Complementary roles for Nkx6 and Nkx2 class proteins in the establishment of motoneuron identity in the hindbrain
Alexandre Pattyn1,*,
,
Anna Vallstedt1,*,
Jose M. Dias1,
Maike Sander2,
and
Johan Ericson1,
1 Department of Cell and Molecular Biology, Karolinska Institute, S-171 77
Stockholm, Sweden
2 Center for Molecular Neurobiology, Martinistrasse 85, 20251 Hamburg,
Germany
Author for correspondence (e-mail:
johan.ericson{at}cmb.ki.se)
Accepted 29 May 2003
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SUMMARY
|
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The genetic program that underlies the generation of visceral motoneurons
in the developing hindbrain remains poorly defined. We have examined the role
of Nkx6 and Nkx2 class homeodomain proteins in this process, and provide
evidence that these proteins mediate complementary roles in the specification
of visceral motoneuron fate. The expression of Nkx2.2 in hindbrain progenitor
cells is sufficient to mediate the activation of Phox2b, a homeodomain protein
required for the generation of hindbrain visceral motoneurons. The redundant
activities of Nkx6.1 and Nkx6.2, in turn, are dispensable for visceral
motoneuron generation but are necessary to prevent these cells from adopting a
parallel program of interneuron differentiation. The expression of Nkx6.1 and
Nkx6.2 is further maintained in differentiating visceral motoneurons, and
consistent with this the migration and axonal projection properties of
visceral motoneurons are impaired in mice lacking Nkx6.1 and/or Nkx6.2
function. Our analysis provides insight also into the role of Nkx6 proteins in
the generation of somatic motoneurons. Studies in the spinal cord have shown
that Nkx6.1 and Nkx6.2 are required for the generation of somatic motoneurons,
and that the loss of motoneurons at this level correlates with the
extinguished expression of the motoneuron determinant Olig2. Unexpectedly, we
find that the initial expression of Olig2 is left intact in the caudal
hindbrain of Nkx6.1/Nkx6.2 compound mutants, and despite this, all
somatic motoneurons are missing. These data argue against models in which Nkx6
proteins and Olig2 operate in a linear pathway, and instead indicate a
parallel requirement for these proteins in the progression of somatic
motoneuron differentiation. Thus, both visceraland somatic motoneuron
differentiation appear to rely on the combined activity of cell intrinsic
determinants, rather than on a single key determinant of neuronal cell
fate.
Key words: CNS, Motoneuron, Hindbrain, Homeodomain protein
 |
INTRODUCTION
|
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The central control of movement and body homeostasis rely on two major
classes of motoneurons generated at defined ventral positions in the spinal
cord and brainstem during central nervous system (CNS) development. These are
somatic motoneurons (sMNs) that innervate somite-derived skeletal muscles and
visceral motoneurons (vMNs) that innervate either autonomic ganglia (general
visceral) or branchial arch-derived muscles (special visceral). Extensive
studies in the spinal cord have provided insight into the molecular pathway
that controls sMN differentiation
(Jessell, 2000
;
Shirasaki and Pfaff, 2002
),
whereas less is known about the genetic program that establish vMN identity in
the hindbrain (Cordes,
2001
).
All motoneurons in the developing CNS depend on Sonic hedgehog (Shh)
signals for their generation (Ericson et
al., 1996
; Chiang et al.,
1996
). Shh is secreted by ventral midline cells of the notochord
and floor plate and acts in a graded fashion, inducing distinct neuronal
subtypes at different concentration thresholds
(Jessell, 2000
;
Briscoe et al., 2001
). A key
activity of Shh in this process is to establish the patterned expression of a
set of homeodomain (HD) and basic helix-loop-helix (bHLH) transcription
factors, so that neural progenitor cells at different DV positions acquire
distinct positional identities (Briscoe et
al., 2000
; Novitch et al.,
2001
). These transcription factors fall into two classes, class I
and class II proteins, based on their regulation by Shh
(Briscoe et al., 2000
). The
class I proteins are constitutively expressed by neural progenitors, and their
expression is repressed by Shh. The class II proteins, in turn, depend on Shh
signalling for their neural expression. Many of these proteins act directly as
transcriptional repressors (Muhr et al.,
2001
; Novitch et al.,
2001
), and their repressor activities underlie selective
cross-repressive interactions between class I and class II proteins necessary
to establish and maintain boundaries between distinct ventral progenitor
domains (Briscoe et al., 2000
;
Muhr et al., 2001
;
Vallstedt et al., 2001
;
Novitch et al., 2001
). Once
established, the expression profile of class I and class II proteins appears
also to control the fate of neurons by directing the activation of specific
downstream determinants that establish the subtype identity of post-mitotic
neurons (Briscoe et al., 2000
;
Muhr et al., 2001
;
Novitch et al., 2001
;
Pierani et al., 1999
;
Pierani et al., 2001
;
Zhou and Anderson, 2002
).
Subsequent to the period of neurogenesis, the patterned expression of these
proteins has been shown to control the spatial generation of oligodendrocytes
and astrocytes in the ventral neural tube
(Zhou and Andersson, 2002
;
Lu et al., 2002
).
Many of the basic features of cell patterning and neuronal differentiation
have emerged from studies of somatic motoneuron differentiation in the spinal
cord (Jessell, 2000
;
Shirasaki and Pfaff, 2002
).
sMNs are generated from a common ventral progenitor domain (referred to as the
pMNs domain in this study) which spans the entire spinal cord and extends also
into caudal levels of the hindbrain
(Novitch et al., 2001
;
Arber et al., 1999
). In the
spinal cord, the pMNs domain is flanked ventrally by p3 progenitors that
generate V3 neurons, and dorsally by p2 progenitors that give rise to V2
neurons (Ericson et al., 1997
;
Briscoe et al., 1999
). Dorsal
to the p2 domain, V1 and V0 neurons are generated from the p1 and p0 domains,
respectively (Ericson et al.,
1997
; Pierani et al.,
1999
; Pierani et al.,
2001
). Within the pMNs domain, the HD proteins Pax6, Nkx6.1,
Nkx6.2 and the pMNs domain specific bHLH protein Olig2 have been shown to
promote the generation of sMNs (Ericson et
al., 1997
; Vallstedt et al.,
2001
; Novitch et al.,
2001
). In particular, the activities of Nkx6.1, Nkx6.2 and Olig2
are central to this process, and each of these proteins is sufficient to
induce sMN differentiation at ectopic positions within the neural tube
(Vallstedt et al., 2001
;
Novitch et al., 2001
;
Mizuguchi et al., 2001
).
Moreover, Nkx6.1 and Nkx6.2 (Nkx6 proteins) are partly redundant, and a
virtual complete loss of sMNs is observed in Nkx6.1/Nkx6.2 compound
mutants (Nkx6 mutants) (Vallstedt
et al., 2001
). A similar deficit of sMNs is also observed in mice
lacking Olig2 function (Rowitch et al.,
2002
; Zhou and Andersson,
2002
; Lu et al.,
2002
).
A remaining issue in sMN fate specification is the relative roles for Olig2
and Nkx6 proteins in this process. Both Olig2 and Nkx6 proteins function as
repressors (Novitch et al.,
2001
; Muhr et al.,
2001
), and one role for these proteins is to prevent other
repressor proteins from being expressed in the pMNs domain. Nkx6.1 and Nkx6.2
are necessary to constrain the expression of Dbx1 and Dbx2 to more dorsal p1
and/or p0 progenitor cells (Sander et al.,
2000
; Vallstedt et al.,
2001
), whereas Olig2 suppresses the p2 determinant Irx3
(Novitch et al., 2001
;
Zhou and Anderson, 2002
). As
Irx3 and Dbx proteins have been implicated in blocking sMN induction
(Briscoe et al., 2000
;
Muhr et al., 2001
;
Vallstedt et al., 2001
;
Novitch et al., 2001
), it is
conceivable that the loss of sMNs in Nkx6 and Olig2 mutant
mice primarily reflects the deregulated expression of these repressor
proteins, or as yet unidentified repressor proteins, in the pMNs domain.
However, Olig2 also has a crucial role in ensuring the progression of sMN
differentiation by mediating the activation of the pro-neural bHLH protein
Ngn2 in the pMNs domain (Novitch et al.,
2001
; Zhou and Anderson,
2002
) and Nkx6 proteins have in turn been shown to be required for
the expression of Olig2 in the spinal cord
(Novitch et al., 2001
). Thus,
the loss of sMNs in Nkx6 and Olig2 mutants could also reveal
a more general requirement for Nkx6 proteins to act upstream of Olig2 in the
progression of sMN fate determination
(Novitch et al., 2001
;
Zhou and Anderson, 2002
).
The generation of vMN subtypes is primarily confined to the hindbrain and
sacral and thoracic levels of the spinal cord
(Jessell, 2000
;
Cordes, 2001
). In the caudal
hindbrain, sMNs and vMNs are generated at distinct DV positions, indicating
that graded Shh signalling underlies the distinction between these MN subtypes
at this level (Ericson et al.,
1997
). vMNs are generated in a position immediately ventral to
sMNs and dorsal to the floor plate, from a progenitor domain that we term
pMNv. Like the pMNs domain, cells in the pMNv domain express Nkx6.1 and Nkx6.2
(Sander et al., 2000
;
Pattyn et al., 2003
). However,
they also express Nkx2.2 and Nkx2.9 (Nkx2 proteins)
(Ericson et al., 1997
;
Briscoe et al., 1999
), but not
the pMNs markers Pax6 or Olig2 (Ericson et
al., 1997
; Novitch et al.,
2001
). In addition to its expression in progenitors, Nkx6.1 has
been shown also to be expressed in several vMN nuclei at advanced stages of
brainstem development (Puelles et al.,
2001
). These patterns of expression imply that Nkx6 and Nkx2 class
proteins contribute to the establishment of vMN identity, but genetic analyses
have not yet uncovered a role for these proteins in this process
(Briscoe et al., 1999
;
Sander et al., 2000
;
Pabst et al., 2003
;
Pattyn et al., 2003
).
We have examined the role of Nkx6 and Nkx2 class proteins in the generation
of MNs in the hindbrain. We provide evidence that Nkx6 proteins and Nkx2.2
mediate distinct and complementary activities at initial stages of vMN fate
specification. Although Nkx2.2 appears to act upstream of the vMN determinant
Phox2b (Pattyn et al., 2000
;
Dubreuil et al., 2000
;
Dubreuil et al., 2002
) in the
vMN differentiation pathway, Nkx6 proteins are necessary to ensure the
molecular integrity of differentiating vMNs, by preventing these cells from
initiating a parallel program of V0 neuron differentiation. Both Nkx6.1 and
Nkx6.2 continue to be expressed in most differentiating vMNs, and consistent
with this the migration and axonal projections of vMNs are severely affected
in Nkx6 mutant mice. We also find that the initial expression of
Olig2 in the pMNs domain is unaffected in the hindbrain of Nkx6
mutants. This is in contrast to the spinal cord where the expression of Olig2
depends on Nkx6 proteins. Despite the persistence of Olig2 expression in the
hindbrain all sMNs are missing, indicating a parallel requirement for Nkx6 and
Olig2 proteins in sMN fate determination. Together, these data provide insight
into genetic pathways that control the generation of these distinct classes of
MNs in the hindbrain, and also important loss-of-function support for the idea
(Briscoe et al., 2000
) that the
combinatorial activities of class I and class II proteins are central in the
specification of ventral neuronal subtypes.
 |
MATERIALS AND METHODS
|
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Mouse mutants
The generation and genotyping of Nkx6.1 and Nkx6.2 mutant
mice have been reported previously (Sander
et al., 2000
; Vallstedt et
al., 2001
).
Chick in ovo electroporation
Full-length Nkx2.2 and Nkx6.1 inserted into a RCASBP(B)
retroviral vectors (Briscoe et al.,
2000
) were electroporated into the hindbrain in HH stage 10 or 11
chick embryos (Briscoe et al.,
2000
). After 36-48 hours, embryos were fixed and processed for
immunohistochemistry and in situ hybridization histochemistry.
Immunohistochemistry and in situ hybridization histochemistry
Immunohistochemical localization of proteins was performed as described
(Briscoe et al., 2000
).
Antibodies used were as follows: mouse (m), rabbit (r) and guinea pig (gp)
Isl1/2, gp Nkx2.9, gp Irx3 (Briscoe et al.,
2000
), gp Nkx6.2, m anti-Evx1/2, r Dbx1, m Hb9
(Vallstedt et al., 2001
), m
Gata3 (Santa Cruz Biotechnology), m and r Nkx2.2, r Chx10, m Pax6
(Ericson et al., 1997
), r Pax6
(Covance), r Phox2b (Pattyn et al.,
2000
), r Nkx6.1 (Briscoe et
al., 1999
), r ß-gal (Cappel), gp Olig2
(Novitch et al., 2001
). In
situ hybridization histochemistry on sections or as wholemounts were performed
as described (Schaeren-Wiemers and
Gerfin-Moser, 1993
; Wilkinson,
1992
) using mouse Isl1, Dbx2, Nkx6.1, Nkx6.2, peripherin,
Sox10, Pdgfra probes and chick Phoxb2 and Shh
probes. Whole-mount X-gal staining was carried out as described elsewhere
(Mombaerts et al., 1996
).
 |
RESULTS
|
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Spatial generation of vMNs and sMNs in the hindbrain
vMNs and sMNs can be defined based on their selective expression of
molecular markers. While both classes of neurons express the generic MN marker
Isl1 (Fig. 1B)
(Ericson et al., 1992
), vMNs
selectively express Phox2b, a HD protein required for the generation of all
vMNs in the hindbrain (Fig.
1B,C) (Pattyn et al.,
1997
; Pattyn et al.,
2000
) and sMNs selectively express the HD protein Hb9
(Fig. 1D,N)
(Arber et al., 1999
;
Thaler et al., 1999
). In the
caudal hindbrain, vMNs are generated from the pMNv domain that expresses
Nkx2.2, Nkx2.9, Nkx6.1, Nkx6.2 and Phox2b
(Fig. 1B,C,H-K) (data not
shown) (Briscoe et al., 1999
;
Pattyn et al., 2000
;
Pattyn et al., 2003
). sMNs, in
turn, are generated dorsal to vMNs and ventral to V2 neurons, from progenitors
that express Nkx6.1, Nkx6.2, Pax6 and Olig2 but not Nkx2.2 or the p2
progenitor marker Irx3 (Fig.
1D,F,H,J,L) (Briscoe et al.,
2000
; Vallstedt et al.,
2001
; Novitch et al.,
2001
). The generation of sMNs and vMNs differ also along the AP
axis of the hindbrain. sMNs are confined to caudal axial levels [rhombomeres
(r) 5, 7-8] (Arber et al.,
1999
), whereas vMNs are produced along the entire AP extent of the
hindbrain (r2-8) (Lumsden and Krumlauf,
1996
; Pattyn et al.,
2000
). The absence of sMNs in the anterior hindbrain prompted us
to examine the patterning of neurons along the DV axis at this level. In r2-r4
at E10.5, no expression of Olig2 or Hb9 could be detected
(Fig. 1E), and like Pax6
(Ericson et al., 1997
), the p2
progenitor marker Irx3 extended down to the dorsal boundary of Nkx2.2
expression (Fig. 1G,M). These
data indicated that V2 neurons might be generated immediately dorsal to vMNs
in the anterior hindbrain. In support of this, no spatial gap could be
detected between Chx10+ V2 neurons and pre-migratory
Phox2b+/Isl1+ vMNs at this level of the hindbrain
(Fig. 1C,N-Q).

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Fig. 1. Spatial generation of somatic- and visceral motoneurons in the hindbrain.
(A) Schematic drawing of the embryonic hindbrain showing the distribution of
visceral motoneurons (vMNs) (red) and somatic motoneurons (sMNs) (orange)
along the AP axis. vMNs are generated in rhombomeres (r) 2-8 while sMNs are
generated in r5 and r7-8 (Lumsden and
Krumlauf, 1996 ; Cordes et al., 2001). (B-O) Transverse sections
through r7 and r3 of wild-type (wt) embryos at E10.5. In r7, sMNs express
Isl1/2 (B) and Hb9 (D), and are generated from the pMNs domain that express
Olig2 (D,F) Nkx6.1 (J), Nkx6.2 (H), Pax6 (F) but not Nkx2.2 or Irx3 (B,L). In
r3 and r7, vMNs express Isl1 and Phox2b (B,C) and are generated from the pMNv
domain that expresses Nkx2.2 (B,C), Nkx6.1 (J,K) and Nkx6.2 (H,I) but not
Olig2 (D,E), Pax6 (F,G) or Irx3 (L,M). sMNs are not generated in r3 and no
expression of Olig2 or Hb9 is detected (E). In r3, the expression of Pax6 (G)
and Irx3 (M) extends ventrally to the dorsal boundary of Nkx2.2 expression.
Chx10+ V2 neurons are detected immediately dorsal to pre-migratory
Isl1+ vMNs in r3 (O). In r7, Chx10+ V2 neurons are
detected dorsal to Is1+/Hb9+ sMNs (N). Ventral brackets
in B,D,F,H,J,L,N indicates the pMNv domain and the dorsal bracket the pMNs
domain. Brackets in C,E,G,I,K,M,O indicate pMNv domain and pre-migratory vMNs.
(P,Q) Summary of ventral progenitor domains and neural subtypes generated in
r7 (P) and r3 (Q).
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vMNs express interneuron characteristics in Nkx6-compound
mutants
The role for Nkx6.1 and Nkx6.2 in the generation of sMNs in the spinal cord
has been characterized, whereas their role in the differentiation of cranial
MNs is less well understood (Briscoe et
al., 2000
; Sander et al.,
2000
; Vallstedt et al.,
2001
; Pattyn et al.,
2003
). To investigate in more detail the role of these proteins in
the hindbrain, we first examined the initial generation of vMNs in
Nkx6.1 and Nkx6.2 compound mutant mice (Nkx6
mutants) (Vallstedt et al.,
2001
; Pattyn et al.,
2003
). In the hindbrain of Nkx6 mutants at E10.5, Nkx2.2
was expressed and essentially normal numbers of neurons that expressed Phox2b
and Isl1 could be detected (Fig.
2A-F,I-J). Analysis of lacZ expressed under the control
of the Nkx6.2 locus (Vallstedt et
al., 2001
) also revealed that the initial dorsal projections of
vMN axons were similar in Nkx6 mutants and in
Nkx6.2+/tlz controls
(Fig. 2C,D). In contrast to
sMNs, V2 and V1 interneurons, which are missing or greatly reduced in number
in Nkx6 mutants (Fig.
2I,J; data not shown; see below)
(Vallstedt et al., 2001
),
these data show that Nkx6 proteins are not required for the initial
establishment of vMN identity. We noted, however, that the ventral expansion
of Dbx2 and Dbx1 expression observed in Nkx6 mutants
(Vallstedt et al., 2001
)
encroached also into the Nkx2.2+ pMNv domain
(Fig. 2E-H). Moreover, most
Isl1+/Phox2b+ neurons generated caudal to r3 in
Nkx6 mutants had also initiated expression of the V0 neuronal
determinant Evx1 (Moran-Rivard et al.,
2001
) at E10.5, a situation not observed in controls or in
Nkx6.1 and Nkx6.2 single mutant mice
(Fig. 2M-Q). Albeit dispensable
for their generation, these data show that Nkx6.1 and Nkx6.2 function in a
redundant manner to prevent differentiating vMNs from initiating a parallel
program of V0 neuronal differentiation.

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Fig. 2. Nkx6 proteins are required to suppress interneuron characteristics in vMNs.
(A-N) Transverse sections at r2 (A-H) and r7 levels (I-N) in wt and
Nkx6 mutants at E10.5. The number of cells that co-express Phox2b and
Isl1 in r2 and r7 in Nkx6 mutants (B,J) is similar to controls (A,I).
The initial dorsal projections of vMN axons (arrowhead) are similar in
Nkx6.2+/tlz controls and in Nkx6 mutants at
E10.5, as revealed by lacZ expression in Isl1+ neurons
(C,D). sMNs, which express Isl1 but not Phox2b and are generated dorsal to
vMNs in r7 (I), are extinguished in Nkx6 mutants (J). The expression
of the progenitor HD proteins Dbx2 (G,H) and Dbx1 (E,F) expands
ventrally into the Nkx2.2+ domain in Nkx6 mutants. The
generation of Evx1+ V0 interneurons is ventrally extended in
Nkx6 mutants (K,L), and the generation of these neurons occur at the
expense of V1 and V2 interneurons and sMNs (IL) (data not shown)
(Vallstedt et al., 2001 ). Most
Isl1+ vMNs generated in Nkx6 mutants also express Evx1 at
E10.5 (M,N). The expression of Evx1 in motoneurons appeared transient and no
Isl1+/Phox2b+ cells detected at E11.5 expressed Evx1
(data not shown). (O-R) Transverse sections through r4 of wt (O),
Nkx6.2tlz/tlz (P), Nkx6.1-/- (Q) and
Nkx6 mutant (R) embryos. In Nkx6 mutants, most vMNs
expressed Evx1 (R). No expression of Evx1 could be detected in vMNs of wt
embryos (O) or in Nkx6.2tlz/tlz (P) and
Nkx6.1-/- (Q) single mutants at this level.
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Nkx2.2 but not Nkx6.1 is sufficient to induce expression of the vMN
determinant Phox2b
The generation of vMNs in Nkx6 mutants raised the issue as to
which factors might be directly involved in mediating the activation of
downstream determinants of vMN fate, notably Phox2b
(Pattyn et al., 2000
;
Dubreuil et al., 2000
). Nkx2.2
and Nkx2.9 are candidates to be involved in this process
(Briscoe et al., 1999
), as
these proteins are co-expressed in vMN progenitors
(Pattyn et al., 2003
) and are
still expressed in the hindbrain of Nkx6 mutants
(Fig. 2E,F)
(Pattyn et al., 2003
). The
generation of vMNs, however, is left largely unaffected in both
Nkx2.2 (Briscoe et al.,
1999
) and Nkx2.9 mutants
(Pabst et al., 2003
) but it is
possible that this may reflect functional redundancy between these proteins
(Briscoe et al., 1999
;
Briscoe et al., 2000
;
Pabst et al., 2003
). We
therefore examined if forced expression of Nkx2.2 or Nkx6.1 was sufficient to
induce expression of Phox2b at dorsal positions of the chick hindbrain. Stage
10 chick embryos were electroporated in ovo with RCAS-Nkx2.2 or RCAS-Nkx6.1
vectors (Briscoe et al., 2000
)
and after 36-48 hours of incubation embryos were harvested and analysed.
Widespread expression of Nkx2.2 in the caudal hindbrain resulted in activation
of Phox2b and Isl1 expression along the entire DV extent of the
hindbrain, albeit the induction of Phox2b appeared to be more
prominent in the ventral hindbrain (Fig.
3A-C). By contrast, Phox2b was not induced in response to
Nkx6.1 (Fig. 3E,F). Consistent
with the established role for Nkx6.1 in the specification of sMN fate
(Briscoe et al., 2000
), Nkx6.1
was sufficient to induce expression of the generic MN marker Isl1
(Fig. 3G) (see
Briscoe et al., 2000
). Forced
expression of Nkx2.2 or Nkx6.1 had no effect on the expression of Shh
(Fig. 3D,H). These data show
that Nkx2.2 but not Nkx6.1 is sufficient to induce the vMN determinant Phox2b
in the hindbrain, and provide evidence that Nkx6 and Nkx2 class proteins
mediate separate and complementary activities at initial stages of vMN
generation.

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Fig. 3. Induction of the vMN determinant Phox2b by Nkx2.2. (A-D) Consecutive
transverse sections through a stage 22 chick hindbrain electroporated with
RCAS-Nkx2.2 construct. Forced expression of Nkx2.2 (A) induces expression of
the vMN markers Phox2b (B) and Isl1/2 at ectopic dorsal positions
(C). The expression of Shh was unaffected (D). No ectopic
Isl1/2+ neurons induced in response to Nkx2.2 coexpressed the V0
neuron determinant Evx1 (data not shown). (E-H) Consecutive sections through
stage 22 chick hindbrain electroporated with RCAS-Nkx6.1 construct.
Misexpression of Nkx6.1 (E) induces ectopic expression of Isl1/2 (G) but not
Phox2b (F) or Shh (H).
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Impaired migration and axonal projections of vMNs in Nkx6
mutants
In addition to their expression in progenitor cells, Nkx6.1 and Nkx6.2 are
also co-expressed in most hindbrain vMNs over the period that these cells
differentiate, migrate and extend axons towards peripheral targets
(Fig. 4C-F and data not shown).
In r4-derived facial branchial (fb) MNs and GATA3+ inner ear
efferent (iee) (Karis et al.,
2001
) neurons, however, only the expression of Nkx6.1 persisted
and Nkx6.2 was downregulated soon after their generation, and prior to the
caudal migration (Studer et al.,
1996
) of fbMNs into r6 (Fig.
4G,H; data not shown). The prolonged expression of Nkx6.2 and/or
Nkx6.1 in vMNs prompted us to examine later aspects of vMN differentiation in
mice lacking Nkx6 function. All vMNs, independent of subtype or
origin along the AP axis, revealed impaired migratory and axonal projection
properties in Nkx6 mutants. In normal conditions, most r2-derived
(trigeminal) MNs have completed their dorsal migration at E12, and settled
close to the point at which MN axons exit the neural tube
(Fig. 4J). In Nkx6
mutants, vMNs in r2 were still dispersed along the migratory route at E12 and
cells eventually settled at around E13.5 in an aberrant ventral position
(Fig. 4L,S). Other vMNs
subtypes that migrate in a strict ventral-to-dorsal fashion, such as vMNs of
the glossopharyngeal and vagal nerve in the caudal hindbrain, revealed a
similar migratory phenotype (data not shown). Moreover, the characteristic
caudal migration of r4-derived fbMNs also failed to occur in Nkx6
mutants; instead of initiating a caudal migration from r4 through r5 and into
r6 (Studer et al., 1996
),
these MNs migrated in a strictly ventral-to-dorsal fashion within r4
(Fig. 4P). A similar, albeit
less dramatic, migratory defect of fbMNs was observed also in Nkx6.1
single mutants (Fig. 4O). In
these mice, fbMNs failed to reach r6 and instead migrated dorsally in either
r4 or r5. Importantly, as Evx1 is not expressed in r4-derived MNs in
Nkx6.1 single mutants (Fig.
2Q), these data show that the impaired migration of vMNs cannot
simply reflect an early requirement of Nkx6 proteins to suppress the V0
determinant Evx1 in vMNs.

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Fig. 4. The migration of vMNs is impaired in Nkx6.1 and Nkx6
mutants. (A-F) Transverse sections through r2 of wild-type embryos at E10.5
and E11.5. The expression of Nkx6.1 (C,D) and Nkx6.2 (E,F) but not Nkx2.2
(A,B) persists in Isl1+ trigeminal vMNs as they migrate from the
pMNv domain towards a more dorsal position of the hindbrain. (G,H) Dorsal view
of flat-mounted hindbrain at E11.5. Caudally migrating fbMNs originate in r4
and migrate caudally through r5 (arrowhead) into r6 where they turn dorsally
and settle in a dorsal position (Studer et
al., 1996 ). Nkx6.1 is expressed in caudally migrating fbMNs (G),
whereas Nkx6.2 only is detected only in pre-migratory fbMNs in r4 (H). Both
Nkx6.1 (G) and Nkx6.2 (H) are expressed in trigeminal vMNs
(indicated as N.V). The color reaction in G,H was underdeveloped to reveal the
expression of Nkx6.1 and Nkx6.2 in post-mitotic neurons over
the expression of these genes in ventral progenitor cells. (I-M) Transverse
sections through r2 at E10.5 (I,K) and E12 (J,L) of
Nkx6.2+/tlz controls (I,J) and in Nkx6 mutants
(K,L) showing impaired dorsal migration of trigeminal MNs in Nkx6
mutants. The position of trigeminal MNs was determined by Isl1/2 expression
and their axonal projections was visualized by the expression of
lacZ. In Nkx6 mutants at E10.5 (K), Isl1/2+ cells
are detected closer to the ventral midline compared with
Nkx6.2+/tlz controls (I). At E12, most trigeminal MNs have
reached their final position close to the trigeminal nerve exit point in
Nkx6.2+/tlz controls (J), whereas many cells are
positioned along the migratory pathway in Nkx6 mutants at this stage
(L). Note that the trigeminal nerve exit point appears unaffected
Nkx6 mutants (arrowhead in J,L), but that cells settle in an aberrant
ventral position in Nkx6 mutants compared with controls (J,L). (M)
Quantification of vMN migratory defects in r2 of Nkx6.1 and
Nkx6 mutant mice. The position of migrating Isl1+ cells
along their migratory route was assessed by arbitrarily dividing r2 into four
equivalent zones (indicated as z1-z4 in K,L) between the site of generation
and the trigeminal nerve exit point (arrowhead in J,L). The percentage of
Isl1+ cells located in each zone at E10.5 and E12 in wild-type
controls, Nkx6.1 single mutants and Nkx6 mutants is
indicated (M). No migratory defects were observed in Nkx6.2 single
mutant mice (data not shown). (N-S) Dorsal view of flat-mounted hindbrains
showing Isl1 expression in wild-type (N,Q), Nkx6.1 mutants
(O,R) and Nkx6 mutants (P,S) at E11.5 and E13.5. In wild-type embryos
at E11.5, Isl+ fbMNs generated in r4 have initiated their
caudal migration through r5 into r6 (N), where they accumulate and settle in a
lateral position at E13.5 (Q, indicated as N.VII). In Nkx6.1 mutants,
fbMNs fail to migrate into r6 and instead migrate to occupy positions in r4 or
r5 (O,R). In Nkx6 mutants, all fbMNs fail to initiate a caudal
migration, and cells instead migrate strictly dorsally within r4 (P,S). In
wild-type embryos at E11.5, Isl1+ trigeminal MNs
(indicated as N.V) in r2 and r3 have migrated away from the ventral midline
towards their final settling position (N,Q). As indicated above in I-M, the
migration of Isl1+ trigeminal MNs occur at a slow pace in
Nkx6.1 mutants (arrowhead in O) and Nkx6 mutants (arrowhead
in P).
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In addition, the axonal projection patterns of vMNs were severely affected
by the loss of Nkx6.1 and Nkx6.2 function. Hindbrain vMNs initially extend
their axons dorsally to leave the neural tube from dorsal exit points in even
numbered rhombomeres, and project towards their peripheral targets
(Cordes, 2001
). Analysis of
tau-lacZ expression (Vallstedt et
al., 2001
) revealed that many different aspects in the projection
pattern of motor axons were impaired in Nkx6 mutants. At caudal
hindbrain levels, the majority of vagal and glossopharyngeal motor axons
failed to recognise their exit points and instead, upon reaching a dorsal
position of the neural tube, axons turned and projected caudally or rostrally
within the CNS (Fig. 5A,B).
These caudal groups of vMNs were eventually eliminated in Nkx6
mutants, as indicated by the absence of lacZ+ axonal
projections in the vagal and glossopharyngeal nerves at E13.5
(Fig. 5C-F) and the complete
lack of cells expressing peripherin in the caudal hindbrain at E16.5
(Fig. 5M,N). vMNs at more
anterior hindbrain levels appeared less dramatically affected, and a
trigeminal and facial nucleus were still detected in Nkx6 mutants at
E16.5. However, the total number of MNs in each nucleus was significantly
reduced (Fig. 5G-L) and,
consistent with the migratory defects, the trigeminal nuclei were displaced
ventrally and the facial nuclei occupied an aberrant anterior position in
Nkx6 mutants at this stage (Fig.
5G-L). The fact that a trigeminal and facial nucleus could be
detected as late as E16.5 in Nkx6 mutants indicated that at least a
subset of vMNs in these nuclei project axons out of the CNS and receive the
necessary trophic support provided by peripheral targets
(deLapeyriere and Henderson,
1997
). In direct support for this, analysis of lacZ
expression revealed that motor axons had projected into the trigeminal and
facial nerve in Nkx6 mutants at E13.5, although many axons at this
stage followed aberrant peripheral routes
(Fig. 5C-F). Together, these
data reveal that Nkx6.1 and Nkx6.2 are required for both the migration and
axonal navigation of vMNs, thus being consistent with a cell-autonomous
requirement for Nkx6 proteins in differentiating vMNs.

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Fig. 5. Abnormal projection patterns of vMN axons in Nkx6 mutant mice.
(A,B) Dorsal view of the hindbrain of Nkx6.2+/tlz controls
(A) and Nkx6 mutants (B) showing lacZ expression (detected
by X-gal staining) at E10.5. On the right side of each micrograph in A,B, a
schematic summary of axonal projections of vMN subtypes is included; blue
indicates trigeminal MNs (V) generated in r2-3, red indicates r4-5 derived
vMNs projecting in the facial nerve (VII), pink: indicates r6-derived vMNs
projecting into the glossopharyngeal nerve (IX) and green indicates vMNs
projecting into the vagal nerve (X). Open circles indicate the axonal exit
point of vMN subtypes. In Nkx6 mutants, most vMNs generated in the
caudal hindbrain fail to project out of the neural tube through their normal
exit points in r6 and r7. Instead, axons turn and extend either in caudal or
rostral directions within the neural tube (A,B; data not shown). Axonal
projections of vMNs into the VIIth and Vth nerve is less affected and most
axons converge at their respective exit point. In contrast to controls, few
axons in Nkx6 mutants have at this stage exited the neural tube
(A,B). A significant number of r3-derived trigeminal MNs at E10.5, arbitrarily
project caudally towards the exit point in r4, rather than their normal exit
point in r2. (C-F) Lateral view of E13.5 embryos showing lacZ
expression in vMN axons in Nkx6.2+/tlz controls (C) and
Nkx6 mutants (D). In Nkx6.2+/tlz embryos, the
normal pattern of peripheral projections of the trigeminal (V), facial (VII),
glossopharyngeal (IX) and vagal (X) nerves is detected. In Nkx6
mutants (D), lacZ+ projections of vMNs into the trigeminal
(V), and facial (VII) nerves are severely impaired, although the overall shape
of the facial nerve resembled that of control embryos. At more caudal levels
of Nkx6 mutants, no lacZ+ axonal projections into
the vagal (X) and glossopharyngeal nerves (IX) are detected. Schematic summary
of axonal projections in control (Nkx6.2+/tlz) and
Nkx6 mutants at E13.5 (E,F). (G,H) Transverse sections through the
brainstem of wild type (G,I,K,M) and Nkx6 mutants (H,J,L,N) at E16.5,
hybridized with a peripherin probe to visualize MN nuclei. Sections are shown
in an anterior (upwards) to posterior (downwards) direction. In Nkx6
mutants, the trigeminal (N.V; H) and facial (N.VII; J) nuclei are reduced in
size compared with controls (G,K) and the facial nucleus is displaced
rostrally (J, compare with wild type in K). The nuclei of the vagal nerve,
nucleus ambiguus (N.A) and the dorsal motor nucleus (dmnX), are absent in
Nkx6 mutants (N, compare with wild type in M). In addition, sMNs of
the abducens (N.VI; I) and the hypoglossal (N.XII; M) nuclei are missing in
Nkx6 mutants (J,N).
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Differential regulation of Olig2 expression by Nkx6 proteins in the
hindbrain
We next turned our attention to sMNs generated dorsal to vMNs at caudal
levels of the hindbrain. Nkx6.1 and Nkx6.2 are required for the generation of
sMNs (Sander et al., 2000
;
Vallstedt et al., 2001
), but
the precise role for these proteins in the specification of sMN fate is
unclear; Nkx6.1 and Nkx6.2 mediate their neural patterning activities by
functioning as transcriptional repressors (Muhr et al., 2000), indicating that
they promote sMN generation by suppressing the expression of other repressors,
such as Dbx1 and Dbx2, in sMN progenitors
(Briscoe et al., 2000
;
Sander et al., 2000
;
Vallstedt et al., 2001
).
However, Nkx6.1 and Nkx6.2 are also necessary for the expression of Olig2 in
the spinal cord (Novitch et al.,
2001
). It is possible, therefore, that the loss of sMNs in
Nkx6 mutants (Vallstedt et al.,
2001
) primarily reflects the loss of Olig2 expression in these
mice (Novitch et al., 2001
;
Zhou and Andersson, 2002
). In
the caudal hindbrain of Nkx6 mutants, the expression of Dbx2 is
derepressed in the sMN progenitor domain
(Fig. 6G,H) in a fashion
similar to that previously reported at the spinal cord level
(Vallstedt et al., 2001
).
Unexpectedly, however, we found that the expression of Olig2 appeared
unaffected in the caudal hindbrain at E10
(Fig. 6A-B,E-F). Even more
strikingly, in ventral positions of the anterior hindbrain (r2-r4), where
Olig2 is not normally expressed over the period that MNs are generated, the
loss of Nkx6.1 and Nkx6.2 function resulted in ectopic activation of Olig2
expression (Fig. 6K,L). In the
caudal hindbrain, the expression of Olig2 in Nkx6 mutants was
progressively lost and could no longer be detected at E11.5
(Fig. 6I,J). By contrast, the
domain of ectopic Olig2 expression in the anterior hindbrain persisted over
this period (data not shown). These data reveal an unanticipated differential
regulation of Olig2 expression by Nkx6 proteins along the AP axis of the
neural tube, and while Nkx6.1 and Nkx6.2 are necessary for Olig2 expression in
the spinal cord (Novitch et al.,
2001
), these same proteins are required to suppress the expression
of Olig2 in the anterior hindbrain. The mechanism by which Nkx6 proteins
suppresses the expression of Olig2 in the anterior hindbrain is unclear, but
does not seem to involve overall changes in AP-identity and Hox-gene
expression in the hindbrain of Nkx6 mutants
(Pattyn et al., 2003
) (data
not shown).

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Fig. 6. Olig2 is expressed but sMNs are missing in the hindbrain of Nkx6
mutants. (A-R) Transverse sections through r7 or r4 of wild type and
Nkx6 mutant embryos at E10 (A-H), E11.5 (I-J) or E10.75 (K-R). (A-H)
In r7, the expression of Olig2 is similar in controls and Nkx6
mutants at E10 (A,B). Hb9+ sMNs are detected in controls (A) but
not Nkx6 mutants (B). The pattern of Nkx2.2, Pax6 and Irx3 is similar
in controls and Nkx6 mutants in r7 at E10 (C-F). The expression of
Dbx2 is expanded ventrally and ectopically expressed in the pMNs domain (G,H).
By E11.5, the expression of Olig2 is extinguished at the level of r7 in
Nkx6 mutants (I,J). (K-R) In r4 of wild-type embryos at E10.75, sMNs
are not generated and no expression of Olig2 or Hb9 can be detected (K). In
Nkx6 mutants, Olig2 is expressed ectopically in r4, but no
Hb9+ neurons are detected (L). Most ectopic Olig2+ cells
in r4 of Nkx6 mutants co-express Pax6 and Nkx2.2 at E10.75 (M,N). No
expression of the oligodendrocyte precursor cell markers Sox10 or
Pdgfra is detected either in controls (O,Q) or in Nkx6
mutants (P,R) at E10.75.
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The detection of Olig2 expression in the caudal hindbrain at E10 in
Nkx6 mutants prompted us to examine the loss of sMNs at this level
(Sander et al., 2000
;
Vallstedt et al., 2001
) in
more detail. Pax6 is required for the generation of sMNs in the hindbrain
(Ericson et al., 1997
),
whereas Nkx2.2 and Irx3 have been implicated to suppress sMN generation
(Briscoe et al., 1999
;
Briscoe et al., 2000
). At E10
in Nkx6 mutants, the majority of Olig2+ cells in the
caudal hindbrain expressed Pax6 but not Nkx2.2 or Irx3
(Fig. 6C-F). Thus, the early
loss of Hb9+ sMNs does not appear to reflect a loss of Pax6
expression, or a deregulated expression of Nkx2.2 or Irx3 proteins in sMN
progenitors. At later stages in Nkx6 mutants, however, the expression
of Nkx2.2 had begun to encroach dorsally into the sMN progenitor domain, and
by E10.5 many cells which co-expressed Nkx2.2 and Pax6 could be detected (data
not shown). This expansion of Nkx2.2 expression coincided with the progressive
loss of Olig2 expression observed in the caudal hindbrain
(Fig. 6I,J; data not shown). As
Nkx2.2 is a known repressor of Olig2 expression
(Novitch et al., 2001
), it is
possible that the progressive loss of Olig2 expression at this level in
Nkx6 mutants reflects a requirement for Nkx6 proteins to constrain
the expression of Nkx2.2 to vMN progenitors.
In the anterior hindbrain, the ectopic expression of Olig2 in Nkx6
mutants was not accompanied by any generation of Hb9+ sMNs
(Fig. 6K,L). In addition, in
contrast to the caudal hindbrain, most ectopic Olig2+ cells
co-expressed Nkx2.2 and Pax6 already by E10
(Fig. 6M,N and data not shown).
A dorsal expansion of Nkx2.2 expression, and the co-expression of Nkx2.2 and
Olig2 in ventral progenitor cells, has previously been linked to the
specification of oligodendrocyte precursors (OLPs) in the spinal cord
(Zhou et al., 2001
). We
therefore considered if Olig2+/Nkx2.2+ progenitors in
the anterior hindbrain resulted in a premature generation of OLPs. However, no
expression of Sox10 or Pdgfra, two early markers of OLP
differentiation (Hall et al.,
1996
; Kuhlbrodt et al.,
1998
), could be detected in the anterior hindbrain between
E10-E11.5 either in controls or Nkx6 mutants
(Fig. 6O-R; data not shown). At
caudal hindbrain levels we instead observed a loss of Sox10 and
Pdgfra expression in Nkx6 mutants compared to controls (data
not shown) (M. Qui, personal communication). This observation is consistent
with the progressive loss of Olig2 expression observed at this level
(Fig. 6I), indicating that Nkx6
proteins are required for the generation, or correct temporal specification,
of OLPs in the caudal
hindbrain.

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Fig. 7. Model for the role of Nkx proteins in somatic- and visceral MN generation.
(A) sMN generation: The combinatorial expression of Nkx6 proteins (Nkx6.1 and
Nkx6.2), Olig2 and Pax6 act to suppress cells in the pMNs domain from
undertaking other ventral differentiation programs; Nkx6.1 and Nkx6.2 suppress
V0 and V1 fate (Briscoe et al.,
2000 ; Vallstedt et al.,
2001 ), Olig2 suppresses V2 fate
(Novitch et al., 2001 ;
Zhou and Anderson, 2002 ) and
Pax6 blocks vMN fate (Ericson et al.,
1997 ). In addition to its role in suppressing V2 fate, Olig2 also
promote cells to progress along the sMN differentiation pathway
(Novitch et al., 2001 ; Zhou
and Andersson). In part, Nkx6.1 and Nkx6.2 promote sMN generation by acting
upstream of Olig2 (Novitch et al.,
2001 ) (this study), but Nkx6 proteins also act in parallel with
Olig2 in the progression of sMN fate determination. (B) vMN generation: The
combinatorial activity of Nkx6 and Nkx2 class proteins in the pMNv domain
suppress more dorsal sMN and interneuron differentiation programs. Nkx6.1 and
Nkx6.2 suppress V0 and V1 fate (Vallstedt
et al., 2001 ) (this study), while Nkx2.2 and Nkx2.9 suppress sMN
and V2 neuronal fate (Briscoe et al.,
1999 ; Briscoe et al.,
2000 ; Pabst et al.,
2003 ). In the pMNv domain, Nkx6 proteins are dispensable for the
progression of vMN differentiation, and Nkx2 class proteins mediate the
activation of the vMN determinant Phox2b
(Pattyn et al., 2000 ). Once
induced, Phox2b and Nkx2 proteins may also cooperate in subsequent steps of
vMN fate determination (Dubreuil et al.,
2002 ). For further details, see text.
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DISCUSSION
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Complementary roles for Nkx2 and Nkx6 class proteins in vMN fate
specification
In this study, we provide evidence that Nkx6 and Nkx2 class proteins
mediate complementary activities in the specification of vMN fate. Nkx6.1 and
Nkx6.2 are not required for the generation of vMNs, and instead these proteins
ensure that differentiating vMNs do not initiate a parallel V0 differentiation
program. The role for Nkx6 proteins to suppress Dbx gene expression and the V0
determinant Evx1 (Moran-Rivard et al.,
2001
) in vMNs appears to be analogous to their role in more dorsal
sMN progenitors (Vallstedt et al.,
2001
). However, while progenitors in the sMN domain in
Nkx6 mutants give rise to V0 neurons at the expense of sMNs
(Vallstedt et al., 2001
), vMNs
are generated in close to normal numbers but with a mixed vMN/V0-neuronal
identity. This difference can most easily be explained by the fact that Nkx6
proteins are necessary, either directly or indirectly, for the activation of
downstream determinants of sMN fate (see below), whereas the initiation of
Phox2b expression in the vMN pathway is mediated by a separate activity.
Nkx2.2 and Nkx2.9 are expressed in the hindbrain of Nkx6 mutants
(Pattyn et al., 2003
), and we
show that Nkx2.2 is sufficient to induce Phox2b expression in the hindbrain.
These data indicate that Nkx2.2 acts upstream of Phox2b in the vMN pathway.
Consistent with this idea, the expression of Nkx2.2 is unaffected in
Phox2b mutants despite the fact that all vMNs are missing in these
mice (Pattyn et al., 2000
).
Nkx2.2, however, is not required for the generation of vMNs
(Briscoe et al., 1999
), but
this may reflect functional redundancy between Nkx2.2 and Nkx2.9
(Briscoe et al., 1999
;
Pabst et al., 2003
). The fact
that Nkx2.2 and Nkx2.9 are induced independently of each other in the
hindbrain (Briscoe et al.,
1999
; Pabst et al.,
2003
) and that the generation of vMNs is also left largely
unaffected in Nkx2.9 mutants (Pabst et
al., 2003
), supports this idea. Recent data, however, has shown
that Nkx2.9 cannot compensate for the loss of Nkx2.2 function by rescuing the
generation of serotonergic (S) neurons in the hindbrain
(Pattyn et al., 2003
), despite
the fact that Nkx2.9, in Nkx2.2 mutants, is expressed in the S neuron
progenitor domain. These data indicate differences in the intrinsic properties
of Nkx2.2 and Nkx2.9. Thus, although data may favour redundancy between these
proteins in vMN fate specification, this idea needs to be established by
future analyses of Nkx2.2 and Nkx2.9 compound mutant
mice.
The persistent expression of Nkx2.2 and Nkx2.9 in Nkx6 mutants
also provides a logic as to why vMNs express V0 neuron characteristics, but
not traits typical of other ventrally generated neurons. Previous studies have
shown that Nkx2.2 suppresses the generation of sMNs in the spinal cord
(Briscoe et al., 1999
;
Briscoe et al., 2000
), most
likely due to its role in repressing the expression of Olig2
(Novitch et al., 2001
). Nkx2.2
and/or Nkx2.9 are also strong candidates to suppress V2 neuronal fate, as
expression of the V2 determinant Irx3 only extends ventrally to the dorsal
boundary of Nkx2.2 expression in both Nkx6 and Olig2 mutant
mice (this study) (Zhou and Anderson,
2002
; Lu et al.,
2002
). Thus, while high levels of Nkx6 activity act to suppress V0
and V1 neuronal fate (Vallstedt et al.,
2001
), Nkx2 class proteins instead appear to block programs of sMN
and V2 neuron differentiation. Considering that Nkx2 class proteins act
upstream of Phox2b, the combined activities of Nkx6 and Nkx2 class proteins
appear sufficient therefore to account for the initial steps of vMN fate
specification.
Sequential roles for Nkx6 proteins in vMN differentiation
The expression of Nkx6.1 and/or Nkx6.2 is maintained in differentiating
vMNs, indicating that these proteins are involved in sequential steps of vMN
differentiation. In support of this, we find that both the migration and
axonal projection properties of vMNs in the hindbrain are affected by the loss
of Nkx6.1 and Nkx6.2 function; the dorsal migration of vMN subtypes occurs at
a slow pace, and r4-derived fb MNs fail to migrate caudally into r6 and
instead migrate strictly dorsally within r4. In addition, the overall
navigation of vMN axons, both within the CNS and in the periphery, is
perturbed. The altered properties of differentiating vMNs are consistent with
a cell-autonomous role for Nkx6 proteins in post-mitotic differentiating vMNs,
but as Nkx6.1 and Nkx6.2 are also broadly expressed in ventral progenitor
cells, we cannot exclude that migratory and axonal pathfinding defects also
involve changes in the environment that vMNs encounter as they differentiate.
In addition, the early role for Nkx6 proteins in vMN specification makes it
difficult to definitively link the requirement for Nkx6.1 and Nkx6.2 to
postmitotic neurons. Our analysis of Nkx6.1 single mutants, however,
show that the impaired migration of vMNs cannot only be a secondary
consequence of their early role to suppress Evx1 expression in vMNs, because
Nkx6.2 alone is sufficient to suppress Evx1 in r4-derived fbMNs, but not to
compensate for the loss of Nkx6.1 by fully restoring the subsequent migration
of these neurons. We have noticed that the expression of cadherin 8
(Garel et al., 2000
;
Korematsu and Redies, 1997
) is
not initiated in differentiating fb MNs in Nkx6 mutants (data not
shown), and Muller and colleagues have found that the expression profile of
netrin receptors are altered in vMNs in Nkx6.1 single mutants (M.
Sander, personal communication). These data provide additional, albeit
indirect, support for a cell-autonomous role for Nkx6 proteins in vMNs, and
raise the possibility that the deregulated expression of these proteins may
contribute to the impaired differentiation of vMNs observed in Nkx6.1
and Nkx6 mutant mice.
A parallel requirement for Nkx6 and Olig2 proteins in sMN fate
determination
Our current analysis provides new insight also into the role of Nkx6 and
Olig proteins in the generation of sMNs. Olig2 has previously been shown to
have a dual role in sMN fate determination; it suppresses the expression of
Irx3 in sMN progenitors, and also promotes cell-cycle exit and neuronal
differentiation by derepression of the pro-neural bHLH protein Ngn2 in the sMN
progenitor domain (Novitch et al.,
2001
; Mizuguchi et al.,
2001
; Zhou and Anderson,
2002
; Lu et al.,
2002
). Nkx6 proteins are required for the expression of Olig2 in
the spinal cord (Novitch et al.,
2001
), and there is a similar deficit of sMNs in Nkx6
mutants, Olig2 mutants and Olig1/2 compound mutants
(Vallstedt et al., 2001
;
Zhou and Anderson, 2002
;
Lu et al., 2002
). Because
forced expression of Nkx6.1 in the chick spinal cord results in the ectopic
activation of Olig2 expression (Novitch et
al., 2001
) and the expression of Nkx6.1 is left unaffected in
Olig mutants (Zhou and Anderson,
2002
), a model in which Olig2 acts downstream of Nkx6 proteins in
the sMN pathway has been proposed (Novitch
et al., 2001
; Zhou and
Anderson, 2002
). In contrast to spinal cord levels, we find that
the initial phase of Olig2 expression is unaffected in the caudal hindbrain in
Nkx6 mutants, and neither the expression of Irx3 nor Nkx2.2 have
encroached into the sMN progenitor domain at this stage. Despite this, all
sMNs are missing. These data reveal a requirement for Nkx6.1 and Nkx6.2 in sMN
fate specification that is unrelated to their role in promoting Olig2 gene
expression, and further indicate that Olig2, in the absence of Nkx6 protein
function, is not sufficient to specify sMN fate in the hindbrain. These
findings seem to exclude the possibility that Nkx6 and Olig proteins operate
in a strict linear pathway. As both Nkx6 and Olig proteins mediate their
inductive activities by acting as repressors
(Muhr et al., 2001
;
Novitch et al., 2001
), it
appears more likely that these proteins act in parallel to exclude different
sets of repressor proteins from the sMN progenitor domain
(Muhr et al., 2001
;
Novitch et al., 2001
). If
expressed in sMN progenitors in either Nkx6 or in Olig
mutant mice, such Olig2 of Nkx6 regulated repressor proteins would be
predicted to act independently of each other to block sMN generation at a step
downstream of Olig2. This idea gains support by the fact that forced
expression of Irx3 within the sMN progenitor domain, is sufficient to inhibit
sMN generation (Briscoe et al.,
2000
).
In previous analyses, the genetic ablation of individual class I or class
II proteins has typically resulted in a transformation of progenitor domain
identity, followed by a predictable switch in neuronal subtype identity
(Ericson et al., 1997
;
Vallstedt et al., 2001
;
Pierani et al., 2001
;
Novitch et al., 2001
;
Zhou and Anderson, 2002
).
Although these data highlight a central role for class I and class II proteins
in the establishment of progenitor domains, the early transformation of
progenitor domain identity has precluded attempts to evaluate the relevance of
the combinatorial expression of these proteins in neuronal fate determination.
It remained possible, for example, that the only role for Pax6
(Ericson et al., 1997
;
Novitch et al., 2001
), Nkx6.1
and Nkx6.2 in the pMNs domain (Vallstedt
et al., 2001
; Novitch et al.,
2001
) was to ensure the expression of Olig2, which in turn
directed all downstream events necessary for the establishment of sMN identity
(Novitch et al., 2001
;
Zhou and Anderson, 2002
). In
our current hindbrain analysis, we provide evidence for a parallel requirement
of Nkx6 and Olig2 proteins in sMN fate specification, and furthermore that
Nkx6 and Nkx2 class proteins mediate complementary activities in the
specification of vMN fate. Importantly, as Nkx6.1 and Nkx6.2 are not required
for the initial establishment of either the pMNs or the pMNv progenitor
domain, these findings suggest that the combinatorial activities of class I
and/or class II protein expression in distinct progenitor domains
(Briscoe et al., 2000
) also is
necessary for the rigid specification of ventral neuronal subtypes.
 |
ACKNOWLEDGMENTS
|
---|
We thank T. Jessell, B. Novitch and A. Pierani for providing Olig2 and Dbx1
antibodies, and B. Richardson for the mouse PDGFR
cDNA. We are also
grateful to P. Bailey, T. Jessell and J. Briscoe for comments on the
manuscript. A.P. was supported by a post-doctoral fellowship from the
Karolinska Institute (KI) and J.M.D. is supported by the graduate program of
basic and applied biology of the University of Porto and the Portuguese
Foundation for Science and Technology. J.E. is supported by the Royal Swedish
Academy of Sciences by a donation from the Wallenberg Foundation, The Swedish
Foundation for Strategic Research, The Swedish National Research Council,
Project A.L.S., the KI and by the EC network grants, Brainstem Genetics
(QLRT-2000-01467) and Stembridge (QLG3-CT-2002-01141).
 |
Footnotes
|
---|
* These authors contributed equally to this study 
Present address: CNRS UMR8542 Ecole Normale Superieure, Departement de
Biologie, 75005 Paris, France 
Present address: Department of Developmental & Cell Biology, University
of California at Irvine, 4228 McGaugh Hall, CA, USA 
 |
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