Howard Hughes Medical Institute, Department of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA
* Author for correspondence (e-mail: mariocapecchi{at}genetics.utah.edu)
Accepted 10 December 2003
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SUMMARY |
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Key words: Somatosensory, Viscerosensory, Proprioceptive, Dorsal interneurons, Rhombomeres, Hindbrain, Homeodomain proteins
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Introduction |
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Hox genes have become prime molecular candidates for providing
AP-positional information to all cells at a given axial level. Together with
other investigators, we have characterized the AP-restricted function of Hox
genes in the developing spinal cord and hindbrain through gain- and
loss-of-function analyses. These studies have focused primarily on the
specification of motoneurons. In the spinal cord, for example, Hoxc8
and Hoxd10 are required for the normal development of motoneurons
controlling movement of the forelimbs and hindlimbs, respectively
(Carpenter et al., 1997;
Tiret et al., 1998
). In the
hindbrain, Hoxb1 and Hox3 genes are necessary and sufficient
for the specification of rhombomere (r) 4-branchial and r5-somatic
motoneurons, respectively (Bell et al.,
1999
; Gaufo et al.,
2000
; Gaufo et al.,
2003
; Goddard et al.,
1996
; Guidato et al.,
2003
; Studer et al.,
1996
). These examples of motoneuron specification illustrate the
phenomenon of spatial colinearity, whereby expression and function of Hox
genes along the AP axis of the organism is correlated with their chromosomal
location (Lewis, 1978
;
McGinnis and Krumlauf,
1992
).
In this study, we examined the role of Hox genes on the specification of
interneurons in the sensory system of the developing hindbrain. In the early
developing hindbrain, Hox genes are generally expressed throughout the
neuroepithelium, from the ventricular to the pial layers, suggesting multiple
roles in neuronal differentiation (Gaufo
et al., 2003). Moreover, the reinforcement of later Hox gene
expression in multiple longitudinal columns that correspond to the positions
of various neuronal lineages suggests the potential dependence of many
neuronal subtypes on Hox gene expression along the DV axis
(Davenne et al., 1999
;
Gaufo et al., 2000
;
Gaufo et al., 2003
;
Pattyn et al., 2003
). To begin
to identify the neuronal subtypes that are dependent on Hox genes, we analyzed
the development of three-distinct first-order sensory interneurons arranged in
non-overlapping domains along the DV axis. These interneurons include
first-order proprioceptive, visceral and somatic sensory relay interneurons
that form contiguous columns along the AP axis
(Bermingham et al., 2001
;
Gross et al., 2002
;
Lee et al., 2000
;
Muller et al., 2002
). Analysis
of Hoxb1, Hoxa3, Hoxb3 and Hoxa2 loss-of-function mutations
in embryonic mice reveal that these Hox genes are required for the
specification of visceral and somatic sensory interneurons via the regulation
of Phox2b and Lbx1, respectively. However, formation of
proprioceptive sensory interneurons expressing LH2A/B appears to be
independent of Hox gene function. Taken together, these findings suggest that
Hox genes contribute to the diversity of the sensory system by regulating the
differentiation of specific subsets of first-order sensory relay interneurons
along the AP axis of the developing hindbrain.
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Materials and methods |
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In situ hybridization and immunohistochemistry
Embryonic days 10.5-11.5 whole-embryos were dissected along the dorsal
midline and processed for in situ hybridization using digoxigenin-labeled
Dbh, Mash1, Phox2b and Rnx probes as previously described
(Gaufo et al., 2000;
Pattyn et al., 1997
;
Qian et al., 2001
). Transverse
sections (10 µm) through r2 to r6 of E10.5-11.5 embryos were processed for
immunohistochemistry using Phox2b (Pattyn
et al., 1997
), Lbx1 (Gross et
al., 2002
) and LH2A/B (Lee et
al., 2000
) rabbit polyclonal antibodies, Lmx1b guinea pig
polyclonal antibody, and Lim1/2 and Isl1/2 mouse monoclonal antibodies
(Developmental Studies Hybridoma Bank). Primary antibodies were detected using
various fluorochrome-conjugated secondary antibodies (Molecular Probes;
Jackson Immunoresearch). Fluorescent images were captured on a BioRad 1024
confocal microscope and processed in Adobe Photoshop and Powerpoint.
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Results |
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Loss of precursors for noradrenergic interneurons results in the expansion of neighboring interneurons
The absence of Phox2b protein expression among the precursors of
noradrenergic interneurons in Hoxb1-/- and
Hoxa3-/-b3-/- embryos suggests an
early regulatory role for Hox genes. To examine a cellular consequence of this
defect, we examined for the presence of neighboring interneurons in the
surrounding environment. Consistent with our observation in younger embryos
(Fig. 3), examination of Phox2b
protein together with Lmx1b, a homeodomain protein that also detects
noradrenergic precursors, is eliminated from the dorsal region of r4 and r5 in
E11.5 Hoxb1-/- and
Hoxa3-/-b3-/- embryos, respectively
(Fig. 4A-D). Analysis of Lim1/2
protein expression, which delineates an interneuron population ventral to
noradrenergic interneurons, shows an expanded domain in r4 and r5 of E11.5
Hoxb1-/- and
Hoxa3-/-b3-/- embryos, respectively
(Fig. 4A-D, bracket). The
expanded domain of Lim1/2 was independently confirmed by Pax2-immunolabeling
(data not shown). As will be shown in the next section, the expression of
Lim1/2 is co-expressed with a population of Lbx1-expressing somatic sensory
interneuron precursors (Fig.
5). As no significant cell death was observed in
Hoxb1-/- and
Hoxa3-/-b3-/- embryos between E10.5
and E11.5 (data not shown), the molecular and cellular alterations in these
mutants suggest a change in cell identity such that the mutant segment
resembles the identity of a more anterior segment, a phenomenon that is
characteristic of many Hox gene loss-of-function mutations
(Gaufo et al., 2003;
Hafen et al., 1984
;
Lewis, 1978
;
McGinnis and Krumlauf, 1992
;
Rozowski and Akam, 2002
;
Struhl, 1981
;
Studer et al., 1996
;
Weatherbee et al., 1998
).
|
|
Presence of proprioceptive and somatosensory precursors in Hox mutant embryos suggest independent or redundant roles for Hox genes in r4 and r5
The expression of Hoxb1, Hoxa3 and Hoxb3 throughout the
neuroepithelium of r4 and r5 suggests that they may regulate other first-order
sensory relay interneurons. We therefore assessed for the presence of
precursors for proprioceptive and somatosensory interneurons. Unlike
noradrenergic interneurons, proprioceptive and somatosensory interneurons have
homologous interneurons in the spinal cord. As in the spinal cord, hindbrain
LH2A/B-expressing precursors for proprioceptive interneurons derive
from progenitors expressing the bHLH gene Math1
(Lee et al., 2000;
Lee et al., 1998
). In both
Hoxb1-/- and
Hoxa3-/-b3-/- embryos, the expression
of LH2A/B appears normal compared with controls
(Fig. 5A-D, arrow). We next
examined for the presence of precursors for somatic sensory interneurons by
assaying the expression of the homeodomain protein Lbx1, a regulator of
somatic sensory interneurons in the spinal cord
(Gross et al., 2002
;
Muller et al., 2002
). Like the
precursors for proprioceptive interneurons, the precursors for somatic sensory
interneurons were intact in Hoxb1-/- and
Hoxa3-/-b3-/- embryos
(Fig. 5E-H, bracket). However,
the domain of Lbx1 and Lim1/2 expression appear expanded in the various Hox
mutant embryos.
Redundant functions of Hox genes in somatic sensory interneuron specification
Several possibilities may explain the loss of noradrenergic visceral
sensory interneurons and the sparing of proprioceptive and somatic sensory
interneurons in Hoxb1-/- and
Hoxa3-/-b3-/- embryos in r4 and r5,
respectively. The simplest explanation is that the specification of
proprioceptive and somatic sensory interneurons is independent of Hox gene
function. Alternatively, redundant functions with other Hox genes in r4 and r5
may compensate for the loss of Hoxb1, Hoxa3 and Hoxb3
functions. Another possibility may be that different combinations of Hox genes
are required for the specification of proprioceptive and somatic sensory
interneurons. To address these issues, we analyzed for the presence of
proprioceptive and somatic sensory interneurons in r2 of
Hoxa2-/- embryos. In r2, Hoxa2 is the only Hox
gene expressed and, thus, the function of a single Hox gene can be addressed
(Davenne et al., 1999).
Analysis of LH2A/B expression in r2 showed no dramatic differences between
E11.5 control and Hoxa2-/- embryos
(Fig. 6A-B). Together with the
observations in Hoxb1-/- and
Hoxa3-/-b3-/- embryos, these data
suggest that the specification of precursors for proprioceptive interneurons
is independent of Hox gene function.
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Discussion |
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The present study also shows that the expression of Rnx, a known
determinant of noradrenergic visceral sensory interneurons important for
gustatory, cardiovascular and respiratory control
(Carpenter and Sutin, 1983;
Qian et al., 2001
), is also
subject to Hox gene regulation. In contrast to Phox2b RNA, however,
the expression of Rnx RNA appears to be completely eliminated in
Hoxb1-/- embryos. The loss of Rnx RNA expression
is consistent with the absence of Dbh RNA. The presence of
Phox2b RNA in Hoxb1-/- embryos, however, suggests
that the identity of r4 is initially intact and therefore, the loss of
noradrenergic visceral sensory interneurons is not solely due to a secondary
effect resulting from changes in rhombomere identity. From these observations,
Hox, Phox2b and Rnx genes may be placed in a hierarchical
order to broadly define a regulatory cascade in the specification of
noradrenergic visceral sensory interneurons within a hindbrain segment.
Furthermore, the convergence of these genes on a common function is supported
by central respiratory defects in mice with targeted mutations for
Hoxa3 and Rnx and in humans with heterozygous mutations for
PHOX2B (Amiel et al.,
2003
; Chisaka and Capecchi,
1991
; Shirasawa et al.,
2000
). Altogether, these observations showing the segment-specific
control of sensory structures and the convergence of genes on a common
physiological function provides evidence for an evolutionary conserved
pathway.
Maintenance of complementary gene expression ensures cellular diversity
An established function of Hox genes is to generate cellular diversity
within multiple tissue types. In the mouse, for example, Hox genes are known
to be essential for the specification of tissues that contribute to the
musculoskeletal, urogenital, hematopoietic and nervous systems
(Alvares et al., 2003;
Arenkiel et al., 2003
;
Bell et al., 1999
;
Davenne et al., 1999
;
Davidson et al., 2003
;
Gaufo et al., 2000
;
Gaufo et al., 2003
;
Goddard et al., 1996
;
Guidato et al., 2003
;
Ivanova et al., 2002
;
Manley and Capecchi, 1998
;
Patterson and Potter, 2003
;
Rossel and Capecchi, 1999
;
Studer et al., 1998
;
Studer et al., 1996
;
Watari et al., 2001
;
Wellik and Capecchi, 2003
).
How Hox genes regulate cellular diversity within these varied tissues remains
to be determined. Owing to the well-characterized expression patterns of genes
in the neural tube (Briscoe et al.,
2000
; Hirsch et al.,
1998
; Qian et al.,
2001
; Qian et al.,
2002
), it is possible to assess a detailed role of Hox genes in
this complex tissue. In the present study, we demonstrate that Hox genes are
required for the specification of visceral and somatic sensory interneurons.
However, proprioceptive sensory interneurons appear to be independent of Hox
gene function. The latter observation suggests that although Hox genes are
ubiquitously expressed in the neuroepithelium, their effects are neuronal
subtype specific. It also suggests that the proprioceptive sensory system is
ancient relative to the use of Hox genes to specify AP identity of the
visceral and somatic sensory systems in the hindbrain region (r2-r5)
examined.
The present study also reveals a duality of Hox gene function in the
regulation of various neuronal subtypes in the hindbrain. For example, the
loss of Phox2b-expressing noradrenergic visceral interneurons in
Hoxb1-/- and
Hoxa3-/-b3-/- mutant embryos is
associated with the expansion of neighboring Lim1/2- and
Lbx1-expressing interneurons into the region normally occupied by
Phox2b. This observation suggests that Hox genes can act either as an
activator or repressor depending on their location along the DV axis. This
appears to be a general Hox mechanism, as we have observed the same phenomenon
in the specification of hindbrain motoneurons in various Hox mutant embryos
(Gaufo et al., 2000;
Gaufo et al., 2003
). How can
this apparent regulatory paradox be rectified? A clue may arise from what has
been observed in the spinal cord. Along the DV axis of the spinal cord,
distinct homeodomain and bHLH proteins show complementary expression domains
and cross-repressive interactions. In the ventral spinal cord, for example,
the loss of Pax6 is associated with the dorsal expansion of the more
ventral Nkx2.2 into the domain normally occupied by Pax6
(Ericson et al., 1997
). In the
dorsal spinal cord, Math1 and Ngn1 also show an inverse regulatory
relationship (Gowan et al.,
2001
). As the Hox proteins are co-expressed with these
DV-restricted homedomain and bHLH proteins, it is possible that the duality of
Hox protein function arise from interactions with either the proteins
themselves or co-factors associated with their pathways. Future work to
identify potential Hox protein binding partners with DV-restricted expression
patterns, may give insight into this dual nature of Hox gene function. Short
of these experiments, the present study shows that Hox genes are required to
maintain the normal complement of gene expression necessary to generate a
diverse group of cells.
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ACKNOWLEDGMENTS |
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