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First published online 11 March 2009
doi: 10.1242/dev.030510
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Department of Genetics and Developmental Biology, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030, USA.
* Author for correspondence (e-mail: jali{at}uchc.edu)
Accepted 11 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Thalamus, Compartment, Lineage restriction, Fate map, Mouse, Transcription factor, Gbx2
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Some
thalamic nuclei project topographically to specific areas of the cortex,
having a primary role in processing and relaying sensory input from the
periphery to the cortex; other nuclei broadly project to the cortex,
regulating the states of consciousness of the cortex
(Jones, 2001;
Jones, 2007). The thalamus
develops from the diencephalic part of the neural tube. Based on morphology
and gene expression, the vertebrate diencephalon is transiently divided into
three transverse segments called prosomeres 1-3 (p1-3), which are believed to
give rise to the pretectum, epithalamus and thalamus, and prethalamus,
respectively (Puelles and Rubenstein,
1993; Puelles and Rubenstein,
2003). Virtually all thalamic neurons in mice are generated from
the alar plate of the p2 segment between embryonic day (E) 10.5 and E16.5
(Angevine, 1970). Between E14.5
and E18.5, the diencephalon is progressively partitioned into discrete
neuronal groups that signify their differentiation into nuclei
(Jones, 2007). Little is known
about the mechanism governing the selective segregation of postmitotic neurons
to form the thalamic nuclear complex.
The p2 segment of the diencephalon is defined by the expression of a
homeobox gene, Gbx2, in the mouse embryo
(Bouillet et al., 1995;
Bulfone et al., 1993;
Miyashita-Lin et al., 1999;
Nakagawa and O'Leary, 2001).
In both mouse and monkey, Gbx2 expression is restricted to a subset
of thalamic nuclei at birth and in adulthood
(Jones and Rubenstein, 2004).
Deletion of Gbx2 in mice leads to an almost complete loss of axonal
connections between the cortex and the thalamus
(Hevner et al., 2002;
Miyashita-Lin et al., 1999).
In addition, Gbx2-deficient mice exhibit severe defects in
histogenesis of the thalamus and loss of a subset of thalamic nuclei,
suggesting that Gbx2 may play an important role in differentiation of
thalamic nuclei (Miyashita-Lin et al.,
1999; Nakagawa and O'Leary,
2001). However, the molecular and cellular basis of the thalamic
defects due to the mutation of Gbx2 is largely unknown.
The present study examines the development of the thalamus by analyzing the behavior of Gbx2-expressing cells in the diencephalon of wild-type and Gbx2-mutant embryos. Using a novel Gbx2-CreER-ires-Egfp knock-in mouse line to carry out inducible genetic fate-mapping study, we determine the cell fate of Gbx2-expressing cells in the diencephalon at different embryonic stages. Our data show that the Gbx2-expressing cells and their descendents contribute to the entire thalamic nuclear complex, but not structures that are derived from the pretectum epithalamus and prethalamus, demonstrating that the thalamus is a developmental compartment. We also show that Gbx2 is essential for maintaining the integrity of the boundaries surrounding the developing thalamus. Finally, we show that Gbx2 acts cell-nonautonomously in controlling the histogenesis and boundary formation of the thalamus.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). The new Gbx2 knock-in allele is designated as
Gbx2CreER.
Mouse breeding and genotyping
Mice were maintained on an outbred CD1 genetic background (Charles River
Lab, Wilmington, MA). Noon of the day on which the vaginal plug was found was
designated as E0.5. For inducible genetic fate mapping,
Gbx2CreER/+; R26R-/- males, homozygous
for the Cre reporter R26R
(Soriano, 1999), were bred
with wild-type or Gbx2+/- females
(Wassarman et al., 1997). Four
to six milligrams of tamoxifen (Sigma) in corn oil (20 mg/ml) was administered
by oral gavage to pregnant females as described previously
(Li and Joyner, 2001).
Genotypes of mice were determined by PCR analysis. For PCR analysis of mosaic
deletion, 200 µm brain slices were obtained by vibratome sectioning, and
the thalamus, which is demarcated by EGFP, of Gbx2CreER/F;
R26R+/- embryos at E16.5 was dissected under a fluorescent
stereoscope. The following primers were used to distinguish the floxed and
deletion Gbx2 alleles (Fig.
7I,J): Gbx2-F1, GTTCGCTCCACAGCCACT; Gbx2-R,
TGCTTGGATGTCCACATCTAGG.
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),
were cultured with a high concentration of G418 (6 mg/ml). We screened 150
colonies and identified four Gbx2flox-neo/flox-neo ES cell
clones by Southern blot analysis. Transient transfection of Cre into
Gbx2flox-neo/flox-neo ES cells resulted in
Gbx2-deficient ES cells (Gbx2-/-). To generate
chimeras, Gbx2-/- or control
Gbx2+/flox-neo ES cells were injected into blastocysts
that carried the ROSA26 gene trap insertion, which expresses
β-galactosidase (β-gal) ubiquitously
(Friedrich and Soriano,
1991).
β-galactosidase, BrdU labeling, immunofluorescence and in situ hybridization
Embryos or brains were processed for in situ hybridization as described
previously (Guo and Li, 2007).
Standard X-gal staining was used to examine β-gal activities
(Nagy et al., 2003). BrdU
labeling was performed as described previously
(Li et al., 2002). Pregnant
females were injected intraperitoneally with 100 µg BrdU per gram of body
weight 1.5 hours before they were sacrificed. Detailed protocols are available
in the Li lab website
(http://www.genetics.uchc.edu/lilab/Pages/Protocols.html).
Antibodies used in the study are the following: rabbit anti-GFP (Invitrogen),
mouse anti-BrdU (BD), mouse anti-TuJ1 (Covance) and Alexa fluorescent
secondary antibodies (Invitrogen).
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RESULTS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Wassarman et al., 1997)
(Fig. 1C,D and see below).
In Gbx2CreER/+ embryos, CreER transcripts and
EGFP proteins were detected in the same domain as the endogenous Gbx2
expression between E10.5 and E16.5 (Fig.
2A-C and data not shown). In the diencephalon of
Gbx2CreER/+ embryos at E12.5, EGFP was detected in cells
in the mantle zone and their axons, which traversed through the prethalamus
toward ventral telencephalon (Fig.
2C,D,F,H). To determine if Gbx2-expressing cells in the
diencephalon are postmitotic, we performed colocalization studies of EGFP and
BrdU in Gbx2CreER/+ embryos with BrdU pulse (1.5 hour)
labeling at E12.5. EGFP and BrdU signals were found largely mutually exclusive
(Fig. 2D-E''). We also
performed colocalization analysis of EGFP and the mitotic marker
phosphorylated histone H3 (pH3) on serial coronal sections of the thalamus of
E12.5 Gbx2CreER/+ embryos by confocal microscopy
(Hendzel et al., 1997). At the
rostral and caudal level, EGFP- and pH3-positive cells were largely segregated
(data not shown). At the middle level, pH3 signals were detected in a broad
domain outside the ventricular layer of the thalamus, with some positive cells
embedding in the EGFP-positive domain (Fig.
2F). We did detect a few pH3-positive cells with weak GFP signals
(Fig. 2G-G''). However,
the majority of the pH3-positive cells were negative for EGFP. Furthermore,
examination of a marker for postmitotic neuronal precursors, neuronal class
III β-tubulin (TuJ1), revealed that EGFP was completely colocalized with
TuJ1 in the diencephalon of Gbx2CreER/+ embryos at E11.5
(Fig. 2F-G''). These data
demonstrate that Gbx2 is primarily expressed in the neuronal
precursor cells that have exited the cell cycle in the diencephalon.
The Gbx2-expressing cells and their descendents form a self-contained compartment corresponding to the entire thalamus
To examine the fate of Gbx2-expressing cells in the diencephalon,
we performed inducible genetic fate mapping by combining
Gbx2CreER and the R26R reporter alleles
(Soriano, 1999). In
Gbx2CreER/+; R26R embryos, Cre-mediated recombination at
the R26R locus in cells that express activated CreER will result in
permanent expression of β-gal in these cells and all their descendents
(Joyner and Zervas, 2006).
Activation of CreER is achieved by administration of tamoxifen to pregnant
females carrying Gbx2CreER/+; R26R embryos. We
administered tamoxifen at E10.5, when Gbx2 is already expressed in
the diencephalon (Nakagawa and O'Leary,
2001), and assessed embryos 24 hours later to delineate the
initially marked cohort of Gbx2-expressing cells. β-Gal-positive
cells were detected in the diencephalon, anterior hindbrain and spinal cord of
Gbx2CreER/+; R26R embryos
(Fig. 3B and data not shown).
This pattern of β-gal expression was remarkably similar to that of
Gbx2 expression at E10.75, demonstrating that the activation of CreER
by tamoxifen faithfully labels Gbx2-expressing cells in
Gbx2CreER/+; R26R embryos
(Fig. 3A). No
β-gal-positive cells were detected in Gbx2CreER/+;
R26R embryos (n
10) without tamoxifen administration,
demonstrating that Cre activity from the Gbx2CreER allele
is tamoxifen-dependent (see Fig. S1A in the supplementary material). In
agreement with previous reports (Hayashi
and McMahon, 2002; Zervas et
al., 2004), we found that labeling of Gbx2-expressing
cells in the diencephalon was largely restricted to a window of 6 to 36 hours
after tamoxifen administration (see Fig. S1B-E in the supplementary
material).
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).
Thalamic cells that express Gbx2 at different stages form distinct groups of thalamic nuclei
Previous data on Gbx2 expression in the developing thalamus
between E10.5 and postnatal day 2 (P2) have suggested that Gbx2 is
specifically expressed and maintained in subsets of thalamic neurons that form
the anterior and medial groups of thalamic nuclei
(Jones and Rubenstein, 2004;
Nakagawa and O'Leary, 2001).
Surprisingly, we found that the marked descendents of Gbx2-expressing
cells labeled by tamoxifen administration at E10.5 broadly contribute to the
thalamus in Gbx2CreER/+; R26R embryos
(Fig. 3C-F). To resolve this
apparent inconsistency, we sought to examine if Gbx2-expressing cells
at other stages might have preferential contribution to particular thalamic
nuclei.
As CreER is active within a window of 6-36 hours after the administration
of tamoxifen, we gave tamoxifen at E9.5, and determined the contribution of
the initial Gbx2-expressing thalamic cells in
Gbx2CreER/+; R26R mice at P15, when
various thalamic nuclei can be identified by Nissl histology
(Caviness and Frost, 1980;
Jones, 2007). The marked cells
were found in the lateral-posterior and ventral thalamic nuclei groups (L,
LGd, VM, VL, VB, Pom, VMb, LP and MG), but not the anterior and medial
thalamic nuclei group (Fig.
5A-C; Table 1). The
fate-mapped cells labeled at E10.5 were present in most of the thalamic nuclei
in the caudal and lateral regions of the thalamus, whereas the
rostromedial-most nuclei contained a dramatically reduced number of
β-gal-positive cells (Fig.
5D-F; Table 1).
When tamoxifen was administered at E15.5, β-gal-positive cells were found
in the anterior (AD, AM and AV) and medial (Ce, Cl, MD, Pc, PT, PV and Re)
thalamic nuclei, and were largely absent from posterior and ventral nuclei
groups except for MG and LP (Fig.
5G-I; Table 1).
Therefore, our data show that, while all thalamic nuclei are derived from the
Gbx2 lineage, the precursors for different groups of thalamic nuclei
display distinct temporal expression patterns of Gbx2.
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We found that the morphology of the thalamus in Gbx2CreER/-;
R26R was severely disrupted after E14.5, similar to that found in
Gbx2-/- mutants
(Miyashita-Lin et al., 1999).
The thalamus, which was demarcated by the marked descendents of
Gbx2-transcribing cells labeled at E10.5, was apparently reduced in
the mediolateral dimension but expanded in the ventrodorsal dimension,
resulting in an abnormal shape (Fig.
4D,F,L,N). A large number of marked descendants of
Gbx2-transcribing cells labeled at E10.5 were across the dorsal and
posterior borders of the thalamus expanding into the epithalamus and the
pretectum, respectively, in Gbx2CreER/-; R26R embryos at
E14.5 (Fig. 4D, and data not
shown). The marked cells that crossed the lineage border were mainly found in
the lateral habenular nuclei (Fig.
4N,P) and the anterior part of the pretectum
(Fig. 4F,L) in the mutants at
E18.5. In contrast to the dorsal and the posterior borders, the anterior and
ventral borders of the thalamus were much less affected, with the fate-mapped
cells demarcating a clear thalamus-prethalamus boundary in
Gbx2CreER/-; R26R embryos at E18.5
(Fig. 4F,N). In agreement with
the cell-fate mapping data, histological analysis by Nissl staining revealed
that the dorsal and posterior, but not the anterior and ventral, borders of
the thalamus were disrupted in Gbx2CreER/-; R26R embryos
(Fig. 4G-H,Q-R). Collectively,
our data demonstrate that Gbx2 is required for the formation of the
dorsal and posterior boundaries separating the thalamus from the epithalamus
and the pretectum, respectively. However, a Gbx2-independent
mechanism is involved in the development of the anterior and ventral
boundaries of the thalamus.
Loss of Gbx2 does not result in a major patterning defect in the diencephalon
The severe disruption in the histogenesis and the dorsal and posterior
borders of the thalamus in Gbx2 mutants prompted us to examine by
marker analysis if Gbx2 is required for maintaining the fate of
thalamic cells. At E12.5, the expression domains of Gbx2 and
Dlx2/5 demarcate the thalamus and prethalamus, respectively, while
Shh is expressed in the zona limitans intrathalamica (ZLI) at the
interface between the thalamus and prethalamus
(Bulfone et al., 1993)
(Fig. 6A-D). In agreement with
a previous study (Miyashita-Lin et al.,
1999), the transcripts of truncated Gbx2 were detected in
the same domain in the lateral wall of the diencephalon in
Gbx2CreER/- embryos at E12.5 as that found in wild-type
embryos (Fig. 6A,E).
Furthermore, in Gbx2CreER/- embryos at E12.5,
Dlx2/5 and Shh were each detected in the same domain as
those in wild-type embryos (Fig.
6E-H). Lhx1, Pax3 and Pax7 are expressed in the
pretectum at E13.5 (Fig. 6I,J;
and data not shown). In addition, Lhx1 is also expressed in the ZLI
(Fig. 6I). Again, no difference
in Lhx1, Pax3 and Pax7 expression was observed in
Gbx2-/- embryos (Fig.
6L,M; data not shown). Examination of another pretectum marker,
Bhlhb4, which encodes a basic helix-loop-helix transcription factor
(Bramblett et al., 2002),
showed that its expression was also normally restricted to the anterior
pretectum in Gbx2-/- embryos at E13.5 as in wild type
(Fig. 6K,N). Together, our data
suggest that the abnormal histogenesis and the disruption of thalamic
boundaries in Gbx2 mutants do not result from obvious defects in
patterning or cell-fate specification in the diencephalon.
Gbx2 plays a cell-nonautonomous function in the formation of thalamic boundaries
Differential affinities for cell-cell interactions have been proposed as a
basic mechanism for separating cells into distinct compartments
(Irvine and Rauskolb, 2001;
Kiecker and Lumsden, 2005).
Indeed, members of the Cadherin family and other cell adhesion molecules are
expressed in stripes or patches in the diencephalon with their expression
coinciding with prosomeric borders or developing thalamic nuclei
(Gao et al., 1998;
Mackarehtschian et al., 1999;
Redies et al., 2000;
Yoon et al., 2000). We
therefore probed the possibility that Gbx2 controls thalamic
boundaries by regulating expression of these cell surface molecules. On serial
coronal sections of E14.5 brain, Cdh6 expression was detected in the
medial part of the thalamus, and its expression domain becomes a narrow band
in the lateral region with its dorsal and posterior limits clearly delineating
the thalamus from the epithalamus and the pretectum, respectively
(Fig. 7A; data not shown). In
Gbx2 mutants at E14.5, the dorsal border of Cdh6 expression
was indiscernible, although diffuse expression of Cdh6 persisted in
the presumptive thalamus (Fig.
7C). Efna5 encodes a member of the EphrinA ligand family.
By interacting with EphA receptors, EphrinA ligands mediate cell segregation
in rhombomeres of vertebrate hindbrains
(Xu et al., 1999). In the
E14.5 diencephalon, Efna5 is expressed in four transverse stripes
flanking the p1-2 and p2-3 borders, respectively
(Fig. 7B). Without
Gbx2, the expression of Efna5 in the thalamus was lost,
whereas the two transverse bands of Efna5 expression in the pretectum
and the prethalamus were unaffected (Fig.
7D). These data appear to be consistent with a possible role of
Gbx2 in regulating cell adhesive properties of thalamic neurons.
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).
Surprisingly, we found that Gbx2-/-, like
Gbx2+/- cells, were present throughout the thalamus and
intermingled with the host cells in the thalamus of the chimeric embryos at
E16.5 (Fig. 7E-H). By contrast,
the mutant cells were segregated from the wild-type cells and aggregated
specifically in the cerebellum of the chimeric embryos
(Fig. 7F). These observations
demonstrate that there are distinct cellular requirements for Gbx2 in
the thalamus and the cerebellum. In the cerebellum, Gbx2 appears to
act cell-autonomously in regulating cell adhesion. However, in the thalamus,
the normal cell mixing suggests that wild-type and mutant cells have similar
cell-adhesive properties. Alternatively, wild-type cells may rescue the defect
of cell adhesion of Gbx2-/- cells in the thalamus.
Consistent with a possible cell-nonautonomous role of Gbx2, we found
that, in sharp contrast to those found in Gbx2-null mutants, the
morphology and the histological borders of the thalamus were remarkably normal
in the chimeras that were composed of Gbx2-/- and
wild-type cells (Fig. 7F).
We next investigated whether the mutant thalamic neurons are prevented from
dispersing into the epithalamus or the pretectum in chimeric embryos. We
performed genetic mosaic analysis by combining the
Gbx2CreER allele with a conditional Gbx2 deletion
allele, Gbx2F (Li et
al., 2002). Taking advantage of the mosaic manner of
CreER-mediated recombination (Joyner and
Zervas, 2006), we expected that administration of tamoxifen at
E10.5 would produce a genetically mosaic thalamus composed of
Gbx2CreER/F (Gbx2 heterozygous - wild type in
phenotype) and Gbx2CreER/- (Gbx2 null) cells in
Gbx2CreER/F; R26R embryos, whereas the
Gbx2CreER/- cells would be probably marked by β-gal
(Fig. 7I). PCR analysis of
microdissected thalamic tissues showed that the administration of tamoxifen at
E10.5 indeed produced a genetically mosaic thalamus composed of
Gbx2CreER/F and Gbx2CreER/- cells in
Gbx2CreER/F; R26R embryos at E16.5
(Fig. 7J). Significantly, the
morphology of the thalamus was largely normal in the genetic mosaic embryos
that contained a significant number of β-gal-positive cells in the
thalamus (n=11) (Fig.
7K and inset in Fig.
7O). The labeled descendants of Gbx2-expressing cells
were restricted to the thalamic compartment in Gbx2CreER/F;
R26R embryos at E14.5 and E18.5, similar to those found in
Gbx2CreER/+; R26R embryos (compare
Fig. 7K, inset in
Fig. 7O with
Fig. 3E,F;
Fig. 4E,F, and
Fig. 4M-P). Mosaic embryos that
contained stronger β-gal activity in the thalamus did exhibit a mild
defect in the morphology of the thalamus in Gbx2CreER/F
embryos (n=7; Fig.
7L). These results suggest that in the presence of wild-type
cells, the dorsal and posterior boundaries of the thalamus are rescued in the
genetic mosaic embryos.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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; Puelles and Rubenstein,
2003). However, it remains controversial whether the prosomeres
represent true development compartments that are units of cell lineage
restriction similar to rhombomeres in the vertebrate hindbrain
(Figdor and Stern, 1993;
Larsen et al., 2001;
Zeltser et al., 2001).
According to the prosomeric model, the thalamus is generated from the alar
plate of the p2 segment, presumably from the population of cells that express
Gbx2 (Bouillet et al.,
1995). In this study, we demonstrate that the
Gbx2-expressing cells and their descendents contribute to the entire
thalamus. Significantly, the fate-mapped Gbx2-expressing cells form
lineage-restriction boundaries delineating the thalamus not only from the
pretectum and prethalamus, which correspond to the p1 and p3 segments, but
also from the epithalamus, which is presumably derived from the same p2
segment. Our results have thus revealed the presence of a hitherto unknown
developmental compartment, which is defined by the expression domain of
Gbx2, within the diencephalon. Interestingly, a few marked cells were
often detected in the lateral habenular nucleus and the nucleus of
Darkschewitsch, which are presumably derived from the epithalamus and basal
plate, respectively, of the p2 segment. These data suggest that the
intra-prosomeric boundaries within p2 may be less stringent compared with
inter-prosomeric boundaries. It is worth noting that our current study primarily analyzed the developmental fate of Gbx2-expressing cells, which are mostly postmitotic neuronal precursors. We are not certain whether the progenitors in the ventricular zone of p2 obey the same lineage-restriction boundaries as Gbx2-expressing cells. However, based on the kinetics of tamoxifen induction, we expect that both Gbx2-expressing cells and the progenitors that are committed to express Gbx2 would be labeled in a window of 36 hours after the administration of tamoxifen. Therefore, the lineage-restriction boundaries revealed by our fate mapping of Gbx2-expressing cells should apply to cells in the mantle zone as well as those committed progenitors in the ventricular zone of the p2 segment.
Compartment boundary restriction and nuclear formation in the thalamus
The developmental compartment defined by the Gbx2 lineage
contrasts with the known compartments in the vertebrate hindbrain and
telencephalon, where the postmitotic cells in the mantle zone are known to be
able to cross rhombomeric or the pallial-subpallial boundaries, although their
progenitors in the proliferating zone are restricted to a cell-tight
compartment (Fishell et al.,
1993; Wingate and Lumsden,
1996). It has been postulated that compartmental boundaries are
mainly required for a proliferating cell population with labile cell fates,
whereas boundary restriction becomes dispensable for postmitotic cells, as
their fates are specified (Kiecker and
Lumsden, 2005). Therefore, the confinement of the
Gbx2-expressing cells and their descendents, which are mainly
postmitotic, within the thalamic compartment may serve a different function
from those compartments. Interestingly, we observed that the borders of the
Gbx2 lineage marked at E10.5 were progressively sharpened between
E14.5 and E16.5 (see Figs 3 and
4), coinciding with the initial
parceling of the dorsal thalamus (Jones,
2007). We speculate that the lineage restriction of postmitotic
Gbx2-positive thalamic cells may underlie the formation of thalamic
nuclei. By fate mapping Gbx2-expressing cells at E9.5, E10.5 or
E15.5, we have identified five groups of the thalamic nuclei (summary in
Fig. 5J-L). The initial
Gbx2-expresing cells (around E10.5) give rise to most of the
principal relay nuclei, such as LGd, VB, LP and MG. However, Gbx2
expression is downregulated in LGd and VB (designated as group I nuclei) after
E10.5, and persists in LP and MG (group II). The second wave of
Gbx2-expressing cells (E10.5-E11.5) gives rise to many association
nuclei, such as AV, L, Cl, MD, Pc, Ce, VMb and Re, and relay nucleus L. Among
these nuclei, Gbx2 expression is maintained in MD and Cl, Pc and Ce
(group III), and lost in L and VMb (group IV). The last wave of
Gbx2-expressing cells (E15.5) gives rise to the most anteromedial
nuclei, AD, AM, PV and Re (group V), where Gbx2 expression persists
into postnatal stages. Therefore, the precursors for distinct groups of
thalamic nuclei display dynamic and distinct temporal patterns of
Gbx2 expression, although all thalamic neurons are derived from the
Gbx2 lineage. These observations suggest that the expression of
Gbx2 itself allows the thalamus as a whole to be segregated from the
neighboring structures, which never express Gbx2. Within the
thalamus, however, the dynamic and differential expression of Gbx2
may lead to segregation of Gbx2-positive neurons from
Gbx2-negative neurons, which have not yet started or have lost
Gbx2 expression.
|
). This
arrangement of thalamic neurons according to their time of origin raises the
question of whether the cell-tight thalamic compartment revealed by the
current study is simply a result of lack of cell movement in the developing
thalamus. To further investigate the distribution of thalamic neurons that are
generated at different stages, we marked Gbx2-expresing cells by
administrating tamoxifen at E9.5 (presumed to label postmitotic thalamic
neurons born between E10 and E12) and performed BrdU pulse labeling at E13.5
(presumed to label neurons born around E13.5) in Gbx2CreER/+;
R26R+/- embryos. In agreement with the previous birth-dating
analysis, X-gal positive cells were mostly found in the lateral region,
whereas the majority of BrdU-positive cells were found in the medial region.
However, these two populations of cells were not exclusively segregated, with
many later-born BrdU-positive cells intermingled with earlier-born
X-gal-positive cells (see Fig. S2 in the supplementary material). These
observations suggest that, at least to a certain extent, cell mingling does
occur within the developing thalamus. Significantly, we demonstrate that,
without Gbx2, cells originating from the thalamus no longer obey the
lineage restriction at the dorsal and the posterior borders of the thalamus,
expanding to the lateral habenular nucleus and the pretectum. Collectively,
these observations strongly support the argument that the postmitotic thalamic
neurons are normally restricted to the dorsal and posterior thalamic
boundaries by an active Gbx2-dependent mechanism, rather than by a
lack of cell movement. In addition to the defect of the boundaries, we observed abnormally widened habenulopeduncular tract and accumulation of neurites under the pial surface of the thalamus in Gbx2 mutant embryos (Fig. 4F,H,P,R). However, as shown on the coronal sections at the anterior and posterior levels, the thalamus-epithalamus and the thalamus-pretectum borders are completely disrupted, including at places distant from the habenulopeduncular tract (Fig. 4L,N,P). These data suggest that the abnormality of the habenulopeduncular tract cannot completely explain the aberrant dispersal of thalamic cells into the epithalamus and the pretectum. Importantly, we have found that Gbx2 acts cell-autonomously in the control of axonal outgrowth of thalamic neurons (L.C. and J.Y.H.L., unpublished data).
In contrast to the dorsal and caudal thalamic boundaries, the lineage
boundaries at the anterior and ventral borders of the thalamus are maintained
in the absence of Gbx2. Therefore, the formation of the anterior and
ventral borders of the thalamus is independent on Gbx2.
Interestingly, it has been demonstrated that the ZLI, which defines the p2/3
border, represents a lineage-restriction boundary and a signaling center,
which expresses secreted factor, such as Shh and Fgf8, to
regulate the development of both thalamus and prethalamus
(Kataoka and Shimogori, 2008;
Kiecker and Lumsden, 2004;
Vieira et al., 2005). A recent
study showed that the ZLI represents a unique lineage restriction compartment
depending on Lfng activity
(Zeltser et al., 2001). We
show that the expression of Shh and Lhx1 at the ZLI is
unaffected in Gbx2 mutants, demonstrating that Gbx2 is not
essential for development of the p2/p3 border
(Fig. 6D,H,I,L).
A cell-nonautonomous role of Gbx2 in the regulation of lineage-restriction boundary of the thalamus
Given that the expression of Cdh6 and Efna5 is disrupted
in the thalamus of Gbx2 mutant embryos, we were surprised to discover
that Gbx2-deficient and wild-type cells intermix normally in the
thalamus of chimeric and genetic mosaic embryos. Our data strongly suggest
that Gbx2 plays a cell-nonautonomous role in the formation of the
thalamic boundaries. First, we found that the morphology and the histological
border of the thalamus are remarkably normal in the chimeric and mosaic
embryos that contain a significant percentage (not less than 50%) of
Gbx2-deficient cells, as judged by PCR and β-gal expression. It
is remarkable that mosaic embryos that contained strong β-gal activity in
the thalamus did exhibit a mild defect in the morphology of the thalamus in
Gbx2CreER/F embryos. These results indicate that
administration of tamoxifen indeed leads to deletion of Gbx2, and
deletion of Gbx2 after E10.5 can still recapitulate the defect of
Gbx2-null cells. Therefore, the rescue observed in the mosaic embryos
is unlikely to be due to the residual Gbx2 proteins produced before
CreER-mediated deletion occurs. The mild phenotype in the mosaic embryo with
strong β-gal activity also suggests that a certain percentage of
wild-type cells may be required for the rescue of the mutant phenotype.
Second, in Gbx2CreER/F; R26R embryos with mosaic deletion
of Gbx2 at E10.5, the marked descendents of Gbx2-expressing
cells become normally restricted to the thalamic compartment. It is reasonable
to assume that a significant number of the marked cells in
Gbx2CreER/F; R26R embryos have lost Gbx2 due to
CreER-mediated recombination. The absence of β-gal-positive cells in
either the epithalamus or the pretectum demonstrates that the presence of
wild-type cells rescues the lineage-restriction boundaries of the thalamus in
mosaic Gbx2CreER/F; R26R embryos. We did not detect a bias
of the wild-type cells being at the boundaries of the thalamus in chimeric
embryos, arguing against the possibility that the wild-type cells may form
border cells to restore the boundary. Finally, we found that Efna5
expression is restored in the thalamus of chimeric and mosaic embryos at
E16.5. Because of the unavailability of suitable antibodies, we were unable to
determine whether Efna5 is expressed in Gbx2-deficient cells
in the chimeric or mosaic embryos. Nevertheless, the restored expression of
Efna5 demonstrates that the dorsal and caudal borders of the thalamus
are rescued in the chimeric and mosaic embryos. Collectively, our data
demonstrate that Gbx2 acts cell-nonautonomously in regulating
formation in the thalamic boundary. As Gbx2 is a transcription factor and
presumably acts within the thalamic cells, we postulate that Gbx2 may
regulate an extracellular signaling pathway, which in turn mediates the
cell-nonautonomous role of Gbx2 in controlling boundary formation in
the thalamus.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1317/DC1
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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