Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: mario.capecchi{at}genetics.utah.edu)
Accepted 17 July 2003
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SUMMARY |
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Key words: Motoneurons, Hox3, Mouse
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Introduction |
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There is also evidence that combinations of various Hox genes can elicit
emergent phenotypes beyond the contributions of individual genes
(Condie and Capecchi, 1994;
Gavalas et al., 1998
;
Rossel and Capecchi, 1999
;
Studer et al., 1998
). For
example, the absence of either Hoxa1 or Hoxb1 function leads
to some degree of rhombomere identity transformation and cell loss is observed
(Carpenter et al., 1993
;
Gaufo et al., 2000
;
Goddard et al., 1996
;
Mark et al., 1993
;
Pattyn et al., 2003
;
Studer et al., 1996
). However,
the combined absence of both genes leads to severe abnormal programmed cell
death resulting in the deletion of multiple rhombomeres and subsequent
reorganization of the hindbrain (Gavalas
et al., 1998
; Rossel and
Capecchi, 1999
). Therefore, the interaction of these genes is
equally important for the determination of segmental specification as is the
contribution of each individual Hox gene to individual rhombomere identity. It
remains to be determined how the various Hox genes expressed in the hindbrain,
r2 to r7, interact with each other to determine distinction of each rhombomere
as well as integration of function across rhombomeres.
Similarly, given that Hox genes are co-expressed with molecules involved
with generic neural specification programs in later stages of development, it
is likely that they can also interact with these programs to establish unique
cell identities within each rhombomere
(Davenne et al., 1999;
Gaufo et al., 2000
;
Osumi et al., 1997
;
Pattyn et al., 2003
;
Takahashi and Osumi, 2002
).
For example, neural progenitors expressing the homeodomain proteins Nkx2.2 and
Phox2b give rise to all BMNs present in various rhombomeres of the hindbrain.
The interaction between these molecules with different combinations of Hox
proteins expressed at different rhombomeres may give rise to functionally
distinct BMNs, such as facial BMNs unique to r4 where Hoxb1 is
expressed (Gaufo et al., 2000
;
Goddard et al., 1996
;
Pattyn et al., 2003
;
Studer et al., 1996
).
Likewise, somatic motoneurons (SMNs) derive from neural progenitors expressing
the homeodomain proteins Pax6 and the bHLH Olig2 are also present at various
rhombomeres. In this case, however, the Hox genes involved in their
differentiation into functionally unique SMNs within each rhombomere have not
been determined (Guidato et al.,
2003
).
In summary, given that Hox gene expression persists throughout hindbrain
development, it is plausible that it plays at least two distinct roles in
neuronal specification. First, Hox genes are required for assignment of
rhombomere identity and in their absence, for example Hoxa1 and
Hoxb, such patterning is highly perturbed
(Gavalas et al., 1998;
Rossel and Capecchi, 1999
;
Studer et al., 1998
). Second,
they may play a role in differentiating functionally unique motoneurons at a
later stage of development (Bell et al.,
1999
; Guidato et al.,
2003
). The goal of this study was to identify what combination of
Hox genes are necessary to distinguish rhombomeres r4 to r6, and how these
genes effect the production of motoneuron subtype programs unique to these
rhombomeres. To achieve this, we first focused on Hox3 paralogous genes
(Hoxa3, Hoxb3 and Hoxd3), expressed in caudal rhombomeres,
and analyzed the affects of different combinations of Hox3 mutations on the
segmental identities of r4, r5 and r6. Then, to test the role of these Hox
genes in neural specification, we looked for the presence or absence of
specific SMNs in r5 with relation to different combinations of null mutations
of the Hox3 genes. We observed that loss of any of the Hox3 genes results in a
r6- to r4-like change in cell fate; different double mutation showed a graded
increase in cell fate change. We also observed a gene dosage dependence of SMN
specification of Hoxa3 and Hoxb3 in r5. This specification
appears to be mediated through the control of the Pax6/Olig2
regulatory pathway for SMN formation, suggesting a direct influence of Hox3
genes on SMN fate. Together, these observations reveal two functions for Hox3
paralogous genes in the developing hindbrain in defining segmental identity
across multiple rhombomeres and controlling cell fate within an individual
rhombomere.
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Materials and methods |
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In situ hybridization and immunohistochemistry
For whole-mount in situ hybridization, E11.5 neural tubes were dissected in
cold PBS and fixed in cold 4% formaldehyde for 2-3 hours and processed for
Hoxa3, Hoxb3 and Hoxd3 RNA in situ hybridization as
previously described (Manley and Capecchi,
1998). Detection of Hoxb1GFP was detected by confocal
microscopy on E11.5, live-dissected flat-mount hindbrain preparations.
E9.25-E11.5 embryos were harvested as described above and processed for
cryostat sectioning. Frozen, 10 µm transverse and coronal sections were
labeled with rabbit anti-Hoxb1 (1:250, Covance), rabbit anti-Phox2b (1:1000, a
gift from C. Goridis), rabbit anti-Olig2 (1:5000; a gift from H. Takebayashi),
mouse anti-TuJ1 (1:1000, Covance), rat anti-Hoxb4 (1:25; Developmental Studies
Hybridoma Bank, DSHB), mouse anti-Nkx2.2 (1:25, DSHB), mouse anti-Isl1/2
(1:25; DHSB), rabbit anti-Chx10 (1:2500; a gift from S. Morton and T.
Jessell), mouse anti-MPM2 (1:8000; Upstate Biotechnology), rabbit
anti-activated Caspase-3 (1:50; NEB Cell Signaling), TUNEL (manufacturer's
protocol, Roche), mouse anti-NeuN (1:250; Chemicon), rabbit anti-ChAT (1:1000;
Chemicon) and
BTX (1:500; Molecular Probes). Immunolabeled sections
were developed with Alexa-fluor (1:1000; Molecular Probes) and Cy5 (1:1000;
Jackson Immunoresearch)conjugated secondary antibodies. Images of
fluorescent-labeled sections were captured with the BioRad MRC 1024 confocal
microscope and processed with Adobe Photoshop and Microsoft Powerpoint
software.
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Results |
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Ectopic Hoxb1 expression is associated with r4-like facial
branchiomotoneuron differentiation and migration pattern in r6
We next examined the functional consequence of the ectopic expression of
Hoxb1 in r6 of Hoxa3-/-d3-/- double
mutant embryos. To address this issue, we focused on two well-defined
functions of Hoxb1 in r4: (1) the expression requirement of Nkx2.2, Phox2b and
Isl1/2 among BMNs; and (2) the posterior migration of Hoxb1-expressing BMNs
from r4 to the ventrolateral region of r6
(Gaufo et al., 2000;
Goddard et al., 1996
;
Pattyn et al., 2000
;
Studer et al., 1996
)
(Fig. 1C-E). In control
embryos, a small population of these r4-derived Hoxb1-expressing BMNs can be
seen adjacent to the Nkx2.2-expressing progenitor domain in ventral r6
(Fig. 3A). In
Hoxa3-/-d3-/- double mutant embryos,
Hoxb1 is ectopically expressed in an expanded domain of Nkx2.2-expressing
cells, indicative of BMN progenitors (Fig.
3B) (Gaufo et al.,
2000
; Pattyn et al.,
2000
). Co-localization of Isl1/2 with these same Hoxb1-expressing
cells suggests that these progenitors are differentiating into motoneurons
(Fig. 3D). Furthermore, the
ectopic expression of Phox2b and Isl1/2 suggest that these neurons are
differentiating specifically into BMNs
(Fig. 3F).
|
To address the possibility that the observed Hoxb1-expressing cells in r7
of Hoxa3-/-d3-/- double mutant embryos
were derived from r4 and migrated aberrantly through r6, we analyzed coronal
sections of younger E10.5 embryos at a period when the Hoxb1-expressing BMNs
have initiated their migration from r4
(Fig. 3K,L). In both control
and Hoxa3-/-d3-/- double mutant
embryos, the expression of Hoxb1 in r4 is normal. In r5, the initial migration
of Hoxb1-expressing cells is also normal in both groups. In r6, the ectopic
expression of Hoxb1 in Hoxa3-/-d3-/-
double mutant embryos is evident compared with control embryos. In r7 of
Hoxa3-/-d3-/- double mutant embryos,
small clusters of Hoxb1-expressing neurons, negative for Hoxb4, are clearly
visible (Fig. 3L). This
observation thus precludes the possibility that the ectopic Hoxb1-expressing
cells in r7 have migrated from the BMN population derived from r4. Together,
these observations provide strong evidence that the ectopic expression of
Hoxb1 in r6 is sufficient to activate a BMN differentiation and migration
program normally unique to r4. These findings are consistent with a previous
report demonstrating that the localized or global misexpression of Hoxb1 in r2
of the chick embryo is sufficient to transform cells in this rhombomere into
r4-like BMNs (Bell et al.,
1999). Moreover, the posterior migration of r4-like BMNs from r6
into r7 suggests that r7, like r5, produces local environmental cues required
for the migration of even-numbered derived `facial' BMNs
(Garel et al., 2000
;
Studer, 2001
).
Combined functions of Hoxa3 and Hoxb3 are necessary
for the specification of r5-derived motoneurons
Among the Hox3 genes, only Hoxa3 and Hoxb3 are expressed
in the ventral r5 region from which SMNs are derived
(Fig. 1D,F-H). The loss of
Hoxa3 alone, results in a reduction in the number of SMNs (data not
shown), whereas the elimination of both Hoxa3 and Hoxb3 in
E11.25-E11.5 mutant embryos lead to the complete loss of SMNs, as identified
by expression of the homeodomain HB9 (Fig.
4A,B) (Arber et al.,
1999). By contrast, r4-derived Phox2b-expressing BMNs and
r5-derived visceromotoneurons labeled by Isl1/2 and Phox2b are unaffected
(data not shown). The specific loss of SMNs in
Hoxa3-/-b3-/- double mutant embryos
closely resembles the phenotype of embryos bearing independent mutations for
Olig2 and Pax6 (Fig.
4C) (Lu et al.,
2002
; Novitch et al.,
2001
; Takebayashi et al.,
2002
; Zhou and Anderson,
2002
). However, in the case of the
Hoxa3-/-b3-/- double mutant embryos,
the loss of SMNs is restricted to r5.
|
|
To determine the cause of the Olig2-specific loss in Hoxa3-/-b3-/- double mutant embryos, we examined two possibilities: (1) programmed cell death; and (2) transformation in neuronal fate. To address the first possibility, we analyzed E10.25 embryos for the expression of TUNEL and activated caspase 3, both markers for programmed cell death (Fig. 5E-H). No differences were observed across experimental groups. Analysis of older Hoxa3-/-b3-/- double mutant embryos (E10.5-E11.25) also showed no differences in TUNEL and caspase 3 expression (data not shown). An assay for cell proliferation using the mitosis marker MPM2, showed normal cell division in Hoxa3-/-b3-/- double mutant embryos compared with controls (Fig. 5E-H). The absence of aberrant cell death among the experimental groups left the possibility that the loss of SMNs in Hoxa3-/-b3-/- double mutant embryos may be the result of a change in neuronal fate. To address this issue, we examined the expression pattern of Pax6, which identifies progenitors of SMNs and V2s. In E10.25 Hoxa3-/-b3-/- double mutant embryos, the normal low Pax6 expression in the pSMN domain is qualitatively similar to the more dorsal high Pax6 expression in the pV2 domain (Fig. 5I,M). The molecular change in the pSMN domain to that of the pV2 domain is substantiated by appearance of Chx10-expressing V2 interneurons immediately dorsal to V3 interneurons, a region normally occupied by SMNs (Fig. 5K,O). The ectopic appearance of Chx10-expressing V2 interneurons in the region normally occupied by HB9-expressing SMNs was also confirmed in E11.5 Hoxa3-/-b3-/- double mutant embryos compared with controls (Fig. 5L,P). In a gene dose-dependent manner, Hoxa3 and Hoxb3 are thus required for the specification of progenitors that will give rise to SMNs of the abducens nucleus.
The loss of SMN precursors of the abducens nucleus was also confirmed in
late stage, E18 Hoxa3-/-b3-/- double
mutant embryos. In the upper medulla, the r5-derived abducens nucleus can be
easily identified by its stereotypic relationship with axons from the
r4-derived facial nucleus. The axons of the facial nucleus, known as the genu,
circumscribe the abducens nucleus in a medial to lateral pattern
(Carpenter and Sutin, 1983).
The genu of the facial nerve is clearly visible in both E18 control and
Hoxa3-/-b3-/- double mutant embryos in
the region devoid of NeuN expression, a nuclear marker for differentiated
neurons (Fig. 6A,B, arrow). The
early embryonic loss of r5-derived progenitors and precursors of the abducens
nucleus is substantiated by the absence of choline acetyltransferase (ChAT)
expression in Hoxa3-/-b3-/- double
mutant embryos (Fig. 6C,D).
These findings are supported by an associated reduction in the expression of
TuJ1, a pan-neuronal marker, and acetylcholine receptors (AChR), as visualized
by
-bungarotoxin (
BTX), in transverse sections of the lateral
rectus muscle, innervated normally by the abducens nerve
(Fig. 6E-J). The binding of
BTX in the lateral rectus of
Hoxa3-/-b3-/- double mutant embryos is
consistent with the finding that prepatterning of AChRs occurs in the target
muscle independent of motor innervation
(Yang et al., 2001
). The
remaining expression of TuJ1 in the lateral rectus of
Hoxa3-/-b3-/- double mutant embryos
may represent peripheral processes from sensory neurons (i.e. trigeminal
ganglia). However, the close proximity of these processes with
BTX
suggests a contribution from a motoneuron source, perhaps the aberrant
innervation by axons from other cranial motoneurons.
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Discussion |
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Work in Drosophila, however, has provided insight into how the Hox
genes may use the mechanism of genetic suppression to determine segmental
identity (Hafen et al., 1984;
Lewis, 1978
;
Struhl and White, 1985
;
Weatherbee et al., 1998
). For
example, Ubx normally suppresses multiple genes in the wing
developmental pathway within the metameric segment that gives rise to the
haltere such that in the Ubx mutant these genes are de-suppressed and
the more posterior haltere is transformed into the more anterior wing
(Weatherbee et al., 1998
). The
suppression of the wing program by Ubx is analogous to our current
observation of the r6-specific suppression of the facial BMN specification
program by the Hox3 genes. This important crossregulatory phenomenon among
members of the Hox3 and Hox1 paralogous members ensures the individual
identity of rhombomeres. Interestingly, the observation that Hoxb1 is
suppressed specifically in r6 may reflect a developmental ground state common
amongst even-numbered rhombomeres. Thus, as in other serially homologous
structures, the periodicity of even- and odd-numbered rhombomeres exhibits
common functions (Casares and Mann,
2001
; Lumsden,
1990
; Trainor and Krumlauf,
2000
; Waskiewicz et al.,
2002
).
Hox3 genes are upstream of Pax6- and
Olig2-dependent somatic motoneurons
The phenotypes of mice harboring independent mutations for the Hox3
paralogs Pax6 and Olig2 suggest that these genes are part of
a common regulatory network necessary for determining the fate of SMNs
(Ericson et al., 1997;
Lu et al., 2002
;
Osumi et al., 1997
;
Takahashi and Osumi, 2002
;
Zhou and Anderson, 2002
).
Although these genes are necessary for the specification of SMNs, the
regulatory process by which they attain this goal is qualitatively different.
As described in this study and in a previous report, Hox3 genes and
Pax6 are upstream of Olig2 expression
(Novitch et al., 2001
). In the
loss of Hoxa3 and Hoxb3, however, Pax6 is expressed
at ectopically high levels in the pSMN domain, suggesting that Hoxa3
and Hoxb3 genetically suppress Pax6 expression levels in the
pSMN domain. Our previous report also demonstrated the de-suppression of
Pax6 expression in the Nkx2.2-expressing BMN progenitor
domain in r4 of Hoxb1 mutant embryos
(Gaufo et al., 2000
). These
observations are analogous to the role of the Drosophila Hox gene,
Antp, in suppressing the activity of eyeless, the homologue
of Pax6 in vivo and in vitro
(Plaza et al., 2001
). However,
a direct interaction between the mammalian Hox and Pax genes remains
to be tested.
In contrast to the combined loss of Hoxa3 and Hoxb3, the
Pax6 mutation results in the expansion or de-suppression of the
ventral Nkx2.2 pV3 domain into the more dorsal SMN progenitor domain
in r5 (data not shown) (Ericson et al.,
1997; Takahashi and Osumi,
2002
). Consequently, the loss of Hox3 and Pax6 genes
leads to ectopic differentiation of V2 and V3 interneurons, respectively. The
comparison of the Hox3 and Pax6 genes demonstrates that they are
necessary for the formation of SMNs, but they differ significantly at the
level of specifying SMN progenitors. Genetically, Hox3 genes suppress the
dorsal Pax6 pV2 domain in the more ventral pSMN domain, whereas
Pax6 suppresses the ventral Nkx2.2 pV3 domain in the more
dorsal pSMN domain. In contrast to Pax6, the functions of
Olig2 appear similar to Hoxa3 and Hoxb3 in its
capacity to specify pSMNs. In both Olig2-/- and
Hoxa3-/-b3-/- mutant embryos, the loss
of SMNs is associated with the de-suppression of Pax6 and the
subsequent ectopic differentiation of V2 interneurons
(Lu et al., 2002
;
Takebayashi et al., 2002
;
Zhou and Anderson, 2002
). In
both mutants, the more ventral Nkx2.2-expressing V3 progenitor domain
appears unaffected, unlike that observed in the Pax6 mutant. The
functional similarities between the Hox3 and Olig2 genes with respect
to the specification of SMN progenitors in r5 suggest interactions between the
pathways mediated by these genes.
The observation that the gene-dose dependent loss of Hoxa3 and
Hoxb3 strongly correlates with the loss of Olig2/Hb9 and ectopic
Chx10 expression suggests a direct role in SMN fate decisions. Furthermore,
the expression of the Hox3 genes among progenitors and differentiating neurons
in r5 (Fig. 1F-H,
Fig. 4I) supports a possible
role for Hox3 genes at later stages of motoneuron differentiation. This
hypothesis has received additional support by the recent report that
Hoxa3 gain-of-function in chick r1-r4 is sufficient to generate SMNs
in these rhombomeres (Guidato et al.,
2003). Roles for the Hox3 genes at different stages of motoneuron
specification would be consistent with the observation for the multi-level
developmental regulation of the Drosophila wing by the Hox gene,
Ubx (Weatherbee et al.,
1998
). However, the question remains whether the effect of Hox3
gene mutations is direct or results from a gradual titration of r5 to a more
anterior rhombomere - a hallmark of a homeotic transformation. The present use
of conditional mouse models should clarify more precisely the role of the Hox3
genes on distinct stages of motoneuron patterning.
A general function for paralogous Hox genes in the coordination of
activation and suppression along the anteroposterior axis
The 13 mammalian Hox paralogs, each containing two to four genes, are
expressed in a nested pattern along the AP axis of the neural tube, from the
caudal-most tip of the spinal cord to the level of r2 of the hindbrain
(Davenne et al., 1999;
Economides et al., 2003
). To
date, the only published reports addressing the role of paralogous Hox genes
on neuronal patterning have been the knockouts of the paralogous Hox1 and Hox2
genes (Davenne et al., 1999
;
Gavalas et al., 1998
;
Rossel and Capecchi, 1999
;
Studer et al., 1998
). The
characterization of the Hox1 paralogous mutants, Hoxa1 and
Hoxb1, highlights the basic requirements for these genes in normal
hindbrain patterning (Gavalas et al.,
1998
; Rossel and Capecchi,
1999
; Studer et al.,
1998
). During early hindbrain patterning, prior to the closure of
the roof plate, Hoxa1 and Hoxb1 are involved in establishing
the AP-restricted identities of at least rhombomeres 3, 4 and 5. Equally
important to this role is the requirement for Hoxa1 and
Hoxb1 in cell proliferation and survival. In the absence of
Hoxa1 and Hoxb1, the hindbrain undergoes abnormal programmed
cell death associated with deletions of multiple rhombomeres and subsequent
reorganization (Rossel and Capecchi,
1999
). The deletion of multiple hindbrain segments precludes
analysis of later patterning events associated with the specification of a
multitude of neuronal subtypes along the DV axis.
In contrast to the paralogous Hox1 mutants, the numbers and periodicity of
rhombomeres appear to be normal in paralogous Hox2, Hoxa2 and
Hoxb2 mutant embryos (Davenne et
al., 1999). The normal features of the rhombomeres thus allow for
the examination of later events in neuronal patterning. Indeed, the analysis
of Hox2 mutant embryos led to the discovery that the program mediated by Hox
genes along the AP axis appear to influence later patterning events along the
DV axis. The present study, however, defines the possible mechanisms by which
Hox genes may control distinct aspects of AP and DV patterning. For example,
the functions of Hoxa3 and Hoxb3 appear to influence early
neuronal fate decisions by regulating a developmental program common with a
SMN-specific determinant, Olig2
(Lu et al., 2002
;
Novitch et al., 2001
;
Takebayashi et al., 2002
;
Zhou and Anderson, 2002
).
However, owing to the extended expression of the Hox3 genes during
embryogenesis, their precise role in neuronal specification remains to be
characterized. Nevertheless, the direct or indirect regulation of
Olig2 expression, a putative transcriptional repressor, by the early
actions of Hoxa3 and Hoxb3 highlights the complex interplay
between mechanisms of activation and suppression in the progressive
specification of motoneurons.
The role of the paralogous Hox1, Hox2 and Hox3 groups in the positive
regulation of segmental formation and DV patterning programs explain only in
part the genetic mechanism by which segmental neuronal identity is achieved
(Davenne et al., 1999;
Gavalas et al., 1998
;
Rossel and Capecchi, 1999
;
Studer et al., 1998
). Combined
with the observations revealing the role of Hox genes in genetic suppression
along the AP axis, a general mechanism emerges of how neuronal identity is
acquired. Remarkably, the fundamental principles of Hox gene function involved
in this developmental process are programmed within a single paralogous Hox
group (where a distinct combination, a `Hox code', may perform distinct
roles). The functions mediated by the Hox3 genes in r5 and r6 may represent a
phenomenon reiterated by other Hox paralogs along the entire AP axis of the
neural tube. Ultimately, this developmental process could lead to the unique
identities of neurons along the entire AP axis of the embryo.
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ACKNOWLEDGMENTS |
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