? MRC Centre for Developmental Neurobiology, 4th Floor New Hunt's House, King's
College, Guy's Campus, London SE1 1UL, UK
1 Present address: Department of Developmental Neurobiology, National Institute
for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
* Author for correspondence (e-mail: sarah.guthrie{at}kcl.ac.uk)
Accepted 18 March 2003
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SUMMARY |
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Key words: Somatic motoneurones, Rostrocaudal axis, Hindbrain, Retinoic acid, Hox genes, Somites, Quail, Chick
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INTRODUCTION |
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Motoneurones later diversify into subpopulations that form discontinous
columns, occupying distinct domains along the rostrocaudal axis. In the
cranial region, motoneurones are subdivided into branchiomotor (BM), visceral
motor (VM) and somatic motor (SM) neurone subtypes, depending on their
columnar organisation and synaptic target. These subpopulations are thought to
originate within particular dorsoventral progenitor domains. For example,
BM/VM neurones are born in a domain immediately dorsal to the floor plate,
which expresses Nkx2.2, whereas SM neurones originate in a domain
dorsal to this, which is Nkx2.2-negative but which expresses
Pax6 at low levels and Olig2
(Briscoe et al., 1999;
Briscoe et al., 2000
;
Novitch et al., 2001
;
Jessell, 2000
).
Patterning of motoneurones along the rostrocaudal axis may depend on
signals from the paraxial mesoderm because, in the trunk region, heterotopic
transplantation of the paraxial mesoderm can alter some aspects of motoneurone
phenotype (Ensini et al., 1998;
Matise and Lance-Jones, 1996
).
Retinoic acid (RA), fibroblast growth factors (FGFs) and the TGFß family
member GDF11 have also been implicated in conferring rostrocaudal identity on
spinal motoneurones (Liu et al.,
2001
; Sockanathan and Jessell,
1998
). The actions of these, and possibly other mesoderm-derived
molecules, appear to repattern the rostrocaudal expression domains of Hoxc
genes, which are expressed in discrete rostrocaudal domains of the neural tube
(Ensini et al., 1998
;
Liu et al., 2001
), conferring
upon Hox genes a central role in the interpretation of extrinsic signals in
motoneurone patterning.
In the hindbrain, motoneurone patterning is linked to segmentation into
rhombomeres, each of which contains a characteristic set of motoneurone
subtypes with distinct axon pathways. BM, VM and SM neurone subtypes are
differentially distributed with respect to the rostrocaudal axis: rhombomeres
2-4 contain only BM neurones (Lumsden and
Keynes, 1989), whereas rhombomeres 5-8 contain BM, VM and SM
neurones in various combinations. Segmentation occurs between stage 9 and
stage 12 in the chick embryo, but a coarse rostrocaudal pattern is established
even earlier, at neural plate stages, providing a substrate upon which later
dorsoventral patterning acts (Lumsden and
Krumlauf, 1996
; Simon et al.,
1995
). Rhombomeres express different combinations of Hox genes,
which play roles in segmentation, rhombomere patterning and neuronal
differentiation (Lumsden and Krumlauf,
1996
). In particular, some Hox genes, including Hoxb1,
are expressed at early neural plate stages and it is plausible that they might
act to establish rhombomeres as territories committed to generate particular
repertoires of motoneurones. Recently, Hoxb1 and Hoxa2 have
been shown to play a direct role in the specification of facial and trigeminal
motoneurones, respectively (Gavalas et
al., 1997
; Bell et al.,
1999
; Jungbluth et al.,
1999
; Studer et al.,
1996
).
As in the spinal cord, patterns of Hox gene expression are responsive to
environmental signals, including those from the mesoderm. Transposition of
rhombomeres from the pre-otic region to the post-otic region at stage 10-12
resulted in respecification of the Hox `code' and neuronal organisation
(Grapin-Botton et al., 1995;
Itasaki et al., 1996
), whereas
transpositions of rhombomeres between axial levels within the pre-otic region
resulted in the maintenance of rhombomere identity and Hox gene expression
(Grapin-Botton et al., 1997
;
Grapin-Botton et al., 1995
;
Guthrie et al., 1992
;
Itasaki et al., 1996
;
Kuratani and Eichele, 1993
).
The discrepancy seems to depend on differences in the paraxial mesoderm, which
is unsegmented in the pre-otic region
(Hacker and Guthrie, 1998
),
but forms somites caudal to the otic vesicle. Somite grafts adjacent to the
neural tube in the pre-otic region were capable of respecifying Hox gene
expression and some aspects of neuronal phenotype, effects that could be
mimicked by RA (Gavalas and Krumlauf,
2000
; Gould et al.,
1998
; Grapin-Botton et al.,
1997
; Grapin-Botton et al.,
1995
; Itasaki et al.,
1996
). RA is known to be produced by the somites, making it a
likely candidate for a caudalising signal in vivo
(Maden et al., 1998
;
Swindell et al., 1999
). An in
vivo role of RA has been demonstrated in a series of studies on avian and
mouse embryos, in which depletion or elimination of RA signalling leads to a
failure in the development of rhombomeres 4-7
(Dupé and Lumsden, 2001
;
Gale et al., 1999
;
Maden, 1996
;
Niederreither et al.,
2000
).
However, in these studies, only selected aspects of rhombomere identity were examined and these did not include cranial motoneurone identities or axon trajectories. No systematic study has been carried out to elucidate the precise relationship between environmental signals, patterns of Hox gene expression and motoneurone specification. It is not clear at which timepoint rhombomeres become committed to generating particular motoneurone subtypes, nor is the source and nature of the inductive signals that control these processes known. In the present work we focus on these issues, and investigate in particular the mechanisms that govern somatic motoneurone generation in the hindbrain and confine it to caudal rhombomeres (5-8). We have performed transplantation experiments in early chick embryos, which indicate that the capacity of a rhombomere to generate SM neurones is labile at the neural plate stage but becomes fixed at stage 10-11, around the time of neural tube closure. Somites grafted rostrally were able to induce ectopic Hox gene expression (including that of Hoxa3) and SM neurones, in particular rhombomeres, in a restricted time period. RA-loaded beads grafted rostrally mimicked this effect, inducing SM neurones in the same region and within the same time window. We also tested a possible involvement of Hoxa3 in defining the territory that generates SM neurones, and found that ectopic expression of Hoxa3 in the rostral hindbrain induced SM neurones in rostral rhombomeres.
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MATERIALS AND METHODS |
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Rhombomere fate mapping
Because rhombomere boundaries are not complete until stage 12
(Vaage, 1969), the territories
corresponding with presumptive rhombomeres were mapped in both chick and quail
embryos at stage 9. Small spots of DiI (Molecular Probes, Oregon, USA) were
injected, using a micrometer graticule, at defined positions within the neural
tube, relative to a caudal limit at the first somite and the rostral end of
the neural tube. Embryos were incubated for 24 hours prior to analysis.
Rhombomere 1 (r1) was mapped at 900 µm from the rostral end of the neural
tube, and thereafter each rhombomere was approximately 100 µm long, with r7
located at level of the first somite. These measurements were used to ensure
accurate dissection of rhombomeres for transplantation.
In ovo electroporation of chick embryos
In the electroporation experiments, we used full-length cDNA for
tauGFP (green fluorescent protein), mouse Hoxa3 and human
HOXB3 (gifts from Dr Andrea Brand, Dr Michael Hofmann and Dr Guy
Sauvageau, respectively) under the control of a chicken ß-actin promoter
(pCAßlink kindly provided by Dr Jon Gilthorpe). The tauGFP
plasmid was co-electroporated with the mouse Hoxa3 or human
HOXB3 plasmid to allow in vivo fluorescent screening of embryos. We
found that a 1:3 ratio of tauGFP/Hoxa3 or HOXB3 gave the
best co-localization of GFP and Hox gene expression. Plasmids were
used at a combined final concentration of 2 µg/µl, whereas in control
experiments the tauGFP plasmid alone was used at a final
concentration of 0.5 µg/µl. Electroporation was performed as previously
described (Itasaki et al.,
1999). Briefly, eggs were windowed and embryos (stage 8-17) made
visible. The vitelline membrane was opened and DNA solution was injected
either into the neural tube (stage 9-17) or on top of the neural plate (stage
8). Two silver electrodes were then placed dorsal and ventral to the embryo at
the rostrocaudal level desired, so that electroporation (10V; 5 pulses of 50
milliseconds) allowed the entry of DNA into the basal plate of the neural tube
where motoneurone progenitors are located. Unilateral entry of DNA was
obtained in most cases. Eggs were then sealed and incubated for 48-96 hours
before embryos were removed for fixation (at E4-E6).
In situ hybridisation
Whole-mount in situ hybridisation was performed essentially as published
(Henrique et al., 1995), using
Islet2 (Tsuchida et al.,
1994
), Hb9 (Tanabe et
al., 1998
), Hoxa3
(Saldivar et al., 1996
),
Hoxb3 (Rex and Scotting,
1994
) and Hoxd4
(Grapin-Botton et al., 1995
)
chick-specific probes. A mouse Hoxa3 probe (full-length cDNA; GenBank
Accession Number Y11717) was used to detect the expression of electroporated
Hoxa3. Briefly, stage 28-30 embryos were fixed, dehydrated in
methanol and stored at -20°C for a maximum of 2 weeks. Prior to
pre-hybridisation and hybridisation at 70°C, embryos were treated with 10
µg/ml proteinase K for 20 minutes at room temperature. Digoxigenin (DIG)-
or Fluorescein (Fluo)-labelled antisense RNA probes were synthesised according
to the manufacturer's instructions (Roche Molecular Biochemicals), and
hybridised at 1 µg/ml. For double in situ hybridisation, probes were added
simultaneously to the embryos, whereas antibody incubations (anti-DIG-AP and
anti-Fluo-AP antibodies) and developing reactions were carried out
sequentially. The NBT/BCIP reaction was always performed first, followed by a
series of washes and a 30 minute incubation at 70°C to destroy residual
alkaline phosphatase activity. Incubation with the anti-Fluo-AP antibody and
Fast Red development followed. All reagents used for in situ procedures were
from Roche Molecular Biochemicals, except for Fast Red (Sigma).
Immunostaining on whole mounts in rhombomere and somite grafting
experiments
In grafting experiments, the correct graft position and integration was
ensured by immunostaining with a monoclonal antibody to quail cells (QCPN).
Following the in situ procedure, embryos were post-fixed, washed extensively
in PBS containing 1% Triton X-100 (TX100), and left in blocking solution
(PBS/10% heat-inactivated sheep serum (HSS)/1% TX100) for a minimum of 2 days
prior to incubation with the primary antibody. After 2-3 days, embryos were
washed extensively in PBS/10% HSS/1% TX100, incubated for further 2-3 days in
secondary antibody, then finally washed overnight, dissected and mounted in
DABCO/glycerol.
Immunohistochemistry on sections
Cryostat sections (10 µm) were immunostained by overnight incubation
with primary antibodies in PBS/1% HSS/0.1% TX100. After several washes in
PBS/0.02% TX100, sections were incubated for 2-3 hours at room temperature
with fluorescent-conjugated secondary antibodies (FITC-, Cy3-, Alexa Fluor
568- or Cy5-labelled goat anti-mouse, goat anti-guinea pig or goat anti-rabbit
antibodies), washed briefly and mounted in DABCO/glycerol prior to analysis.
Vibratome sections (80 µm) were incubated with primary antibodies for 2
days in PBS/10% HSS/1% TX100. Sections were then washed extensively in PBS/1%
TX100 and incubated overnight with secondary antibodies. After final washes,
sections were mounted in DABCO/glycerol.
Primary antibodies used were polyclonal anti-neurofilament heavy chain (AB1991, Chemicon International), monoclonal anti-quail axons or quail cells (QN or QCPN, respectively; Developmental Studies Hybridoma Bank), monoclonals 4D5 (anti-Islet1/2), 4H9 (anti-Islet2), and polyclonals anti-Chx10 and anti-Lim3 (all kind gifts of Dr T. Jessell). We also used monoclonals anti-Nkx2.2, 81C10 (anti-Mnr2/Hb9) and anti-Pax6, and polyclonals anti-Irx3 and anti-Olig2 (also kind gifts of Dr T. Jessell). Finally in misexpression experiments, a rabbit anti-GFP antibody (A-6455, Molecular Probes) was used to identify electroporated regions. All fluorescently-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories, except for Alexa Fluor 568, which was from Molecular Probes.
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RESULTS |
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Both in chick and mouse embryos, Hox3 paralogues have been reported to be
expressed from the r4/5 boundary caudally
(Grapin-Botton et al., 1995;
Lumsden and Krumlauf, 1996
;
Rex and Scotting, 1994
).
However, although this was the expression pattern of Hoxa3 in the
chick (Fig. 1D), we found that,
at E6, Hoxb3 was also expressed in r4, with a rostral cut-off at the
r3/4 boundary (Fig. 1A,E).
Double in situ hybridisation for Hoxb3 and Islet2 confirmed
this localisation, because the abducens neurones of r5 and r6 were completely
contained within the Hoxb3-expressing territory
(Fig. 1E). Hoxd4 was
expressed from the r6/7 boundary caudally, in accordance with previous reports
(Grapin-Botton et al., 1995
),
and double in situ hybridisation with Hoxd4 and Islet2
showed that hypoglossal neurones lay within the Hoxd4-expressing
domain, whereas abducens neurones lay rostral to this region
(Fig. 1F). Thus Hoxa3,
Hoxb3 and Hoxd4 were used as markers of more caudal rhombomere
identity.
In pre-otic rhombomere transplants, Lim gene expression and
motoneurone identity is fixed from stage 10 onward
To ascertain the timing of rhombomere commitment to generate SM neurones,
r5 to r3 (r5/r3) quail to chick rhombomere transplants were performed at stage
10-12 (Fig. 2A), and operated
hindbrains were assessed at stage 26-29
(Table 1A). Whole-mount in situ
hybridisation on operated embryos showed that the transposed r5 maintained
Islet2 expression in the ectopic r3 position
(Fig. 2B), which suggests that,
from stage 10 onwards, motoneurone progenitors within r5 are committed to
express SM markers. Conversely, when r3 was transposed to the r5 position
(r3/r5) at stage 10-12, the transposed r3 failed to express Islet2
(Fig. 2L,
Table 1A), implying that r3 has
maintained its rostrocaudal identity and failed to generate SM neurones. Thus,
both r5/r3 and r3/r5 grafts after stage 10 showed rhombomere autonomy in gene
expression patterns and SM neurone production.
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Do SM neurones within the r5 transplanted to r3 join the pathway of the
endogenous abducens nerve? During normal development, abducens axons emerge
from r5 and r6 as multiple rootlets, which fasciculate ventral to the brain,
and extend rostrally to innervate the lateral rectus eye muscle, and the small
pyramidalis and quadratus nictitans (P/Q) muscles
(Wahl et al., 1994). In
immunostained transverse sections of r5/r3 embryos, quail-derived axons
exiting ventrally joined the host abducens nerve as it extended beneath the
brain (Fig. 2Q), whereas in
parasagittal sections quail-derived axons fasciculated with the endogenous
abducens and accessory abducens axons to extend towards the LR and P/Q muscles
close to the orbit (Fig. 2R,S;
n=4). It appears therefore that as early as stage 10, motoneurone
progenitors in r5 are committed to express SM markers and to pursue an
abducens pathway.
In pre-otic transplantations, rhombomeres show plasticity at stage
9
To pinpoint the stage at which rhombomeres become committed to generate SM
neurones, transplantation experiments were performed at stage 9
(Table 1A). Following r5/r3
transplants at this stage, the transposed rhombomere failed to express
Islet2, even though QCPN immunostaining showed that the graft was
correctly located and integrated (Fig.
2F,G). Thus, SM neurones were not generated in the transplanted
r5, which behaved according to the new location. Conversely, in r3/r5
transplants at stage 9, Islet2 expression was induced, reflecting SM
neurone production (Fig. 2M,N).
Hence, in both caudal to rostral and rostral to caudal transpositions in the
pre-otic region, cranial motoneurone rostrocaudal identity is susceptible to
environmental cues at stage 9, but is fixed by stage 10.
Caudal to rostral rhombomere transpositions in the chick as early as stage
8+ have shown that the expression of Hox genes is maintained autonomously
(Guthrie et al., 1992;
Kuratani and Eichele, 1993
;
Simon et al., 1995
), and it
has been suggested that other aspects of rhombomere identity are also fixed
(Grapin-Botton et al., 1995
).
However, our experiments show that, up to stage 9, Islet2/Hb9
expression changes in pre-otic rhombomere transplants according to their new
position. We therefore investigated the relationship between Hox genes and SM
neurone formation using whole-mount single and double in situ hybridisation
with Islet2 and/or Hoxa3/Hoxb3 probes on r5/r3 embryos
(Table 1A). In r5 to r3
transplants performed at stage 9 and at stage 10, the transposed r5 maintained
expression of Hoxa3 and Hoxb3
(Fig. 2D,E,H-K). Because in
grafts at stage 9, the transplanted r5 did not express Islet2
(Fig. 2F,G), there is a
discrepancy between Hox gene expression and cranial motoneurone identity at
this stage. However, Hoxa3 and Hoxb3 expression in the transposed r5
was lower than in the host r5, at both stage 9 and stage 10.
In pre-otic to post-otic rhombomere transplants, SM neurone
production can be induced
Previous studies have shown that rostral rhombomeres transposed caudally
(to post-otic levels) show caudalised Hox gene expression profiles due to
mesoderm-derived signals (Grapin-Botton et
al., 1997; Grapin-Botton et
al., 1995
). To further investigate SM neurone production in
relation to other aspects of rhombomere phenotype, we grafted r3 or r4 into
the rostral or caudal part of r8 (r8a or r8p; level of somite 2 or 4,
respectively) and analysed Hox gene expression
(Fig. 3A;
Table 1B). For grafts at stage
10-11, Hoxa3, Hoxd4 and Hoxb3 were all induced in the
ectopic r3 in r8a or in r8p (Fig.
3B-D; data not shown), whereas Islet2 and Hb9
were not (Fig. 3E-G). Only at
stage 9 did r3 express Islet2; the induced level of Islet2
expression was higher in grafts in r8p than in grafts placed in r8a, but
overall the expression level was lower than on the control side
(Table 1B; data not shown).
These data suggest that r3 is capable of responding to post-otic environmental
signals by producing SM neurones, but only up to stage 9, reflecting a similar
time dependence to its behaviour in r3 to r5 grafts.
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In order to characterise further the putative SM neurones in r4
transplanted at stage 10, r4/r8p chimaeras were double-immunostained with
various combinations of antibodies (n=7;
Fig. 3K-O). Quail cells in the
ectopic r4 expressed Islet2 protein, and quail axons exited ventrally from the
neuroepithelium to fasciculate with chick hypoglossal axons
(Fig. 3K,N,O). Some quail axons
also exited the neuroepithelium dorsally, which suggests that some r4
motoneurones projected along vagus and cranial accessory axon pathways (data
not shown). To confirm the hypoglossal identity of the newly induced SM
neurones, the Lim homeobox `code' of the transposed r4 was also investigated.
Previous studies showed that r8 contains two distinct populations of
motoneurones: vagus and cranial accessory neuronal somata are located
laterally and express Islet1, whereas hypoglossal neurones lie more medially
and express Islet1, Islet2 and Lim3
(Varela-Echavarría et al.,
1996). We examined the Lim homeoprotein expression profiles of
hypoglossal neurones in more detail at E6 in caudal r8, using antibodies
against Islet1/2, Islet2 and Lim3 proteins. Because all motoneurones still
express Islet1 at this developmental stage
(Varela-Echavarría et al.,
1996
), we can conclude that hypoglossal neurones express Islet1
and Islet2 or Lim3, with a subset co-expressing Islet2 and Lim3
(Fig. 3L-P). Each of these
neuronal subpopulations occupied specific locations, with respect to the
dorsoventral and mediolateral axes, that were distinct from those of abducens
neurones at r5/6 level. For example, Islet2/Lim3-positive neurones were
located medially and close to the ventral (pial) side of the neuroepithelium
(Fig. 3O). Double-immunostaining on adjacent sections showed that the Lim homeobox code
of the grafted r4/r8p mirrored perfectly that of the control r8 side
(Fig. 3L-P), which shows that
signals at r8p level could repattern r4 so that at least a proportion of
motoneurones adopted a hypoglossal identity.
SM neurones can be induced by signals from the paraxial mesoderm
Because somites transplanted into the pre-otic region could repattern Hox
genes and neuronal differentiation
(Grapin-Botton et al., 1997;
Itasaki et al., 1996
), we
tested a possible influence of the caudal paraxial mesoderm on somatic
motoneurones. Quail somites were grafted into isochronic chick hosts (stage
9-11) at various levels within the cranial paraxial mesoderm and beneath the
neuroepithelium (Fig. 4A;
Table 2A), and induction of
Islet2, Hb9, Hoxd4 and Hoxa3 was analysed at E6. For grafts
of rostral somites (s1 to s3) beneath the rostral hindbrain (r2-4) none of the
markers assayed were induced, which indicates that these somites are devoid of
inductive capability at the stages tested (9-12), which is consistent with
previous data (Itasaki et al.,
1996
). When caudal somites (s5 or more caudal) were grafted
underlying r2 and r3 at stage 9-11, no Islet2 induction was observed,
whereas Hoxd4 induction was observed in the alar plate of r2 and r3
(Table 2A; data not shown).
Grafting of caudal somites underlying r3 and r4 at stage 11 led to a similar
failure of SM neurone differentiation, and induction of Hoxd4 in the
r3 and r4 alar plate only (Table
2A; data not shown). However, for transplants at stage 9-10, both
Islet2 and Hb9 were induced in r4, although in some cases
this was limited to the caudal region (Fig.
4B; data not shown). In these grafts, Hoxa3 and
Hoxd4 expression was, in some cases, induced in the alar plate of r3
(data not shown) but was induced in r4 extending ventrally as far as the floor
plate, corresponding with the region of SM neurone induction
(Fig. 4C,D).
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The apparent abducens identity of ectopic SM neurones following somite
transplants presents a paradox, because the expression of Hoxa3 and
Hoxd4 together (in embryos analysed 76 hours post-grafting;
Table 2A) is consistent with a
hypoglossal rather than an abducens phenotype. A possible reconciliation of
these data lies in previous studies showing that following rostral somite
transplants, caudal Hox genes take longer to be induced in rostral rhombomeres
than more rostral Hox genes (Grapin-Botton
et al., 1997; Itasaki et al.,
1996
). We therefore analysed Hox gene expression 24, 36 and 48
hours after somite grafting. After 24 hours, neither Hoxa3 nor
Hoxd4 were expressed, whereas after 36 hours Hoxa3 but not
Hoxd4 was expressed, and after 48 hours both Hoxa3 and
Hoxd4 were expressed (Table
2B). These data demonstrate that there is indeed a timelag in
Hoxd4 induction in response to somite-derived signals, so that
although Hoxa-3 is switched on at the equivalent of stage 15/16,
Hoxd4 expression is not initiated until approximately stage 20. This
may be consistent with the acquisition of an abducens phenotype by ectopic SM
neurones in r4.
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Taken together, these somite-grafting experiments indicate that there are rostrocaudal differences in the time-window during which cranial motoneurone progenitors are capable of responding to patterning signals from the somites. Rhombomere 7 is still capable of responding to these signals as late as stage 11, whereas r4 is refractory to these signals by stage 11, and r2 and r3 are not sensitive to the signal even as early as stage 9. These observations could imply either a complete inability of the rostral hindbrain to respond to somitic signals, or an earlier fate commission of rostral rhombomeres relative to more caudal levels. Finally, it is also possible that r2 and r3 require a higher level of inductive signal than a single grafted somite can supply.
Retinoic acid beads mimic the caudalising action of the somites
It has been proposed that retinoic acid (RA) mediates a part of the ability
of the paraxial mesoderm to caudalise the rostrocaudal axis
(Gavalas and Krumlauf, 2000;
Gould et al., 1998
). In the
early chick embryo, the somitic mesoderm generates RA, with younger, more
caudal somites generating higher levels of RA than older ones
(Berggren et al., 1999
;
Maden et al., 1998
;
Swindell et al., 1999
). As our
somite grafting experiments showed that only caudal somites could induce SM
neurones, this implies that RA is a candidate in SM neurone induction.
When beads treated with RA [10-4 M] were grafted in chick hosts at various axial levels within the cranial paraxial mesoderm and beneath the neuroepithelium (Fig. 4A; Table 3), Islet2 and Hb9 were induced exclusively in r4, and only in grafts at stage 9-10 at r3/4 level (Fig. 4I; Table 3). No induction was observed in grafts done at stage 11 or when beads were implanted at more rostral positions (data not shown; Table 3). When a higher concentration of RA was used (5x10-4 M), SM neurone induction was still exclusive to r4, although some Hb9 induction was observed in the r4 contralateral to the bead implantation (Fig. 4J). We also analysed Hox gene expression following RA bead implantation. No induction of Hoxa3 or Hoxd4 was observed in transplants performed at stage 11. However, for transplants of beads treated with RA [10-4 M] at stage 9/10, five out of five cases showed Hoxa3 induction, whereas three out of seven cases showed Hoxd4 expression (Table 3). This lower frequency of Hoxd4 expression was observed even when 5x10-4 M RA was used to soak the beads, in which case one out of three embryos showed induction (Table 3). The Lim homeobox gene expression of the ectopic r4 SM neurones was assessed in E6 embryos grafted at stage 10 (RA 10-4 M, n=3; RA 5x10-4 M, n=2), and Islet2 positive/Lim3 negative motoneurones were found on the grafted side (Fig. 4K,L). Hence, ectopic RA-induced SM neurones have an abducens phenotype, as in somite grafting experiments. The invariant induction of Hoxa3 and lower frequency of induction of Hoxd4 are broadly consistent with the abducens phenotype of the induced neurones.
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Misexpression of Hoxa3 is sufficient to induce ectopic SM
neurones in the rostral hindbrain
Although most of our data indicate a correlation between rostrocaudal
motoneurone identity and Hox gene expression, there were examples to the
contrary. For example, in rostral to caudal grafts (r3/r8a), r3 expressed Hox
genes typical of r8 identity, but failed to produce SM neurones. In order to
test directly the role of Hox genes in specifying rostrocaudal identity of
motoneurones, in ovo electroporation was used to misexpress the full-length
Hoxa3 cDNA in the rostral hindbrain of stage 8-16 embryos (r1-r4
region). Hoxa3 is expressed from the rhombomere 4/5 boundary caudally
(Fig. 1A,D) and is therefore a
good candidate to impose an abducens phenotype on r5 and r6 motoneurones
(Capecchi, 1997). To allow
identification of the ectopic region of Hoxa3 expression in the chick
hindbrain, a mouse Hoxa3 cDNA was used
(Fig. 5A,C), and to facilitate
the screening of electroporated embryos, a tauGFP plasmid was
co-electroporated (Fig. 5B,C).
The results of electroporation of the two constructs were compared with
control electroporations of the tauGFP plasmid alone. GFP expression
was observed in embryos 3 hours after electroporation, as previously reported
(Momose et al., 1999
). After
48 hours (equivalent of E4), embryos showing unilateral GFP expression in the
rostral hindbrain were processed by whole-mount in situ hybridisation for
Hb9. Ectopic Hb9 motoneurones were found only in those
embryos that had received both tauGFP and Hoxa3 plasmids
(n=21; Fig. 5D-G), but
never in the control group (n=5; data not shown). Interestingly, each
of rhombomeres 1 to 4 was found to produce Hb9-positive motoneurones
in some cases, in contrast to the inability of r1-3 to generate SM neurones in
somite or RA bead grafting experiments. In all embryos examined
(n=5), some GFP-positive axons left the neuroepithelium from ectopic
ventral exit points in the GFP/Hoxa3 positive region, consistent with
the induction of SM neurones (data not shown). To assess Lim gene expression
patterns of these ectopic SM neurones, embryos were grown up to E5, and GFP
fluorescence was combined with double-immunostaining for anti-neurofilament
antibodies and anti-Lim antibodies on serial sections
(Fig. 6A-D). These experiments
showed that a similar number of motoneurones were present in both the control
and electroporated sides (Fig.
6A,B), but ectopic Islet2-positive motoneurones were present only
on the GFP-positive electroporated side
(Fig. 6A,C,D). The majority of
Islet2-positive motoneurones were Lim3 negative, Which is indicative of an
abducens phenotype, but occasional Islet2/Lim3-positive neurones were detected
(Fig. 6C,D). Because in these
experiments, embryos were fixed earlier (E5) than in other experiments (E6),
this may reflect the fact that accessory abducens neurones (destined to be
Islet1/Lim3) are in the process of migrating laterally and have yet to
downregulate Islet2. Hence, misexpression of Hoxa3 at stage 8-16 is
sufficient to induce SM neurones in the rostral hindbrain. However, the number
of motoneurones within the Hoxa3 misexpression domain was relatively
small compared with those at axial levels that normally generate SM neurones.
Electroporation of Hoxa3 at stage 17 or later failed to induce
Hb9 expression (Fig.
5H,I), showing that the window of competence to generate SM
neurones extended only up to stage 16. As the domain of expression of
Hoxb3 also coincides with the region that generates SM neurones, we
also tested whether misexpression of Hoxb3 in r1-4 could induce
ectopic SM neurones. However, co-electroporation of a human HOXB3
plasmid and a tauGFP plasmid at the same stages as for Hoxa3
(stage 8-16) resulted in a few ectopic Hb9-positive cells in the
rostral hindbrain in only two out of seventeen cases (data not shown), and in
the majority of cases no SM neurone induction was observed
(Fig. 5J,K). Thus the effect of
Hoxb3 in SM neurone induction appears to be weak or absent compared
with that of Hoxa3.
|
|
In Hoxa3 electroporated embryos, we performed double
immunostaining on transverse sections showing GFP expression, to visualise the
domains of expression of various dorsoventral markers of progenitor domains or
cell fate. In particular, we focused on patterning changes in regions in which
ectopic SM neurones were present, as shown by immunopositivity using an
antibody against the SM markers Mnr2/Hb9 (and which we shall refer to as
Hb9-positive cells). In embryos analysed at stage 27, Hb9-positive induced SM
neurones were located ventrally within the hindbrain, and were in some cases
interspersed with or ventral to Chx10-positive interneurones
(Fig. 6E-H). As the location of
these cells would be consistent with the conversion of the p2 domain into an
ectopic pMN domain, we analysed whether at earlier stages (e.g. stage 18)
Olig2 had been induced in the GFP/Hb9-expressing domain. At this stage,
ectopic Hb9-psostitive cells were observed laterally as well as ventrally,
which suggests that some more laterally-located cells may die, leading to the
ventral localisation of Hb9-postitive cells observed at stage 27
(Fig. 6I-K). Only a few of the
most ventral of these Hb9-positive cells were Islet1/2-positive, which is
consistent with this idea (Fig.
6K; data not shown). However, no ectopic Olig2 was detected at
r2-4 levels (Fig. 6O), despite
the presence in the same embryo of an Olig2-postitive domain at rhombomere
levels 5-8 where resident Hb9-positive motoneurones were also detected
(Fig. 6P). However, Olig2
expression was induced coincident with ectopic Hb9-postitive cells in caudal
r1 in response to Hoxa3 overexpression
(Fig. 6S,T), perhaps reflecting
an enhanced tendency for r1 to produce SM neurones (the SM trochlear nucleus
lies in rostral r1 although it is Hb9-negative; see
Fig. 1). The p2 marker Irx3 was
repressed in GFP-expressing regions within r2-4
(Fig. 6L-N,S,T). Irx3
and Olig2 have been shown to be mutually repressive
(Novitch et al., 2001), and
yet these data suggest that Hoxa3 is capable of repressing
Irx3 and inducing SM neurones without induction of Olig2.
The localisation and level of Nkx2.2 expression failed to change following
Hoxa3 overexpression (Fig.
6L), whereas Pax6 expression was repressed to a modest extent, and
particularly in dorsal regions (Fig.
6Q,R). Taken together, the downregulation of both Irx3 and Pax6
would be consistent with the induction of an ectopic pMN domain that generates
ectopic SM neurones, although this domain apparently lacks Olig2
expression.
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DISCUSSION |
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Cranial motoneurone identity is plastic prior to neural tube
closure
Our data indicate that cranial motoneurone commitment to a particular
rostrocaudal identity must occur in a very restricted time period at stage
10-11 and coincident with the time of neural tube closure. These results are
broadly comparable to those for the spinal cord, in which motoneurone
progenitor identity is labile at stage 11 but becomes fixed just a few hours
later, at around stage 12 (Ensini et al.,
1998; Lance-Jones and
Landmesser, 1980
; Matise and
Lance-Jones, 1996
). In the hindbrain the first post-mitotic
motoneurones are detected using Sc1 and Islet1 markers at late stage 13
(Ericson et al., 1992
;
Guthrie and Lumsden, 1992
;
Varela-Echavarría et al.,
1996
). As the acquisition of a generic motoneurone fate remains
sensitive to local Shh signalling until late in the final cell cycle of
motoneurone progenitors (Ericson et al.,
1996
), rhombomere commitment to generate a particular repertoire
of motoneurones occurs relatively early in the process of motoneurone fate
specification.
Rostrocaudal patterning of cranial motoneurones by the paraxial
mesoderm and RA
Our results from transplantation experiments are consistent with the
existence of a mesoderm-derived caudalising activity that is capable of
eliciting somatic motoneurone differentiation from at least a subset of
progenitors. The timing and modality of action of such a caudalising activity
differs along the rostrocaudal axis, with caudal rhombomeres being more
susceptible than rostral ones. Prior to stage 10, both r3 and r4 were
competent to express Hb9/Islet2 and produce SM neurones in response
to somite signals when transplanted into r8 caudal position. However, by stage
10 only r4, and not r3, could generate SM neurones following transposition to
r8, whereas at stage 11, r7 was still capable of responding to caudal signals.
This suggests that the competence to respond to somite signals by SM neurone
differentiation decreases with time, and is switched off more rapidly in r3
than in r4 and more caudally. Moreover, there appears to be a gradient of
signals along the rostrocaudal axis, because for the same rhombomere (r4)
transplanted at the same stage (10/11), Islet2 was induced in grafts
made into the caudal but not the rostral part of rhombomere 8.
In caudal rhombomere transpositions, caudalising signals are likely to come
from the somites, an idea supported by the results of somite grafting
experiments. Somites transplanted beneath the neural tube at r2-4 levels
induced SM neurone differentiation in r4, as well as a caudalisation of Hox
gene expression. Perhaps surprisingly, somite grafts did not induce SM
neurones in r3, even at a stage (stage 9) at which transplantation of r3
tissue to caudal r8 (adjacent to s5) would have resulted in SM neurone
differentiation. This may suggest the existence of factors capable of
repressing the caudalising agent, localised in rostral rhombomeres and/or the
rostral (pre-otic) paraxial mesoderm. We found that r4 also manifested a
differential response to caudal transplantation or juxtaposition of a somite,
as in the former case hypoglossal neurones were induced, whereas in the latter
case abducens neurones formed. The explanation for this finding appeared to be
that Hoxa3 expression was induced in r4 more rapidly than
Hoxd4, which is consistent with previous studies
(Grapin-Botton et al., 1997;
Itasaki et al., 1996
).
Expression of Hoxa3 in the absence of Hoxd4 is consistent
with an abducens motoneurone phenotype, and by the time Hoxd4 was
switched on, motoneurone fates and axon pathways might already have been
established.
We have found that the rostral implantation of RA beads mimicked the action
of the somite, inducing SM neurones in r4, but not r3, and with an identical
stage dependence. Indeed, it has been proposed that RA mediates a part of the
caudalising ability of the paraxial mesoderm in patterning the rostrocaudal
axis (Berggren et al., 1999;
Gavalas and Krumlauf, 2000
;
Gould et al., 1998
;
Maden et al., 1998
;
Swindell et al., 1999
), and RA
is produced by the somites (Maden et al.,
1998
). Our finding that only somites caudal to s5 were capable of
inducing SM neurones is also consistent with the idea that RA is involved,
because RA is generated at a high level in younger somites
(Maden et al., 1998
;
Swindell et al., 1999
) and the
ability of the somites to repattern Hox gene expression is lost in a rostral
to caudal wave (Itasaki et al.,
1996
). It is thus possible that RA influences SM neurone
patterning through a rostral (low) to caudal (high) gradient in the somitic
mesoderm and possibly the hindbrain (for a review, see
Maden, 1999
). Application of
RA beads reliably induced Hoxa3 but only induced Hoxd4 in a
proportion of cases, in contrast to the invariant induction of Hoxd4
in somite grafts. This observation may be consistent with studies showing that
somitic signals capable of inducing Hoxd4 are not entirely accounted
for by RA; a higher molecular weight factor is involved
(Gould et al., 1998
). In view
of the lack of RA-mediated induction of SM neurones in r3, higher doses of RA
were applied: this did not induce ectopic SM neurones in r3, but instead
induced SM neurones on both sides of r4. These results also tend to favour the
idea that r3 or its surrounding mesoderm contains an inhibitory factor that
modulates the action of RA, preventing SM neurone differentiation. Indeed, our
recent results from in vitro experiments have shown that the ability of RA to
induce SM neurones in the rostral hindbrain is modulated by the RA-degradative
enzyme Cyp26, and that Cyp26 expression in the neuroepithelium is
upregulated by factors derived from the mesoderm and endoderm
(Guidato et al., 2003
).
Hox genes as determinants of rostrocaudal motoneurone identity
Taken together, the results of our grafting experiments showed that
rostrocaudal motoneurone identity and Hox gene expression profile are
intimately linked. Nevertheless there were examples to the contrary. For
example, r3 grafts in r8a expressed Hoxa3, Hoxb3 and Hoxd4
but failed to produce hypoglossal SM neurones, which are normally
characteristic of neuroepithelium with that Hox `code'. In addition, somite
grafts induced expression of all three Hox genes in r4, but the neurones
produced were abducens rather than hypoglossal. Therefore in grafts of r3 to
r8a, by the time Hox gene expression is initiated, the time-window for
motoneurone specification is already over, whereas for somite grafts,
Hoxa3 has an earlier onset of expression than Hoxd4, leading
to an abducens phenotype. Another example of the mismatch between Hox
expression and motoneurone fate concerns stage 9 r5 grafts to the r3 position;
these grafts expressed Hoxa3 and Hoxb3 but did not generate
SM abducens neurones. In this case, Hox expression was at a lower level than
at more caudal axial levels and so levels of Hox protein may not have been
high enough to maintain segment identity
(Greer et al., 2000).
In a more direct test of the role of Hox genes, we misexpressed Hoxa3 rostral to its normal expression limit and obtained SM neurone induction in r1-4, which indicates that driving a high level of Hoxa3 expression could overcome any inherent inhibition of SM neurone production in the rostral hindbrain. Induction of SM neurones by Hoxa3 could be accomplished in electroporations performed up to stage 15/16. This data is broadly consistent with that from somite grafting experiments in which induction of Hoxa3 in r4 level occurred approximately 36 hours after grafting, when embryos were approximately stage 15/16, and this was sufficient to induce differentiation of SM neurones with abducens phenotype. Hoxa3 was found to repress Irx3 and Pax6, and Hb9-expressing SM neurones were found adjacent to, or interspersed with, Chx10-positive cells, which are derived from the Irx3-expressing domain. This suggests that ectopic SM neurones are induced at the expense of presumptive Chx10-positive interneurones. However, no induction of Olig2 was seen in r2-4 of the hindbrain, but it was detected in r1. As Olig2 has been shown to play an important role in SM neurone generation, this implies that Hoxa3 can function in parallel with, or downstream of, Olig2 in hindbrain SM neurone differentiation, at least at r2-4 axial levels.
In Hoxa3 electroporated embryos, SM neurones were present in
relatively large numbers in embryos analysed at stage 18, but were present in
small numbers at stage 27, when they were restricted to a ventral column on
either side of the floor plate and had a phenotype more consistent with
abducens identity. These data are thus consistent with Hox3 paralogues being
involved with SM specification, and the abducens phenotype in particular.
However, the maintenance of these motoneurones following initial induction
seems to occur only for more ventrally located cells, and thus may depend on
other local factors. In both mouse and chick there are three Hox3 paralogues,
Hoxa3, Hoxb3 and Hoxd3, which, based on a number of studies,
are expressed to a rostral limit at the r4/5 boundary
(Capecchi, 1997;
Lumsden and Krumlauf, 1996
).
Although little is known about the early expression pattern of Hoxd3
in the chick hindbrain, Hoxa3 and Hoxb3 are expressed to
this rostral limit in the early neural plate
(Lumsden and Krumlauf, 1996
;
Rex and Scotting, 1994
). Hence
the early expression domains of Hoxa3 and Hoxb3 genes
coincide with the territory of SM neurone differentiation, making them good
candidates for patterning SM neurones. It therefore remains to be determined
which Hox genes, and in which combinations, give rise to SM neurones of
particular phenotypes. The analysis of various Hox-null mutant mice implicates
Hox genes in conferring rostrocaudal identity upon motoneurones
(Gavalas et al., 1997
;
Gavalas et al., 1998
;
Goddard et al., 1996
;
Lumsden and Krumlauf, 1996
;
Studer et al., 1996
). In
Hoxa3 mutants, there are defects in the formation of the ganglion of
the IX cranial nerve (glossopharyngeal), which may relate to aberrant neural
crest migration, and in addition glossopharyngeal (BM/VM) motoneurones in r6
project incorrectly, possibly as a result of the ganglionic defect
(Watari et al., 2001
). To our
knowledge no defects in the abducens or hypoglossal SM neurone populations
have been reported in Hox3 paralogue mutants, although neural crest and
skeletal patterning are affected (Chisaka
and Capecchi, 1991
; Condie and
Capecchi, 1993
). However, because mice mutant for both
Hoxa3 and Hoxd3 show defects not found in either of the
single mutants (Condie and Capecchi,
1993
), SM neuronal patterning might require further analysis in
these or other double or triple Hox3 mutants.
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ACKNOWLEDGMENTS |
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