). PHOX2A (Fig.
2E) and BRN3A (Fig.
2F) gene expression combined with retrograde tract-tracing
demonstrates that these identified RN neurons lie in the BRN3A+/EMX2+
territory of the medial arc.
These data establish that the medial arc contains multiple cell types distinguished by unique axonal connections and patterns of transcription factor gene expression. These cell types are not, however, spatially intermixed, but form discrete territories. Along the anteroposterior axis, the pRN, the OMC and the medial arc TH neurons appear in succession, with the OMC overlapping both the pRN and the TH neurons when viewed from the ventricular surface. Along the ventricular-pial axis, the three cell-groups are fully segregated, with OMC lying closest to the ventricular layer. Thus, at E5 when midbrain neurogenesis is still under way (T. A. Sanders, PhD thesis, University of Chicago, 2000), the mantle layer of the ventral midbrain is patterned in all three dimensions.
Ventral midbrain origin of pRN neurons
Previous studies have suggested that neurons of the RN, like the neurons of
hindbrain precerebellar nuclei, are produced by dorsal neural tube
(Cooper, 1946;
Hamilton et al., 1959
;
Sidman and Rakic, 1982
;
Streeter, 1912
). Indeed,
modern textbooks illustrate red nucleus neurons that originate in dorsal
midbrain and translocate to ventral midbrain in migratory streams
(Haines, 2002
;
Sadler, 1995
). In light of our
connectional findings indicating that RN neurons are found in the medial arc
of the ventral midbrain by E5, we further investigated the origin of the RN in
an explant culture system. Ventral midbrain tissue was harvested for
organotypic culture at stage 12-15, a time when postmitotic neurons detected
in dorsal midbrain are massed in the prospective mesencephalic trigeminal
nucleus adjoining the roof plate (not illustrated)
(Easter et al., 1993
). After
3-4 days in vitro, the ventral midbrain cultures displayed strong expression
of the pRN marker BRN3A (Fig.
3). Moreover, based on PHOX2A mRNA expression, the
explant pRN formed in a medial arc-like territory whose three-dimensional
architecture closely matched that observed in vivo, with a BRN3A+ pRN
lying anterior and pial to a PHOX2A+ OMC
(Fig. 3C,D; data not shown). We
detected a medial arc pRN in all explants examined, even when no
PAX3+/BRN3A+ dorsal midbrain tissue remained attached to the ventral
midbrain explant (Fig. 3A,C,
residual tectum attached, n=54;
Fig. 3B,D, no residual tectum,
n=5). We conclude that BRN3A+ pRN neurons originate in
ventral midbrain, and that the generation of a medial arc properly organized
in three dimensions is an autonomous property of E3 ventral midbrain.
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Emx2 is essential for the development of the red nucleus in
mice
The homeobox gene EMX2 identifies the RN anlage in the medial arc.
We sought genetic evidence for an EMX2 role in RN development by
analyzing mice with targeted deletions of the Emx2 gene
(Pellegrini et al., 1996).
Emx2 gene expression establishes that, as in the chick, the mouse
ventral midbrain is organized into arcuate stripes
(Fig. 4A). The most medial
Emx2+ arcuate stripe, when viewed in whole-mount preparations,
overlaps with Isl1 and Brn3a gene expression territories,
indicating the presence of both an OMC (Isl1+) and a pRN
(Emx2+, Brn3a+) within the mouse medial arc
(Fig. 4B-D).
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In homozygous Emx2 mutant mice, Brn3a gene expression in ventromedial midbrain is retained between E12.5-E16.5, suggesting that pRN neurons are generated in the absence of Emx2 function (Fig. 5A,B). At E18.5, however, no RN can be identified with Brn3a or Class III ß-tubulin gene expression (Fig. 5C-F), whereas Isl1+ and Th+ cells appear unaffected (Fig. 5G,H). Thus, within the medial arc, Emx2 is specifically expressed by the RN pronucleus and appears to be essential for RN development.
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The OMC and pRN are coordinately regulated by SHH
The first arc is flanked medially by the rostral floor plate, a source of
the signaling molecule SHH. As illustrated in
Fig. 6, the relationship
between the SHH source and the medial arc is dynamic between E2, the
onset of arc pattern formation, and E5, when both the pRN and the OMC can be
readily identified by their signature transcription factors. At E2,
SHH gene expression in the midbrain is confined to the midline
(Fig. 6A), but by E5 it has
fanned out into the ventricular region overlying the medial arc
(Fig. 6B-D). Previous work has
shown that SHH can induce motoneurons and dopaminergic neurons in the ventral
midbrain (Agarwala et al.,
2001; Hynes et al.,
1995
; Wang et al.,
1995
; Watanabe and Nakamura,
2000
). Given the proximity of the BRN3A+ cells to the
SHH source, we tested by in ovo electroporation whether SHH
can also elicit pRN neurons. We were particularly interested in the spatial
relationship between any induced pRN and OMC neurons. As SHH is thought to
specify multiple cell-types in a concentration- and position-dependent manner
(Agarwala et al., 2001
;
Ericson et al., 1997
), the
observation that the pRN and the OMC are equidistant from the ventral midline
SHH source at E2 would appear to predict that any induced pRN neurons would be
intermixed with OMC neurons. What we found, though, was very different.
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SHH misexpression in the ventral midbrain of stage 8 to 15 chick
embryos produced by E5 expanded expression of SHH
(Fig. 7A) and its
transcriptional targets FOXA2/HNF3B and PTC1
(Fig. 7B)
(Agarwala et al., 2001).
SHH overexpression also expanded the population of PHOX2A+
oculomotor neurons and the pRN territory identified by BRN3A and
EMX2 labeling (Fig.
7C-F). Remarkably, the normal spatial relationship between the OMC
and the pRN territories, seen in the control (left) hemitegmentum, was
maintained in the enlarged territories along all three dimensions. The
expanded OMC and pRN were coextensive along the mediolateral axis
(Fig. 7D,F), partially
overlapping along the anteroposterior axis
(Fig. 7C,E) and completely
segregated along the ventricular-pial axis
(Fig. 7D,F). These results
demonstrate that SHH misexpression does not produce an intermixture
of OMC and pRN cell-types, but rather a coordinated expansion of discrete OMC
and pRN territories.
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SHH misexpression in lateral and dorsal midbrain can elicit
ectopic midbrain arcs (Agarwala et al.,
2001). We tested whether SHH misexpression in lateral
tegmentum or in tectum would also produce pRN cell-types. SHH
electroporation at the tectal-tegmental border
(Fig. 7G) or in dorsal midbrain
(Fig. 7H), where arcs are
normally never seen, reproduced the entire pattern of midbrain arcs, including
the EMX2+/BRN3A+ pRN and the PHOX2A+ OMC territories of the
medial arc. The ectopic OMC and pRN occupied discrete positions within the
ectopic medial arc, such that their spatial relationship always resembled that
of the normal OMC and pRN in all three dimensions
(Fig. 7H insert). Thus, each of
a variety of SHH manipulations in midbrain development coordinately affect the
size and shape of the pRN and the OMC, indicating that the patterning element
regulated by SHH is the medial arc and not simply the induction of its
constituent cell types.
Distance from the isthmus also regulates medial arc
nucleogenesis
An ectopic SHH signal in dorsal or ventral midbrain produces OMC and RN
anlagen that are offset in the anteroposterior axis. This finding suggests
that midbrain cells, in interpreting the SHH signal, rely on independent
positional information regarding their anteroposterior position [see also
Fedtsova and Turner (Fedtsova and Turner,
2001)]. We have tested the role of the isthmus in supplying this
positional information by shifting its location rostrally, into prospective
midbrain. To shift the isthmus forward, we drew on previous findings that
ectopic expression of FGF8 can transform prospective midbrain into hindbrain
tissue (Irving and Mason,
1999
; Liu et al.,
1999
; Sato et al.,
2001
) and that the juxtaposition of hindbrain and midbrain tissue,
even at ectopic sites, induces a new isthmus
(Irving and Mason, 1999
). This
manipulation, which contracts the size of the midbrain tegmentum, allowed us
to ask whether it is the position of the isthmus caudally or the distance from
the forebrain rostrally that controls the anteroposterior patterning of the
medial arc. If it is distance from the isthmus, then rostral midbrain
cell-types should be lost as the isthmus is moved rostrally. If it is distance
from the forebrain, then moving the isthmus forward should delete caudal
midbrain territories. If position relative to both boundaries is important,
then rostral advance of the isthmus may delete central elements.
Fgf8 delivery by electroporation to midbrain at E2 (HH stages 8-15) produced by E5 a rostral expansion of the GBX2+ hindbrain territory, a rostral regression of the OTX2+ midbrain territory and a concomitant rostral shift of the isthmus, identified by WNT1 and FGF8 gene expression (Fig. 8B,D; data not shown). In addition, the electroporated midbrain often took on the tubular appearance of rostral hindbrain (Fig. 8D). In the Fgf8 electroporated hemitegmentum, the hindbrain trochlear nucleus (a rhombomere 1 derivative) was elongated and the midbrain arcs, identified by PHOX2A, PAX6 and EVX1 labeling, appeared compressed (Fig. 8A,E). Within the medial arc, PHOX2A and BRN3A gene expression territories were both shifted rostrally (Fig. 8E-H). The extent of this rostral shift was related to the anteroposterior location of the ectopic isthmus. Thus, progressively more rostral Fgf8 electroporations resulted in progressively more rostrally located isthmi, which in turn produced more rostrally located OMC and RN pronuclei (Fig. 8E-H).
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Remarkably, the positional relationships of the shifted PHOX2A+ and BRN3A+ territories to the ectopic isthmus remained constant: the OMC territory always abutted the isthmus, the pRN territory appeared at a distance, and the rostral OMC and the caudal pRN territories overlapped. However, with very rostral Fgf8 electroporations that converted the midbrain tegmentum entirely to GBX2+/OTX2- hindbrain, BRN3A gene expression was severely reduced (n=4) or could not be detected (n=2; data not shown), with PHOX2A labeling entirely restricted to the subthalamus of caudal forebrain. These results demonstrate that manipulations that shift the medial arc rostrally produce corresponding rostral shifts in the OMC and the pRN that invariably maintain the spatial relationships between these pronuclei. In addition, our results show that the pronuclei of the medial arc are specified according to their distance from the caudally located isthmus.
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DISCUSSION |
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Origin of the red nucleus
Although many populations of neurons remain within the region of brain
where they are generated, some migrate great distances to reach their final
locations (Rakic, 2000).
Notable examples include dorsal hindbrain neurons that migrate ventrally to
populate precerebellar nuclei in the pons and medulla
(Harkmark, 1954
;
Rodriguez and Dymecki, 2000
;
Wingate and Hatten, 1999
).
Previous studies have held that neurons of the midbrain RN, like neurons of
the hindbrain cerebellar system, are initially generated in dorsal neural tube
(Cooper, 1946
;
Haines, 2002
;
Hamilton et al., 1959
;
Sidman and Rakic, 1982
;
Streeter, 1912
). These
conclusions were primarily based on histological descriptions of streams of
cells that appeared to be migrating from dorsal to ventral midbrain
(Sadler, 1995
). In this study,
we found that SHH, a ventral midline positional signal, can induce a RN anlage
and precisely regulate its size. In addition, we found that a BRN3A+
pRN could develop in ventral midbrain explant cultures that had no dorsal
midbrain attached. These observations lend strong support to the idea that,
unlike many other cerebellar system neurons, neurons of the RN are ventral
neural tube derivatives.
The homeobox gene Emx2 is required for the maturation of the
red nucleus
Two homeobox genes, BRN3A and EMX2, identify the pRN
territory of the medial arc. Analysis of mutant mice indicates that both genes
are needed for RN development. In Emx2-deficient mice, the
Brn3a-positive neurons of the pRN are generated, but are not detected
beyond E16.5. By E18.5, RN neurons are also lost in mice lacking
Brn3a (McEvilly et al.,
1996; Xiang et al.,
1996
). Thus, both Emx2 and Brn3a are essential
for the proper maturation of the red nucleus, although they appear not to be
required for its initial specification (this report)
(McEvilly et al., 1996
). That
Emx2 and Brn3a are both expressed in the pRN suggests that
the genetic requirement for these transcription factors is autonomous to pRN
neurons, but it will be important to test this possibility directly in
chimeric mouse experiments (Alvarez-Bolado
et al., 2000
).
Previous studies of Emx2 function have indicated a role for this gene in
the growth and initial areal patterning of the cerebral cortex
(Bishop et al., 2000;
Cecchi and Boncinelli, 2000
;
Mallamaci et al., 2000
). Our
findings suggest a broader role for Emx2 in brain development, one
that includes the regulation of neuronal survival and the development of
nuclei. In addition, the finding that the development of the RN requires at
least two homeodomain transcription factors, Emx2 and Brn3a, coupled with
previous work indicating that multiple homeodomain proteins are needed for the
generation of oculomotor neurons and midbrain dopamine neurons
(Pattyn et al., 1997
;
Simon et al., 2001
;
Smidt et al., 2000
), suggests
the general hypothesis that the development of distinct nuclei within specific
arcs is regulated by multiple homeodomain transcription factors.
Regulation of medial arc nucleogenesis by SHH and FGF8
Our in ovo electroporation experiments establish that SHH
misexpression eliciting an expansion or induction of the medial arc produces
both oculomotor and pRN cell-types. With all SHH manipulations, the spatial
relationships of the ectopic OMC and pRN neurons remain constant and resemble
those within the unmanipulated medial arc: the OMC and pRN form distinct
pronuclei that are co-extensive in the mediolateral axis and partially
separated along the anteroposterior axis. We tested whether this
anteroposterior segregation of the medial arc would be maintained if the
medial arc were moved rostrally within the ventral midbrain. We found that as
an ectopic isthmus is induced at progressively more anterior positions by
ectopic FGF8 delivery, the medial arc is shifted rostrally as well. In these
brains, the OMC and the pRN appear at progressively more anterior sites, until
first the pRN, and eventually both the pRN and OMC territories are lost, and
the entire electroporated half of the midbrain is converted to hindbrain.
These results demonstrate that rostral shifts in the location of the medial
arc result in corresponding shifts in the OMC and the pRN. They also show that
anteroposterior patterning of medial arc pronuclei is controlled by caudal
positional signals emanating from the midbrain-hindbrain junction. These
signals may include FGF8 itself, as well as other isthmus-specific FGF and WNT
proteins (Carl and Wittbrodt,
1999; Crossley et al.,
1996
; Wurst and Bally-Cuif,
2001
). Interestingly, whatever the molecular character of these
anteroposterior patterning cues, our results indicate that they are present
throughout the midbrain, as SHH misexpression anywhere in midbrain
results in the production of an ectopic medial arc within which the ectopic
pRN and the OMC retain appropriate anteroposterior polarity.
Patterning of midbrain nuclei along the ventricularpial axis
An emerging view in studies of neural development is that a two-dimensional
Cartesian coordinate system specifies cell fates within the developing
neuroepithelium. According to this `grid' model, orthogonal signaling centers
provide positional information for the medial-lateral and anteroposterior
patterning of the nervous system (Jessell
and Lumsden, 1997; Simon et
al., 1995
; Wolpert,
1969
; Ye et al.,
1998
). However, this model is not sufficient to account for the
segregation we have found of the midbrain pronuclei along the ventricular-pial
axis. What might be the mechanism for patterning midbrain nuclei along this
third axis? In the rat, although the OMC and the RN are born between E12-E14,
the peaks of their neurogenesis are staggered by 0.5-1.0 day
(Altman and Bayer, 1981
;
Marchand and Poirier, 1982
).
Thus, cells that leave the ventricular zone could be specified successively to
be OMC neurons (born E12-E13) then RN neurons (born E13-14). The mechanism
underlying such a temporal switch in cell-type generation could be the length
of time a progenitor cell spends in the proliferative zone. Alternatively, the
switch may be mediated by specific signaling molecules
(Desai and McConnell,
2000
).
One possibility, prompted by our observations on the changing profile of
midbrain SHH gene expression, is that SHH itself could participate in
ventricular-pial patterning. As Fig.
6 illustrates, although the source of SHH in the ventral
midbrain is a strip at its onset, by E3 it fans out to assume a wedge-shaped
profile (Ericson et al., 1995;
Marti et al., 1995
). Based on
the rat midbrain neurogenesis studies and the onset of PHOX2A in the
chick OMC by E2 and BRN3A in the pRN by E4, SHH gene
expression overlies the medial arc at a time when the fates of medial arc
neurons are still being specified (Altman
and Bayer, 1981
). That the same signaling systems can be involved
sequentially in the control of patterning along different axes has been shown
in studies of early Drosophila development
(Gonzalez-Reyes et al.,
1995
).
Conclusions
We have examined the relationship between midbrain arc pattern formation
and the specification of midbrain nuclei, using the medial arc as a
prototypical arc. Our results indicate that a single arc can harbor multiple
spatially segregated neuronal territories that by connectional and molecular
criteria correspond to specific midbrain nuclei. These findings suggest that
one role for the arcuate patterning of the ventral midbrain is the generation
of nuclei.
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ACKNOWLEDGMENTS |
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