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First published online November 21, 2006
doi: 10.1242/10.1242/dev.02694
1 Gene Expression Laboratory, The Salk Institute, La Jolla, CA 92037, USA.
2 Department of Medicine, University of California, San Diego, 9500 Gilman
Drive, La Jolla, CA 92093, USA.
Author for correspondence (e-mail:
pfaff{at}salk.edu)
Accepted 12 October 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Tbx20, Migration, Branchiomotor, Hindbrain, Wnt, Planar cell polarity, Mouse
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Marin and Rubenstein, 2003).
Neuronal migration increases the cellular complexity within each region of the
brain and presumably facilitates the assembly of more complex circuits.
Members of the T-box transcription factor family (Tbx) have been linked to the
proper development of migrating cells (reviewed by
Naiche et al., 2005;
Smith, 1999). During
gastrulation, brachyury and Tbx2b control the cellular reorganization of the
embryo that drives convergent extension and notochord formation
(Fong et al., 2005;
Wilkinson et al., 1990).
Similarly, Tbx5 and Tbx20 have been implicated in the cardiac cell migration
associated with the formation of heart chambers
(Cai et al., 2005;
Hatcher et al., 2004;
Singh et al., 2005;
Stennard et al., 2003;
Stennard et al., 2005), Tbx1
in neural crest migration and proper middle/inner ear development
(Moraes et al., 2005), and
Tbr1 in cellular organization within the cerebellum
(Fink et al., 2006).
Tbx20 is expressed by migratory branchiomotor (BM) and visceromotor (VM)
neurons in the hindbrain (Ahn et al.,
2000; Kraus et al.,
2001). BM neurons innervate branchial arch-derived muscles that
control jaw and eye movement, facial expression, and muscles within the
pharynx and larynx. VM cells innervate parasympathetic ganglia to control
lacrimal gland activity and salivation. During hindbrain development,
trigeminal (V) BM cell bodies migrate dorsolaterally within rhombomeres 2-3
(r2-3) (summarized in Fig.
1I-L). Facial (VII) VM cells move dorsolaterally and settle in a
unique location near the pial surface of r5, whereas the BM component of the
facial nucleus is generated within r4 but moves tangentially along the
anteroposterior axis of the hindbrain into r6 and then migrates
dorsolaterally. Interestingly, vestibuloacoustic (VIII) neurons, which provide
efferent input to the inner ear, are also generated within r4 and exhibit a
very unique migratory pattern in which their cell bodies cross the midline to
the contralateral side of the hindbrain
(Fritzsch, 1996;
Simon and Lumsden, 1993;
Tiveron et al., 2003). Thus,
multiple subclasses of motor neurons are generated in specific locations
within the hindbrain. Although each of these motor neuron subtypes displays a
unique pattern of cell migration, all of them are apparently related by virtue
of expressing Tbx20. Nevertheless, the function of Tbx20 in these distinct
populations of migratory cells is unknown.
Several observations indicate that facial neurons are responsive to
extrinsic cues within their local environment. In Kreisler
(Mafb - Mouse Genome Informatics) and Krox20 (Egr2
- Mouse Genome Informatics) mutant mice, r5 progenitors acquire an r6-like
identity (Manzanares et al.,
1999; Schneider-Maunoury et
al., 1993; Seitanidou et al.,
1997; Swiatek and Gridley,
1993). Despite their respecification, facial cells initiate their
typical pattern of caudal migration but prematurely move radially once they
encounter the new environment adjacent to r4
(Garel et al., 2000;
Schneider-Maunoury et al.,
1997). Although these studies demonstrate that r5 is not required
to initiate tangential migration, transplants of mouse r5 tissue placed
homotopically into chick embryos trigger ectopic facial cell migration
(Studer, 2001). Vascular
endothelial growth factor (VEGF164) mutant mice exhibit tangential
migration defects of facial motor neurons indicating that this factor may
selectively attract facial BM cells
(Schwarz et al., 2004).
The intrinsic factors controlling facial motor neuron migration have begun
to emerge through extensive genetic studies. A transcriptional cascade
including Hoxb1, Gata2, Gata3 and Phox2b regulates their identity and
migration (Bell et al., 1999b;
Goddard et al., 1996;
Nardelli et al., 1999;
Pata et al., 1999;
Pattyn et al., 2000;
Studer et al., 1996).
Transcription factors expressed at later stages, such as Nkx6.1 (Nkx6-1 -
Mouse Genome Informatics) and Ebf1, are dispensable for facial motor neuron
specification but are necessary for their tangential migration
(Garel et al., 2000;
Muller et al., 2003). Cell
surface molecules and intracellular signaling components have been implicated
in facial neuron migration. Mouse mutants of the VEGF164 receptor gene
neuropilin 1 exhibit tangential migration defects comparable to
VEGF164 mutants (Schwarz et al.,
2004). Zebrafish mutants trilobite and
strabismus (vang-like 2 - Zebrafish Information Network),
van gogh (vang-like 1 - Zebrafish Information Network) and
prickle1 (pk1), which are components of the planar cell
polarity pathway (PCP), arrest facial neuron migration
(Bingham et al., 2002;
Carreira-Barbosa et al., 2003;
Jessen et al., 2002).
Nevertheless, the relationship between facial cell migration and that of the
other hindbrain motor neuron classes is not well understood, and it is unclear
whether shared gene regulatory networks are used for the migration of distinct
motor neuron subtypes.
Here, we show that Tbx20 is selectively expressed by divergent classes of migratory motor neurons within the hindbrain. The regional patterning of the rhombomeres and generation of post-mitotic motor neurons were normal in Tbx20 conditional mutants, consistent with the relatively late but selective onset of Tbx20 expression in post-mitotic BM/VM neurons. Tbx20 mutants displayed an assortment of cell migration defects including abnormal dorsolateral migration of BM trigeminal cells and VM facial neurons, arrested tangential migration of facial BM neurons, and a lack of trans-median migration of vestibuloacoustic cells. We found that hindbrain motor neurons lacking Tbx20 retained the ability to extend neurites into the periphery; thus, it is unlikely that the severe disruption of neuronal migration in these mutants arises indirectly from a loss of axons that might provide a substrate for soma translocation. Deletion of Tbx20 resulted in the downregulation of Tbx2 and key components of the PCP pathway. Together, our results demonstrate that Tbx20 is shared by a variety of BM/VM neurons to regulate their proper cell body migration.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Shirasaki et al., 2006).
Neural-specific Tbx20 conditional mutant mice were obtained by
crosses between a heterozygous animal carrying a null allele of Tbx20
and a Nestin::Cre allele (Jackson Laboratory), and a homozygous
animal carrying a Tbx20fl conditional allele, to produce
animals with the genotype: Tbx20fl/KO;
Nestin::Cre+/-. Ret mutant mice have been described
previously (Schuchardt et al.,
1994) and were kindly provided by F. Costantini (Columbia
University, New York, USA).
Immunohistochemistry
Embryos were obtained and processed for immunohistochemistry as previously
described (Thaler et al.,
1999). To generate a Tbx20-specific antibody, a peptide
corresponding to amino acids 426-445 of mouse Tbx20 was synthesized, coupled
to hapten and injected into rabbits. Additional antibodies used in this study
are: guinea pig anti-Isl1 (Thaler et al.,
2004), rabbit anti-Isl1/2
(Ericson et al., 1992), rabbit
anti-Phox2b (Pattyn et al.,
2000), rabbit and guinea pig anti-Hb9
(Thaler et al., 1999), rabbit
anti-Lhx3 (Sharma et al.,
1998), rabbit anti-GFP (Invitrogen), monoclonal anti-HA (Babco),
monoclonal anti-MNR2 (Developmental Studies Hybridoma Bank)
(Tanabe et al., 1998), rabbit
anti-Nkx6.1 (Beta Cell Biology Consortium) and goat anti-GATA2 (Santa Cruz)
antibodies. Fluorophore-conjugated species-specific secondary antibodies were
used as recommended (Jackson Laboratory and Invitrogen). For whole-mount
neurofilament staining, embryos were fixed, permeabilized with a graded
methanol series and incubated with rabbit anti-neurofilament antibody
(Chemicon) followed by HRP-conjugated secondary antibody (Jackson Laboratory)
for DAB staining.
In situ hybridization
Embryos were fixed in 4% paraformaldehyde, mounted and cryosectioned for in
situ hybridization. Transverse sections were hybridized with
digoxigenin-labeled probes specific for individual genes. Each cDNA sequence
used to generate probes was amplified from mouse or chick embryonic cDNA using
the Advantage cDNA PCR Kit (Clontech) and TOPO Cloning Kit (Invitrogen). For
flat-mounted hindbrain in situ hybridization, hindbrains were dissected and
processed as previously described with minor modification
(Garel et al., 2000;
Ohsawa et al., 2005). Briefly,
dissected hindbrains were fixed, permeabilized with methanol and proteinase K
treatment, hybridized with digoxigenin-labeled probes, incubated with
anti-digoxigenin alkaline phosphatase-conjugated antibody (Roche), and
developed with NCIP/NBT substrates (Roche). Specimens were cleared with
glycerol and flat-mounted for visualization.
Flat-mount hindbrain culture
Embryonic day (E) 11.5 mouse embryos were used. Flat-mounted preparations
of the hindbrain were prepared as previously described
(Shirasaki et al., 1995). The
neural tube of the hindbrain region was dissected, cut along the dorsal
midline and flat-mounted in ice-cold DMEM/F12 (Invitrogen). The hindbrain was
then placed on a collagencoated membrane in a 6-well tissue culture plate
(Yamamoto et al., 1989)
(Transwell Collagen, Corning Costar) with the ventricular side facing down,
and cultured in the medium for 30 hours as described
(Shirasaki et al., 1998).
After culture, the preparations were fixed in 4% paraformaldehyde in PB for 2
hours at room temperature, followed by several rounds of washing with PBS. The
preparations were then detached from the membrane filter and
immunohistochemistry was performed as described above.
Chick in ovo electroporation
Chick eggs (Charles River and McIntyre Farms) were incubated in a
humidified chamber. DNA constructs were injected into the lumens of chick
embryonic spinal cords at HH stage 10 to 12. Electroporation was performed
using a square wave electroporator (BTX)
(Nakamura et al., 2000).
Coelectroporation resulted in >80% of cells expressing all constructs.
Incubated chicks were harvested and analyzed at HH stage 20 to 25.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The pattern of Tbx20 expression was similar to
that of transcription factors Isl1 and Phox2b, whose overlapping expression
marks BM/VM neurons (Fig. 1E)
(Pata et al., 1999;
Pattyn et al., 2000). As
development progressed from E10.5 to E13.5, the location of
Tbx20+ cells closely matched the position of
migrating Isl1+/Phox2b+ BM/VM neurons
along the entire anteroposterior axis of the hindbrain
(Fig. 1A-H).
Next, we generated an antibody against a unique peptide sequence at the
carboxy terminal end of the protein and performed immunocytochemistry. From
the onset of expression at E10.5, Tbx20 was restricted to
Isl1+ motor neurons (see Fig. S1 in the supplementary
material). Medially-located cells emerging from the ventricular zone expressed
Isl1 but not Tbx20 (Fig. 1M,
and see Fig. S1 in the supplementary material), suggesting that Tbx20
expression occurs shortly after Isl1. Since Isl1 expression is closely
associated with the post-mitotic birth of motor neurons
(Ericson et al., 1992), Tbx20
expression is likely to be initiated after neuroepithelial cells have exited
the cell cycle. The location of the
Isl1+/Tbx20+ cells corresponded to the
position of facial BM neurons (VII) in r4
(Fig. 1M). However, Tbx20 was
not detected in the somatic motor (SM) population of abducens (VI) motor
neurons (Fig. 1P, and see Fig.
S1 in the supplementary material), although it was found in the adjacent
facial BM neurons at r5. Similarly, SM hypoglossal (XII) neurons did not
express Tbx20, whereas the adjacent vagal (X)/cranial accessory (XI) BM
neurons were labeled at r8 (see Fig. S1 in the supplementary material).
Vestibuloacoustic (VIII) neurons are generated together with facial cells in
r4 and express Gata2 and Gata3 which distinguishes them from the facial
population which downregulate these factors upon differentiation
(Bell et al., 1999a;
Tiveron et al., 2003). At
E11.5, Tbx20 was detected in the ventral
Gata2+/Isl1+ vestibuloacoustic cells
(Fig. 1M-O). Based on these
labeling patterns, we conclude that trigeminal (V), facial (VII),
vestibuloacoustic (VIII), glossopharyngeal (IX), vagal (X) and spinal
accessory (XI) motor neuron groups express Tbx20
(Fig. 1I-L). Thus, the
dorsal-settling cranial motor neurons comprising the BM/VM cell groups express
Tbx20 during their cell migration period, whereas ventral-settling SM cells
within the abducens (VI) and hypoglossal (XII) nuclei lack this T-box
factor.
|
;
Singh et al., 2005;
Stennard et al., 2005). We
thus generated conditional Tbx20 mutant mice selectively lacking
Tbx20 expression in neurons (Cai et al.,
2005). Tbx20fl/fl mice were mated to
Tbx20ko/wt; Nestin::Cre animals. Embryos with the
genotype Tbx20fl/ko; Nestin::Cre (abbreviated to
Tbx20 cKO) were used for our analysis. This genetic strategy was
chosen because nestin is broadly expressed in neuronal progenitor cells and
only one allele of Tbx20 required recombination to generate
Tbx20-null cells. Tbx20 cKO embryos lacked Tbx20 protein
expression from the outset of motor neuron development in the hindbrain
(Fig. 2A,B, and see Fig. S2 in
the supplementary material). In situ hybridization using exon 2-specific
probes demonstrated that this segment had been deleted as expected (see Fig.
S2 in the supplementary material). These findings indicate that the
recombination of Tbx20fl is efficient and occurs prior to
the normal expression of Tbx20 in motor neurons. The development of
Tbx20 cKO embryos appeared to be normal up to E12.0. After E12.5,
however, Tbx20 mutants began to die due to apparent heart defects
caused by transient expression of Nestin::Cre in developing cardiac
cells. Thus, we focused our analysis of hindbrain development on stages from
E10.5-E12.0.
We first examined whether the absence of Tbx20 prevented the specification
of BM/VM neurons, focusing on the r4 level of the hindbrain. The truncated
Tbx20 transcript remaining in the conditional mutants was used
as a lineage marker to identify Tbx20-null cells. We found that the
motor neurons normally destined to express Tbx20 were still present in E11.5
Tbx20 cKO embryos (Fig.
2C,D). At the r4 level, a detailed genetic cascade has been
defined for facial and vestibuloacoustic neuron specification. Hoxb1 is
thought to confer rhombomere 4 cell identity, leading to the subsequent
activation of Mash1 and Math3 (Ascl1 and Neurod4, respectively - Mouse Genome
Informatics), Gata2, Phox2b, Isl1 and Nkx6.1
(Muller et al., 2003;
Ohsawa et al., 2005;
Pata et al., 1999;
Pattyn et al., 2000).
Transverse sections taken at r4 of E11.5 Tbx20 cKO embryos revealed a
normal distribution of Hoxb1, Math3 and Mash1 expression in
progenitor cells and newly formed motor neurons
(Fig. 2E-J). Furthermore, the
detection of Nkx6.1+/Isl1+ post-mitotic
motor neurons suggested that the initial specification of these cells was
unaffected in Tbx20 cKO embryos
(Fig. 2K,L). Likewise, Phox2a,
whose expression is restricted to post-mitotic facial cells, was present in r4
from Tbx20 cKO embryos (Fig.
2M,N). Similarly, the general pattern of Gata2 labeling
was essentially unchanged in flat-mounted hindbrains from Tbx20
mutants, although the medial nVIII cells within r4 failed to express
Gata2 when Tbx20 was deleted
(Fig. 2O,P, and data not
shown). Although many features of hindbrain development appeared normal in
Tbx20 cKO embryos, we found that the anteroposterior distribution of
facial BM neurons marked by Phox2b was altered in the mutants
(Fig. 2Q,R). Taken together,
these findings indicate that the elimination of Tbx20 from r4-derived
facial motor neurons does not disrupt their specification nor initial
generation, but does appear to hinder the tangential migration of their cell
bodies.
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Motor neuron soma migration is disrupted in Tbx20 mutants
Although BM/VM neurons appeared to be specified properly based on their
expression of marker genes, we found that the cells became ectopically located
in Tbx20 cKO mutants. In control embryos at E11.5,
Isl1+ facial BM neurons have begun to migrate caudally past
the Hb9+ abducens cells generated at r5
(Fig. 4A). Facial neurons
failed to migrate caudally in Tbx20 mutants
(Fig. 4B). The dorsolateral
migration of trigeminal neurons was also disrupted in Tbx20 mutants
(Fig. 4A,B). Normally,
trigeminal cells at r2-3 have migrated dorsolaterally by E11.5
(Fig. 4A), but in
Tbx20 mutants the cells were still visible adjacent to the midline
(Fig. 4B). A day later in
development, at E12.5, trigeminal cells were found scattered along the
mediolateral axis of Tbx20 mutants, stalled in their
laterally-directed migration (see Fig.
5C,D,G,H). These results were further confirmed by examining the
distribution of Phox2b+/Isl1+ trigeminal
neurons in transverse sections taken at specific rhombomere levels. By E11.5,
the majority of trigeminal neurons had migrated into the lateral half of r2-3
in control embryos, whereas most of the
Phox2b+/Isl1+ cells were located medially
in Tbx20 conditional mutants (Fig.
4C,D,I). Similarly, at r4 levels, the lateral migration of the VM
subclass of facial neurons initiated their lateral migration but failed to
reach their final destination (Fig.
4E,F). Furthermore, the caudal migration of facial BM neurons
resulted in a large number of
Phox2b+/Isl1+ cells entering r6 in control
embryos, whereas no facial neurons were observed in r6 E11.5 Tbx20
mutants (Fig. 4G,H,J).
|
; Uemura et al.,
2005). The reporter mice revealed migrating vestibuloacoustic cell
bodies and their axonal processes within the rhombomere 4 midline
(Fig. 4K). The absence of GFP
labeling in the r4 midline indicated that vestibuloacoustic neurons failed to
migrate to the contralateral side of the hindbrain in Tbx20 mutants
(Fig. 4L). Thus, our results
indicate that multiple types of migration defects arise in Tbx20
mutants: (1) the dorsolateral migration of trigeminal and facial VM cells is
disrupted; (2) the caudal (tangential) migration of facial BM neurons is
blocked; and (3) the trans-median migration of vestibuloacoustic neurons is
defective (Fig. 4M,N).
Tbx20 mutants display subtle defects in peripheral motor axon projections
Migration defects in BM/VM neurons in the absence of Tbx20 prompted us to
examine whether axons of these cells innervate their peripheral targets
normally. To examine the axon projections of trigeminal and facial neurons, we
used the SE1::gfp line to selectively label the cells. Despite the
abnormal cell body settling of trigeminal and facial neurons in Tbx20
mutants, we found that their axons selected the correct exit points from r2
and r4, respectively (Fig.
5A-H). Next we labeled the peripheral projections of cranial
neurons by performing neurofilament staining on E11.5 embryos. The overall
pathfinding of both trigeminal and facial neurons appeared normal
(Fig. 5I,L). However, the axon
terminals of facial neurons were not normal in Tbx20 mutants. The
distal ends of wild-type facial motor axons exhibit a characteristic
triangular pattern at this stage, whereas facial neurons in Tbx20
mutants extended axons beyond their normal stopping point and occasionally
were misrouted and looped back (Fig.
5J,M). Similarly, subtle defects in the routing and fasciculation
of vagal axons were noted in Tbx20 mutants
(Fig. 5K,N). These data confirm
that the overall ability of BM/VM neurons to properly extend axons into the
periphery is preserved in Tbx20 mutants, but that subtle axon
pathfinding errors exist.
Cell body migration defects in Tbx20 mutants are not due to developmental arrest
In principle, the soma migration defects in Tbx20 mutants could
arise because embryos develop more slowly. To exclude this possibility and
establish that migration defects are autonomous to the neuroepithelial cells,
we used organotypic culture of the hindbrain to extend the survival of the
mutant tissue. Hindbrains from SE1::gfp transgenic mice were
dissected from E11.5 embryos and cultured as flat-mounts. GFP and Isl1
labeling were used to monitor the cell body position of motor neurons after
culturing for 30 hours. During the culture period we found a marked increase
in the number of Isl1+/GFP+ motor neurons
that migrated caudally from r4 into r5/6
(Fig. 6A-D). Facial BM neurons
within hindbrains derived from Tbx20 mutant embryos failed to migrate
caudally after 30 hours, in contrast to the migration observed with controls
(Fig. 6E,F). Likewise, the
dorsolateral migration of trigeminal neurons also failed to occur in explants
derived from Tbx20 mutants, whereas trigeminal cell migration was
observed with controls (Fig.
6G,H). These findings indicate that the lack of BM/VM neuron
migration observed in Tbx20 mutants is unlikely to be due to a
general delay in the development of these embryos.
|
;
Singh et al., 2005;
Stennard et al., 2005). To
determine whether similar genetic interactions operate within hindbrain motor
neurons, we examined the expression of Tbx2 in Tbx20 mutants
using in situ hybridization. Unlike in the heart, Tbx2 and Tbx20 are
co-expressed by BM/VM neurons rather than being mutually exclusive
(Fig. 7A). Tbx2 mRNA
levels were significantly reduced in trigeminal cells (V) and almost
undetectable in facial BM neurons (VII) from Tbx20 mutants
(Fig. 7A-F). Thus, Tbx20
appears to activate Tbx2 expression in motor neurons and repress it in
myocardial cells. Similarly, Tbx20 represses Isl1 in myocardial cells
(Cai et al., 2005), whereas
Isl1 and Tbx20 are co-expressed in cranial motor neurons (see
Fig. 1M). Furthermore, Isl1 is
not upregulated in cranial motor neurons in Tbx20 mutants
(Fig. 4A,B,
Fig. 8Y). To examine the
genetic interaction among transcription factors defining BM neurons, we
ectopically expressed Phox2a in the chick embryonic neural tube using in ovo
electroporation. Previous studies have shown that the closely related Phox2b
transcription factor is sufficient to trigger ectopic BM/VM neuron
differentiation (Dubreuil et al.,
2000). We found that Phox2a induced the ectopic expression of the
motor neuron marker Isl1 within the dorsal neural tube
(Fig. 7G). Adjacent sections
revealed that the Isl1+ cells also expressed Tbx20
and Tbx2 (Fig. 7G-I).
However, misexpression of Tbx20 failed to induce Tbx2 (data not shown),
indicating that Tbx20 is not sufficient to induce Tbx2 in the neural tube.
These findings suggest that Phox2a/b are upstream regulators of T-box
transcription factors in cranial motor neurons, and that Tbx20 and Tbx2 have a
different regulatory relationship in neurons compared with that in heart cells
(Cai et al., 2005).
Next we screened for changes in the expression of a battery of genes
implicated in facial motor neuron development, migration and axon navigation
to better understand the molecular basis for the migration defects in
Tbx20 mutants. We focused on BM facial neurons originating from r4
because numerous studies have identified genes required for their migration
(reviewed by Chandrasekhar,
2004). In Tbx20 mutants, we observed ectopic expression
of Ret in r4 facial neurons, whereas Ret is expressed by
wild-type facial cells after they migrate caudally from r4
(Fig. 8A-F). However, facial
neurons migrated normally in Ret mutants (see Fig. S4 in the
supplementary material), indicating that this gene is dispensable. Thus, the
ectopic expression of Ret in Tbx20 mutants is likely to
occur because facial motor neuron cell bodies are unable to depart from r4 in
Tbx20 mutants but they still initiate their normal expression of Ret
despite their ectopic location. Similarly, we also detected ectopic expression
of neogenin (Neo1) within r4 of Tbx20 mutants, probably due
to the stalled migration of facial cells. In the case of genes whose
expression normally begins prior to facial cell migration, including Cdk5,
Ebf1, Unc5h3 (Unc5c - Mouse Genome Informatics), neuropilin 1
and neuropilin 2, their expression was maintained within the ectopic r4 motor
neurons of Tbx20 mutants (Fig.
8Y; data not shown). Our results suggest that Tbx20 is dispensable
for the expression of these genes, and that these proteins are insufficient to
promote the tangential migration of facial neurons in the absence of
Tbx20.
|
|
; Carreira-Barbosa et
al., 2003; Jessen et al.,
2002). This prompted us to examine whether PCP genes are regulated
by Tbx20 in facial neurons; these included Wnt4, Wnt5a, Wnt11, Fzd7,
Prickle1, Prickle2, Celsr3/flamingo, Vang1 (Vangl1 - Mouse
Genome Informatics), Vang2/Stbm/Tri
(Vangl2 - Mouse Genome Informatics), dishevelled (Dsh1),
dishevelled 2 (Dsh2) and dishevelled 3 (Dsh3) (Dvl1,
Dvl2 and Dvl3, respectively - Mouse Genome Informatics)
(Fig. 8G-Y, and data not
shown). Among the genes examined, Wnt11, Fzd7, Pk1, Vang1, Vang2,
Celsr3 and Dsh3 were detected in wild-type facial cells as they
migrated from r4 to r6. We found that Wnt11, Fzd7, Vang1 and
Vang2 were significantly downregulated and Pk1 was
moderately reduced in facial neurons located within r4 of Tbx20
mutants. Dsh3 and Celsr3 were more broadly expressed in many
neural tube cells, including facial neurons, and were unaffected by the loss
of Tbx20 (Fig. 8G-Y, and see
Fig. S6 in the supplementary material). We also observed a loss of cadherin 8
expression (Fig. 8Y); however,
we cannot exclude the possibility that this is an indirect consequence of
facial neurons being trapped in r4 as cadherin 8 is normally only expressed
after cells enter into r6 (Garel et al.,
2000). Our results reveal that Tbx20 is required for the
coordinate expression of multiple components of the PCP pathway within
migrating facial neurons. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hierarchy of transcription factors for branchiomotor and visceromotor neuron development
During BM/VM neuron development the serial actions of multiple
transcription factors are believed to build a hierarchal cascade that ensures
the proper cell specification and subsequent differentiation of motor neuron
subtypes (reviewed by Chandrasekhar,
2004). Naturally, the roles of these transcription factors and the
degree of defects that arise in their absence vary. Phox2b is
essential for assigning BM/VM neuronal fates and consequently mutations in
this gene result in the loss of the entire BM/VM cell population
(Pattyn et al., 2000). In the
absence of Mash1 and Math3, which are restricted to the
progenitors of BM/VM cells, hindbrain motor neurons are generated but their
identity is mis-specified and therefore later features of their maturation,
such as cell body migration, fail to occur properly
(Ohsawa et al., 2005). By
contrast, the elimination of Nkx6.1, whose expression extends into
the post-mitotic BM/VM neurons, does not appear to switch motor neuron subtype
identity but is needed to regulate their migration
(Muller et al., 2003). Thus,
depending on the timing and location of expression, each factor performs
specific roles in controlling BM/VM neuron development.
|
;
Jessell, 2000), it is not
unexpected that this factor is dispensable for the generation of these
neurons. We found that BM/VM cells are generated, survive, extend axons and
appropriately express a wide array of genes in Tbx20 mutants.
Importantly, the overall transcription factor profile of these neurons was
intact. These findings indicate that the identity of BM/VM cells is properly
established independently of Tbx20 function. However, we did find that
Tbx2 expression was downregulated in Tbx20 mutants. Since
Phox2a induced Tbx20 and Tbx2 expression, we propose that
Tbx20 and Tbx2 are relatively late post-mitotic end products of the genetic
cascade controlling BM/VM neuron development
(Fig. 8Z).
Our findings differ somewhat from those reported by Takeuchi et al. using
RNA interference to knock down Tbx20 expression
(Takeuchi et al., 2005). These
authors report that Isl2 and Hb9 are downregulated following interference of
Tbx20 expression. However, we did not detect Tbx20 expression in the motor
neuron subtypes that express Isl2 and/or Hb9 (SM cells) at either hindbrain or
spinal cord levels (Fig. 1, and
data not shown), nor did we find a change in the expression of these markers
in our mutants. Likewise, the phenotype in the heart of Tbx20-null
mutants differs from that described using RNA interference
(Cai et al., 2005;
Singh et al., 2005;
Stennard et al., 2005;
Takeuchi et al., 2005). The
basis for these differences remains to be determined, but might have arisen
from non-specific RNAi effects or a general delay in development reported with
the embryos (Takeuchi et al.,
2005).
Tbx20 regulates cell body migration of trigeminal, facial and vestibuloacoustic neurons
Neuronal migration is observed in many regions of the brain and is thought
to facilitate the formation of more complex circuits comprising multiple
neuronal types (Hatten, 1999;
Marin and Rubenstein, 2003).
Thus, a variety of human disorders have been identified that arise due to
neuronal migration defects, including lissencephaly and Kallmann syndrome
(reviewed by Gleeson, 2001).
Numerous signaling molecules and cytoskeletal proteins have been identified
that are crucial for neuronal migration and it has long been appreciated that
neuronal interactions with glial cells represent one of the mechanisms that
guides the migration process (reviewed by
Bielas et al., 2004;
Hatten, 1999). The most
striking defect found in Tbx20 mutants was a lack of BM/VM cell
migration. Although these two motor neuron classes are functionally related,
they comprise diverse motor neuron subtypes that undergo different patterns of
cell soma migration. Facial BM neurons migrate tangentially from r4 into r6
orthogonal to the radial glial fibers, whereas trigeminal BM neurons in r2
migrate dorsolaterally along a non-radial pathway. Vestibuloacoustic cells
display a third type of cell body movement, migrating across the midline at r4
to the contralateral side of the hindbrain
(Chandrasekhar, 2004;
Fritzsch et al., 1993;
Simon and Lumsden, 1993). The
last step in the migration of these motor neuron subtypes is shared,
comprising the radial migration of the cell bodies toward their final settling
position near the pial surface of the neural tube.
Genetic studies of facial motor neuron migration have found that separate
signaling pathways are involved in the initial tangential and later radial
migration of these cells (Garel et al.,
2000; Muller et al.,
2003; Rossel et al.,
2005; Schwarz et al.,
2004). Despite defects in tangential migration in VEGF164
mutants, the radial migration of facial cells is preserved, suggesting that
other signals are responsible for this migratory process
(Schwarz et al., 2004).
Accordingly, Reeler mutant mice, lacking the reelin extracellular
matrix protein, exhibit defects in the radial pattern of BM/VM cell movement,
but the tangential path of soma migration is intact
(Rossel et al., 2005). Our
analysis did not focus on the late phase of radial migration because the
earlier steps in motor neuron migration were found to be defective, making it
unclear whether radial migration depends on Tbx20. Despite the expectation
that each migration pathway will rely on different signaling, we found that
all BM/VM neurons share a common requirement for Tbx20. This finding makes it
less likely that Tbx20 is alone sufficient to specify the directionality of
neuronal migration in response to extrinsic cues. Consistent with this, we did
not observe changes in the expression of cell surface molecules, such as
neuropilin 1 and neuropilin 2, implicated in controlling the tangential
migration of facial BM neurons (Fig.
8) (Schwarz et al.,
2004). Although it is possible that Tbx20 controls the expression
of components that comprise the general machinery required for neuronal
migration, this seems unlikely for several reasons. First, we did not observe
a change in expression of components of the general machinery for cell
migration such as Cdk5 (Fig. 8)
(Ohshima et al., 2002).
Second, Tbx20 is not expressed by all migrating neuronal populations, but is
restricted to BM/VM cells. Third, the overall ability to extend axons appeared
normal in motor neurons lacking Tbx20, arguing against a major cytoskeletal
defect.
In principle, we would predict that the mis-expression of Tbx20 in SM
neurons in the hindbrain might lead to ectopic cell body migration. We
attempted to test this by electroporating Tbx20 expression constructs into the
neural tube of chick embryos; however, we found that this triggered the
ectopic formation of motor neurons (see Fig. S5 in the supplementary
material). This activity made it difficult to assess whether motor neurons
residing in ectopic locations had differentiated at this site or migrated
there following the mis-expression of Tbx20. We did not expect Tbx20 to be
sufficient to promote motor neuron differentiation in chick embryos because
our loss-of-function analysis demonstrated that Tbx20 is not required to
generate motor neurons in mice. We discovered that Tbx20 mis-expression
induces HNF3ß+ floor plate cells (see Fig. S5 in the
supplementary material). Since the floor plate is a source of sonic hedgehog
for motor neuron differentiation (Jessell,
2000), it is possible that the ectopic appearance of motor neurons
following Tbx20 mis-expression occurs through a non-cell-autonomous pathway.
The basis for this neomorphic activity of ectopicallyexpressed Tbx20 is
unknown, but might reflect the ability of Tbx20 to mimic the activity of other
Tbx factors, such as brachyury, in the notochord
(Wilkinson et al., 1990).
Tbx20 controls planar cell polarity signaling in facial neurons
Our search to find downstream targets of Tbx20 led to the identification of
components of the PCP pathway: Wnt11 is a ligand; Fzd7 is a receptor; Vang1,
Vang2 and Celsr3 are cell surface molecules; and Pk1 and Dsh are intracellular
molecules (Montcouquiol et al.,
2006; Saburi and McNeill,
2005). Among these, zebrafish mutants lacking
stbm/tri/vang2 and pk1 have facial cell
migration defects (Bingham et al.,
2002; Carreira-Barbosa et al.,
2003; Jessen et al.,
2002). We observed downregulation of multiple components of the
PCP pathway including Stbm/Tri/Vang2 and
Pk1, providing compelling evidence that Tbx20 controls facial cell
migration by regulating PCP signaling in these cells
(Fig. 8Z). Studies from
zebrafish have found evidence for both cell-autonomous and non-autonomous
functions of PCP components in mediating facial cell migration
(Bingham et al., 2002;
Carreira-Barbosa et al., 2003;
Jessen et al., 2002). Thus, it
is somewhat surprising to find that a motor neuron-specific regulator such as
Tbx20 is required to target PCP gene expression specifically to facial motor
neurons. This might be interpreted to suggest that PCP genes function cell
autonomously within mammalian facial motor neurons. Nevertheless, we found
that Vang2 is normally expressed in a heterogeneous salt-and-pepper pattern
among migrating facial motor neurons, consistent with it having non-autonomous
functions. Although further studies are needed to confirm this, PCP signaling
might occur between neighboring motor neurons such that cells that form the
facial motor nucleus might influence the migration of one another.
Despite the strong correlation between Tbx20 and the PCP pathway in facial cells, we did not observe the presence of PCP components in trigeminal and vestibuloacoustic motor neurons. Thus, the transcriptional activity of Tbx20 appears to be highly context-dependent, and capable of controlling different cell migration programs in a cell type-specific manner. In facial motor neurons, Tbx20 regulates the caudally-directed migration of cells via control over the PCP pathway, whereas in trigeminal and vestibuloacoustic cells Tbx20 regulates cell migration by a different means.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: Biomedical Sciences Graduate Program, University of
California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
Present address: Department of Molecular and Cellular Biology, Huffington
Center on Aging, Baylor College of Medicine, Houston, TX 77030, USA
|
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