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The Journal of Neuroscience, August 1, 2001, 21(15):5637-5642
Generation of a Novel Functional Neuronal Circuit in
Hoxa1 Mutant Mice
Eduardo Domínguez
del
Toro1,
Véronique
Borday1,
Marc
Davenne2,
Rüdiger
Neun2,
Filippo M.
Rijli2, and
Jean
Champagnat1
1 Neurobiologie Génétique et
Intégrative, Unité Propre de Recherche 2216, Centre
National de la Recherche Scientifique (CNRS), 91198 Gif-sur-Yvette,
France, and 2 Institut de Génétique et de
Biologie Moléculaire et Cellulaire, CNRS/Institut National de la
Santé et de la Recherche Médicale/Université Louis
Pasteur, Collège de France, BP 163-67404 Illkirch,
Centre Universitaire de Strasbourg, France
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ABSTRACT |
Early organization of the vertebrate brainstem is characterized by
cellular segmentation into compartments, the rhombomeres, which follow
a metameric pattern of neuronal development. Expression of the homeobox
genes of the Hox family precedes rhombomere formation, and analysis of mouse Hox mutations revealed that they
play an important role in the establishment of
rhombomere-specific neuronal patterns. However, segmentation is a
transient feature, and a dramatic reconfiguration of neurons and
synapses takes place during fetal and postnatal stages. Thus, it is not
clear whether the early rhombomeric pattern of Hox
expression has any influence on the establishment of the neuronal
circuitry of the mature brainstem. The Hoxa1 gene is the
earliest Hox gene expressed in the developing hindbrain.
Moreover, it is rapidly downregulated. Previous analysis of mouse
Hoxa1 / mutants has focused on
early alterations of hindbrain segmentation and patterning. Here, we
show that ectopic neuronal groups in the hindbrain of
Hoxa1/ mice establish a
supernumerary neuronal circuit that escapes apoptosis and becomes
functional postnatally. This system develops from mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with r2
identity, and integrates the rhythm-generating network controlling
respiration at birth. This is the first demonstration that changes in
Hox expression patterns allow the selection of novel
neuronal circuits regulating vital adaptive behaviors. The implications
for the evolution of brainstem neural networks are discussed.
Key words:
homeobox genes; Hoxa1 knock-out; respiration; suction; rhythm generation; rhombomeres; neural progenitors; migratory pathways; neuronal networks, reticular formation; pons; hindbrain; brainstem; newborn mice
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INTRODUCTION |
In the hindbrain of the vertebrate
embryo, Hox genes are segmentally expressed and loss-
and gain-of-function mutations revealed their involvement in neuronal
patterning (Carpenter et al., 1993 ; Mark et al., 1993; Goddard
et al., 1996; Lumsden and Krumlauf, 1996; Studer et al., 1996; Gavalas
et al., 1997, 1998; Helmbacher et al., 1998; Rijli et al., 1998; Bell
et al., 1999; Davenne et al., 1999; Jungbluth et al., 1999; Rossel and
Capecchi, 1999). Expression of Hoxa1 is one of the earliest
signs of regionalization within the developing hindbrain. As early as
7.5 d postcoitum (dpc), the Hoxa1 expression domain
extends from the posterior end of the mouse embryo up to the
presumptive rhombomere 3 (r3)-r4 border and is downregulated before
rhombomere boundary formation (Murphy and Hill, 1991). This transient
expression has a profound impact on hindbrain patterning, because
Hoxa1-targeted inactivation results in a severe reduction of
r4 and r5 and their derived structures (e.g., the motor nucleus of the
facial nerve) and in lethality shortly after birth (Carpenter et al.,
1993; Mark et al., 1993). However, it is unclear how transient
Hox expression before segment formation may influence the
generation of functional neuronal networks in the postsegmental
hindbrain (Fortin et al., 1999) and affect vital behaviors during
postnatal life (Fortin et al., 2000). By examining hindbrain neural
networks in Hoxa1/ mice, we now
identify ectopic groups of mis-specified neurons that escape apoptosis
(Rossel and Capecchi, 1999) during development and control the
respiratory rhythm-generating neural network (Champagnat and Fortin,
1996) after birth.
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MATERIALS AND METHODS |
Mouse lines and genotyping. Hoxa1 mutant
mice (Mark et al., 1993), embryos, and newborns were genotyped by PCR
as described previously (Gavalas et al., 1998). The r2-lacZ
transgenic line was obtained by injection of a construct carrying a 2.5 kb BamHI Hoxa2 genomic fragment (Frasch et al.,
1995) cloned in a non-native orientation into the BGZ40 plasmid (Studer
et al., 1996), containing the human  -globin promoter driving
lacZ expression. Transgenic r2-lacZ mice were
bred with Hoxa1+/ mice to produce
Hoxa1+/, r2-lacZ
animals. The latter were bred with
Hoxa1+/ animals to produce embryos
with the desired genotype. Detection of the transgene was performed by PCR.
Whole-mount in situ RNA hybridization,
immunohistochemistry, and 5-bromo-4-chloro-3-indolyl
-D-galactoside
staining. Whole-mount in situ RNA hybridization
was performed as described previously (Davenne et al., 1999) using the
Phox2b (Pattyn et al., 1997) and Hoxb1 (Studer et
al., 1996) probes. Whole-mount immunohistochemistry using the anti-ISL1
monoclonal antibody (4D5) (Developmental Studies Hybridoma Bank, Iowa
City, IA) and 5-bromo-4-chloro-3-indolyl -D-galactoside staining was performed as
described previously (Davenne et al., 1999). Hindbrains were dissected
out and flat-mounted before being photographed. Postnatal neuronal
groups (Jacquin et al., 1996) were identified on coronal, horizontal,
and parasagittal 40-µm-thick sections processed alternatively using
cresyl violet and polyclonal antibodies to choline acetyltransferase
(1:1000 in PBS, pH 7.4; Chemicon, Temecula, CA) and to tyrosine
hydroxylase (1:1000 in PBS; Boehringer Mannheim, Mannheim, Germany) in
the presence of Triton X-100 and were subsequently revealed using the
Vectastain avidin-biotin complex kit (Vector Laboratories, Burlingame,
CA) as described previously (Jacquin et al., 1996). To study axonal
pathways, the trigeminal motor root or the bulbar reticular area
ventral to the ambiguus nucleus was pressure injected with DiI (5 mg/ml
in DMSO) after brain fixation. Incubation times (at 37°C) were 3 d after trigeminal injections and 4 d after bulbar injections.
Plethysmograph recordings and naloxone treatment in
vivo. We used 231 mice from 34 Hoxa1 litters.
Sixty mice were wild type (WT), 124 mice were heterozygous mutants, and
47 mice were homozygous mutants, a proportion close to the Mendelian
expectation. Respiratory activity was measured every 6 hr using a
modified barometric method used previously in neonates (Jacquin et al.,
1996). The whole-body plethysmograph chamber (20 ml) equipped with a
temperature sensor (LN 35 Z) was connected to a reference chamber of
the same volume. The pressure difference between the two chambers was
measured with a differential pressure transducer (DP 103-12; Validyne, North Ridge, CA) connected to a sine wave carrier demodulator (CD15;
Validyne). Neonates were removed individually from the litter and
placed in the plethysmograph chamber, which was kept hermetically
closed and maintained at 31°C during the recording session (2 min).
During quiet breathing, a computer-assisted method was used to measure
the duration of inspirations and expirations from which the respiratory
frequency is derived. Naloxone was administered (3.33 mg/kg, s.c., in
50 µl of saline) using a Hamilton syringe at the end of the first
plethysmographic recording (1-2 hr after birth), and the stimulatory
effect on respiration was controlled 0.5-1 hr later.
Network analysis in vitro. The brainstem was
removed as described previously (Jacquin et al., 1996, 1999) and cut
horizontally (see Fig. 3F) under visual
control with a vibratome (series 1000; Technical Products
International, O'Fallon, MO). The 1200-µm-thick slice was
transferred, dorsal side up, into a recording chamber and perfused with
artificial CSF, pH 7.4, containing (in mM)
130 NaCl, 5.4 KCl, 0.8 KH2PO4, 26 NaHCO3, 30 glucose, 1 MgCl2, and 0.8 CaCl2,
saturated with carbogen (90% O2, 10%
CO2). Motor activities were recorded from the
motor trigeminal roots using suction electrodes. Previous experiments
(Jacquin et al., 1999) have demonstrated that the respiratory activity
in vitro propagates to this nerve. The selected root was
contralateral to the studied pontine neuronal structure (see Fig.
3G) to avoid stimulating directly the recorded motoneurons.
Other electrodes were located on the dorsal surface under the visual
guidance of a microscope (ACM; Zeiss, Thornwood, NY), and
locations were identified histologically. Neurons were recorded in the
whole-cell configuration with patch-clamp electrodes as described by
Fortin et al. (1999). Electrodes containing 0.1-0.5 mM AMPA in artificial CSF were used for pressure
application (0.1 bar, 20 msec). Experiments were performed according to
authorization by the ministry of Research Technology and Agriculture,
which provides authorization to have the animal facilities.
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RESULTS |
A supernumerary neuronal structure in the dorsal pons of
Hoxa1/ mice
Morphological analysis of the pons at birth indicates a rather
extensive cellular reorganization in
Hoxa1/ mutants, affecting
different cell types. First, in keeping with the heterogeneous
anteroposterior (A-P) pattern of the ventricular zone, the anterior
fourth ventricle exhibits a characteristic morphological abnormality in
newborn mutants (Fig. 1, compare A-D). Moreover, the size of the reticular formation is
affected both dorsally and ventrally. Ventrally, a 40% reduction in
the length of the ventral pons (vP) (Fig. 1E,F,
rectangle) results from the elimination of r4- and
r5-derived structures. In contrast, dorsally, a 6% increase in the
postnatal A-P length of the dorsal pons (dP) (Fig.
1E,F, arrow) was observed, so that the
ratio of dP to vP in Hoxa1/
animals, although variable (average ± SEM, 1.45 ± 0.08;
n = 18), is much larger than in wild-type animals
(0.77 ± 0.02; n = 18).
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Figure 1.
Distinct dorsal and ventral anatomical phenotypes
in the Hoxa1/ brainstem at birth.
A-D, Adjacent horizontal sections showing the ependymal
epithelium at the anterior end of the fourth ventricle;
A and C are dorsal to B
and D, respectively. Scale bar, 250 µm. Also see
E and F [caudal to dorsal tegmental area
(DTg)] and Fig. 2B for location.
The epithelium forms a single invagination in WT mice
(A, B, E), closing in the
dorsal pons medially to the trigeminal motor nucleus and forms multiple
invaginations (2-5) in Hoxa1/
mice (C, D, F).
E, F, Parasagittal sections of the hindbrain at P0 in WT
(E) and
Hoxa1/ (F)
littermates; the A-P length of the pons is affected differently
dorsally [arrow, from the rostral limit of the
hypoglossal nucleus (12) to the caudal limit of the
DTg] and ventrally [rectangle, from the
rostral pole of the inferior olive (IO) to the caudal
pole of the pontine nuclei (Pn)]. IP,
Interpeduncular nucleus; Sol, solitary nucleus.
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We have localized in the dorsolateral pons the anatomical modifications
underlying this dP increase. In wild-type mice, caudal to the
trigeminal motor nucleus, the parvocellular reticular formation (Rpc- ) (Fig. 2A,B,
pc) normally contains trigeminal premotor interneurons
involved in feeding behaviors (Lund et al., 1998). The Rpc- is
likely derived from r3, because it is eliminated in
Krox-20/ mutants (Jacquin et
al., 1996), in which pontine defects lead to an abnormal suction
behavior after birth. In all
Hoxa1/ mice (n = 10), the anatomy of the Rpc- is reorganized (Fig. 2) and extended
along the A-P axis, in keeping with the abnormalities of the r3-r4
region at early developmental stages (described below) (Carpenter et
al., 1993; Mark et al., 1993; Gavalas et al., 1998; Helmbacher et al.,
1998; Rijli et al., 1998; Rossel and Capecchi, 1999). In particular,
radial stripes of reticular formation and ectopic motoneurons
alternate, forming a compound reticular and motor supernumerary
neuronal structure (SNS). Most extensive labeling of ectopic SNS
motoneurons included three distinct subnuclei (Fig. 2A,B) identified by analysis with anti-choline
acetyltransferase antibodies. In addition, injecting the fluorescent
marker DiI into the trigeminal motor root (Fig.
2C-F) revealed that these ectopic subnuclei form a
distinct dorsoventral trigeminal motor fasciculus running laterally in
the SNS (Fig. 2D,F, asterisks) caudal to
the normal root (Fig. 2E, asterisk).
Therefore, a dP increase in
Hoxa1/ mice results from the
generation of three additional trigeminal subnuclei alternating with
stripes of reticular formation at the same location as the wild-type
Rpc-.
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Figure 2.
The dorsal anatomical phenotype in
Hoxa1/ mice at birth:
identification of motoneurons showing location of the SNS.
A, Sagittal sections of the brainstem, cut parallel to
Figure 1E,F. The drawings on the
left (including the analysis of 5 Hoxa1/ mice) include both lateral
and medial structures. Scale bar, 1 mm. Medial sections (in
gray, showing the ventricular surface) are illustrated
in Figure 1E,F. Note that supernumerary motor
(lateral) and ventricular (medial) structures are at the same
anteroposterior level of the dorsal pons. Lateral sections (on the
right) show choline acetyltransferase-immunoreactive WT
(+/+) ventral facial structures eliminated by the mutation: the
branchial motor nucleus (VII), the preganglionic
nucleus (pg), and accessory nuclei (between VII
and pg, extending close to the descending facial root,
VIIn). In Hoxa1/
mice, caudal to the trigeminal nucleus
(V), the SNS includes three dorsal motor
subnuclei (outlined and numbered) alternating with two unstained
stripes of reticular formation. IO, Inferior olive;
IP, interpeduncular nucleus; pc,
parvocellular reticular formation; Pn, pontine nuclei;
SO, superior olive; X,
XII, dorsal-vagal and hypoglossal motor nuclei.
B, Horizontal sections cut parallel to the
arrow in Figure 1E,F. Drawings on
the left (including the analysis of 5 Hoxa1/ mice) show the left part
of the pons (scale bar, 1 mm) and the relative positions of the V and
VII nuclei and trigeminal nerve root (Vn). Close to the
midline (dotted line), note the appearance of a
supernumerary ventricular structure (illustrated in Fig.
1D) and elimination of the abducens motor nucleus
(VI). The right part superimposes
choline acetyltransferase-immunoreactive pontine neurons in WT
(black) and Hoxa1/
(red) littermates from four horizontal sections
sampling, in each littermate, the entire V nucleus and adjacent areas.
Supernumerary motor nuclei (1, 2, and
3) are at the same place as the WT Rpc-
(pc), VIIn, and pg,
respectively. C-E, Horizontal sections showing
retrograde DiI labeling of trigeminal and SNS motoneurons in a WT
(C; arrow, 200 µm) and a
Hoxa1/ (D, E)
mouse. Labeling of the SNS shows the three ectopic trigeminal subnuclei
(compare D with C), and a more ventral
view (E) shows a supernumerary dorsoventral
fasciculus located laterally in subnucleus 2 (asterisk
in D and E) and distinct from the WT-like Vn.
F, Medial half of subnucleus 2 at higher magnification
(arrow, 67 µm, oriented as in C; the
border of the V is in the upper left corner; subnucleus
1 is lacking). The supernumerary motoneuron (open
triangle) shows an axon (asterisks) running in
the direction of the lateral fasciculus.
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Function of ectopic reticular neurons in the dorsolateral pons of
Hoxa1/ mice
To further characterize the reticular cells of
the SNS, we investigated their functional connectivity (Fig.
3G). The hindbrain was
isolated in vitro during the first postnatal days
[postnatal day 0 (P0) and P1], and the dorsal pons was exposed in a
thick horizontal slice (Fig. 3F) and made accessible
to dorsal approach under microscopic control. This slice preparation
also included the bilateral ventral respiratory group (VRG) (Fig.
3G, asterisks), which generates a persisting
rhythmic activity propagating to cranial (e.g., trigeminal) motor
neurons from which it can be recorded (Jacquin et al., 1996, 1999).
Neuronal populations immediately caudal to the trigeminal nucleus
(which in wild type include the Rpc- premotor neurons) (Fig.
3G, rectangle) were stimulated by pressure
application of the glutamatergic agonist AMPA. The contralateral trigeminal nerve root was recorded to avoid direct stimulation of motoneurons.
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Figure 3.
Functional connectivity of reticular
neurons in the Hoxa1/
supernumerary neuronal structure at birth. A-D,
Modification of the contralateral trigeminal nerve activity
(Vn) induced by exciting SNS neuronal cell bodies using
brief (25 msec) pressure applications of AMPA in WT (A,
B) and Hoxa1/ (C,
D) hindbrain slices in vitro. A,
C, Four samples of integrated Vn activity (2 min
long) starting (from top to bottom) at
 2, 0, 3, and 5 min after AMPA application (time indicated on the
left). In both WT and
Hoxa1/ littermates, the
rhythm generator produces bursts of activity (fast upward deviations),
and AMPA generates background nonrhythmic activity starting at 0 min.
B, D, Temporal evolution (calibration, 2 min) of
average (±SE) burst frequency from five experiments. A significant
increase followed by inhibition (p < 0.001)
indicates a functional connection to the rhythm generator in
Hoxa1/ mice but not in WT mice.
E, Vn, Integrated nerve activity;
Em, membrane potential of a single
(Hoxa1/) neuron located in the
SNS area (scale bars, 20 mV, 1 sec). A connection from the rhythm
generator results in a simultaneous Vn burst and
neuronal depolarization inducing firing of action potentials. F,
G, Schematic presentation of the slice preparation in sagittal
(F, arrowhead indicates the top
side) and horizontal (G) sections. The
rectangle in G indicates the approximate
extent of the area affected by AMPA applications, indicated by the
arrowhead; more medial applications were ineffective.
Thin arrows indicate WT projections, preserved in
mutants; these are either rhythmic, from the bilateral rhythm generator
(asterisks) to the contralateral trigeminal nucleus
(VMo) and Vn (recorded), or nonrhythmic
premotor neurons from Rpc-/SNS to VMo.
Thick arrows indicate supernumerary connections in
mutants, including those from the SNS to the rhythm generator and the
trigeminal axons of SNS motoneurons. H-J, Sagittal
sections (location in J, rostral to the
left) of the most lateral 300 µm of the pons showing
in mutant (I) but not in WT
(H) animals, an axonal fasciculus stained
after DiI injection in the area of the rhythm generator (bottom
right corner). Scale bar, 200 µm.
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AMPA-induced nonrhythmic trigeminal activities recorded from the
contralateral trigeminal motor rootlet (the upward noisy deflection of
the traces in Figs. 3A,C) indicate that normal premotor Rpc- inputs to the trigeminal motoneurons (Lund et al., 1998) persist in Hoxa1/ mutants. The
Rpc- normally lacks respiratory-related functions. AMPA application
had no effect on rhythm frequency in the wild-type preparations (Fig.
3B). In the mutants, a robust increase in rhythm frequency
is followed in all cases by a transient inhibition of the rhythm (Fig.
3C,D). This effect strongly suggests the presence of
supernumerary functional efferent connections of the SNS to the rhythm
generator, resembling the wild-type ventral pontine respiratory
connections, located rostrally to the SNS and originating in r2 and r3
(Jacquin et al., 1996; Borday et al., 1997). Moreover, rhythmic
activity recorded from single neurons in the SNS area (Fig.
3E) also indicated afferent connections from the rhythm generator. In addition, abnormal axonal pathways were found in the
lateral pons by injecting the fluorescent marker DiI into the VRG area
(Fig. 3H-J). In the mutants, labeling from the VRG revealed a robust axonal pathway (Fig. 3I); this
pathway was not present in the wild-type (Fig. 3H)
and ran laterally in the pons. Thus, in
Hoxa1/ mice, the SNS exhibits a
novel relationship with the respiratory rhythm generator, while
preserving premotor connections with the trigeminal system.
Embryological origin of the supernumerary neuronal system
The appearance of this ectopic neuronal system prompts the
question of its embryological origin. We investigated the expression of
rhombomere-specific molecular markers in
Hoxa1/ mutant hindbrains (Fig.
4). Rhombomere-restricted gene expression persists in the ventricular zone after the segmentation period (Wingate
and Lumsden, 1996). In 11.5 dpc mutants, expression of the r4
marker Hoxb1 is drastically reduced and patchy along the dorsoventral axis (Fig. 4, compare A,B). To assay for r2
features, we generated a transgenic line containing the lacZ
reporter under the control of a Hoxa2 r2-specific enhancer
(Frasch et al., 1995) (Fig. 4C). In
Hoxa1/ mutants, ectopic patches
of cells expressing the r2 marker are present at the r4 axial level
(Fig. 4, compare C,D); this is remarkably similar to what is
observed in Hoxb1/ mice (Studer
et al., 1996). In addition, patches of r2-like cells are also present
at the r3 level, as described previously (Helmbacher et al., 1998).
Thus, in the absence of Hoxa1, some neural precursors at the
presumptive r3-r4 levels fail to activate or properly maintain their
appropriate molecular programs and acquire an r2 identity.
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Figure 4.
Molecular and morphological patterning defects in
a Hoxa1 mutant hindbrain. A dorsal view of 11.5 dpc
WT (A, C) and
Hoxa1/ (B, D)
mutant hindbrains hybridized with the r4-specific Hoxb1
(A, B) or carrying a lacZ reporter under
the control of an r2-specific enhancer (C, D) is shown.
Vertical arrows indicate the location of the motoneuron
progenitor columns.
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To investigate the developmental fate of these ectopic r2-like
precursors, we examined motoneuron development in the hindbrain of
Hoxa1/ mice. In wild-type 11.5 dpc embryos, the Phox2b gene is expressed in migrating
motoneurons (Pattyn et al., 1997). Phox2b expression in
ventral r4 identifies facial motoneurons migrating caudally through r5
into r6 (Fig. 5A, bent arrow) to form the
facial (VIIth) motor nucleus, whereas strings of
Phox2b-positive cells in r2 are indicative of dorsal
migration of trigeminal motoneurons (Fig. 5A, straight
arrows). In the Hoxa1/
mutant r4 region (Fig. 5B), a much reduced, although not
abolished, Phox2b expression identifies a small number of
facial motoneurons migrating caudally (Fig. 5B,
bent and dashed arrow). In
addition, an abnormal trigeminal-like lateral migration of cells can be detected (Fig. 5B, straight arrows) that is
completed at ~12.5 dpc (Fig. 5D) and results in a
characteristic dorsolateral accumulation of ectopic
Phox2b-positive cells (Fig. 5, rectangle, compare
C,D). This population includes ectopic motoneurons as
assessed by anti-Islet1 immunohistochemistry (Fig. 5,
arrows, compare E,F). Remarkably, lack of
caudal migration of facial motoneurons and lateral trigeminal-like migration are also observed in
Hoxb1/ mice (Studer et al.,
1996). Thus, together with the above molecular analysis (Fig. 4), these
data suggest facial-to-trigeminal changes in motoneuron subtype
identity in Hoxa1 mutants that could be induced by lack of
Hoxb1 activation in pre-r4 cells.
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Figure 5.
The Hoxa1/
supernumerary motoneurons: migration and final postnatal location. A
dorsal view of 11.5 (A, B) and 12.5 (C-F) dpc WT and
Hoxa1/ mutant hindbrains,
respectively, flat-mounted and hybridized with Phox2b
(A-D) or Isl1 (E,
F) probes is shown. The bent white arrow
in A and B indicates caudal
migration of facial (VII) motoneurons. The straight
arrows in A and B indicate dorsal
migration of trigeminal (V) motoneurons and, in
Hoxa1/ mice
(B), of supernumerary motoneurons from r4. The
rectangles in C and D and
the arrows in F indicate ectopic,
dorsolateral accumulation of Phox2b- and
Isl1-positive cells, which was not present in WT
mice.
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Persistence and functional role of the supernumerary neuronal
system after birth
To investigate the functional role of the SNS in controlling
respiratory and feeding rhythms in vivo, we have compared
mutant and wild-type behaviors in relation to the anatomical
modification of the pons. Although irregular after birth, the wild-type
minute ventilation increases progressively and stabilizes at the end of
the first day (Fig. 6A,
left). In contrast, mutants exhibited a variable neonatal
respiratory frequency (NRF) 2-4 hr after birth and eventually apneic
breathing and death (Fig. 6A, right). A correlation was found in mutants between the NRF and the hindbrain anatomical index dP/vP (r = 0.83 vs r = 0.32 in wild-type mice) (Fig. 6B), indicating that
there are pontine abnormalities accelerating spontaneous breathing at
birth. In contrast, the suction behavior, estimated by the frequency of
jaw openings induced by a buccal stimulus (Jacquin et al., 1996), was
normal in the mutants and unrelated to dP/vP (r = 0.25). In addition, Hoxa1/
newborns with a low NRF (<35 breaths/min; n = 7) died within 2.5 ± 0.8 hr (Fig. 6C, bottom
left triangles), whereas those exhibiting a higher NRF
(n = 15) progressively increased their respiratory rate
to normal values (Fig. 6A,C) and survived for 18 ± 7 hr. Thus, one possibility is that the appearance of the SNS may
result in enhanced survival rates by significantly increasing NRF
values, so that the rhythm promoting action of the SNS seems to
compensate for the lethal apneic breathing resulting from vP
hypoplasia. To further investigate this hypothesis, animals with the
highest NRF were submitted to naloxone administration, a treatment
known to be effective on life-threatening pathologies resulting from the vP hypoplasia (for example, in
Krox-20/ mutants) (Jacquin et
al., 1996). A striking effect of naloxone administration was obtained
in two of the five treated
Hoxa1/ newborns (Fig.
6C, filled circles); one of them survived
4 d, whereas the other was killed 12 d after birth.
Interestingly, in this animal, histological analysis revealed the same
pattern of SNS motoneurons (Fig. 6D) that was
observed at birth (Fig. 2). The survival of these motoneurons is
noteworthy, considering the wave of apoptosis that normally removes
abnormal motoneurons in the fetal hindbrain before birth
(deLapeyrière and Henderson, 1997). Altogether, the present
in vitro and in vivo observations demonstrate
that the Hoxa1 mutation results in the incorporation of a
SNS, which originates from the mutant r3-r4 region, into the hindbrain
neural network. As a consequence, the animal acquires a novel
respiratory-related function enhancing survival, while not affecting
suction, a function that is under the control of neuronal populations
from the same region.
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Figure 6.
The
Hoxa1/ breathing pattern after
birth. A, Samples of plethysmographic recording
(inspiration upwards) 2, 6, 12, 18, and 24 hr after birth [postnatally
(p.n.)] showing normal maturation in a WT mouse
and transient increase of frequency in a mutant mouse. Calibration: 20 µl, 1 sec. The mutant animal typically exhibits irregular breathing
at birth (top trace) and eventually apneic breathing and
death (bottom trace). B, Individual
Hoxa1/ (open
triangles) and WT (filled squares) mice
identified by their respiratory rate at birth (ordinates) and the dP/vP
index quantifying the abnormality of the pontine A-P distances [see
arrow (dP) and rectangle (vP) in Fig.
1E,F]. A correlation exists in
Hoxa1/ mice but not in WT mice.
C, Temporal evolution of average respiratory
frequency (±SEM) in Hoxa1/
animals breathing faster or slower than 35 breaths/min at birth
(open triangles). The slowest animals lack the rhythm
stimulation shown in A (6-18 hours p.n.); the fastest
animals survive longer; death was delayed by >3 days postnatally
(d.p.n.)] in two animals (filled
circles) treated with subcutaneous naloxone
(NLX). D, Supernumerary
motoneurons in a NLX-treated animal killed 12 d
after birth. The sagittal section (rostral to the left)
shows choline acetyltransferase-immunoreactive motoneurons
(arrowheads) caudal to the trigeminal motor nucleus
(located in the top left corner). Scale bar, 100 µm.
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DISCUSSION |
These results allow a hypothesis that is compatible with the
involvement of developmental control genes in the assembly of functional neuronal circuits (Tanabe and Jessell, 1996; Brunet and
Ghysen, 1999). In fact, this work provides the first formal evidence
that selective modification of the expression pattern of a
Hox gene whose expression is transient in the presumptive hindbrain, namely Hoxa1, is sufficient to incorporate a
novel functional neural circuit in the mature hindbrain. This striking finding prompts the question of the cascade of regulatory events triggered by Hoxa1 loss-of-function, leading to long-term
modification of hindbrain neural networks. Previous work demonstrated a
role for Hoxa1 in the activation of Hoxb1
expression in the presumptive r4 (Studer et al., 1998). Thus, some of
the long-term effects of the Hoxa1 mutation could be
attributable to the lack of Hoxb1 activation in a
subset of presumptive r4 cells, leading to r2-like specification.
However, Hoxa1, unlike Hoxb1, appears to control both r4 and, indirectly, r3 development (Helmbacher et al., 1998) (this
study). Thus, it is tempting to speculate that regulatory changes in
two adjacent rhombomeres may be required for the generation of a SNS.
Interestingly, we have shown recently that assembling of a
rhythm-promoting respiratory network also requires a two-segment functional unit in the chick (Fortin et al., 1999). In this respect, it
will be interesting to compare the physiology of neuronal networks in
Hoxb1/ mutants with that of
Hoxa1/ mutants.
Because hindbrain neurons control adaptive behaviors, these findings
have considerable significance both on developmental and evolutionary
grounds. The evolution of neural networks of multisegmental origin may
be facilitated by the partitioning of the early hindbrain in a number
of metameric units initially developing as independent modules
(Lumsden, 1990; Clarke and Lumsden, 1993; Champagnat and Fortin, 1996).
As a result, subsets of neurons may be developmentally isolated from
each other and allowed to evolve independently. Our present data
suggest that Hox genes may provide a genetic basis for
segment-specific modulation of neuronal development and connectivity.
Changes in Hox cis-regulatory modules and
downstream targets have been suggested to underlie morphological
changes of segmented structures in animal evolution (Gellon and
McGinnis, 1998). Similarly, local changes in the regulation of
Hox genes within the segmented hindbrain of vertebrates may offer novel opportunities for the evolution of distinct subsets of
neurons, without affecting the function of others, eventually resulting
in novel functional features (Brunet and Ghysen, 1999). In this
respect, studies of conditional segment-specific Hox
mutations, which may not result in lethality of the animal, will be
important to further investigate adaptive mechanisms in the development of hindbrain neuronal networks.
| |
FOOTNOTES |
Received Sept. 28, 2000; revised April 11, 2001; accepted May 1, 2001.
Work in J.C.'s laboratory was supported by Human Frontier Science
Program Research Grant 101/97, Action Concertée
Incitative (Biologie du Développement et Physiologie
Intégrative) #57 the Centre National de la Recherche
Scientifique, and the Fondation pour la Recherche Médicale (FRM).
E.D.T. was supported by The European Community (BIO4-CT975-096) and FRM
(EP001227/1) training grants. Work in F.M.R.'s laboratory was
supported by the CNRS, the Institut National de la Santé et de la
Recherche Médicale, the Hôpital Universitaire de
Strasbourg, the Ligue Nationale Contre le Cancer (LNCC), the
Association pour la Recherche sur le Cancer, and the Programme
Génome du CNRS. M.D. was supported by fellowships from the
LNCC and FRM. R.N. was supported by Deutscher Akademischer
Austauschdienst and FRM fellowships. We thank P. Chambon, G. Fortin, C. Goridis, R. Krumlauf, and A. Lumsden for valuable discussions and
comments on this manuscript. We also thank T. Jacquin for his
participation in some in vitro experiments and M. Poulet
for excellent technical assistance. We acknowledge the following
colleagues for kind gifts of reagents: P. Chambon (Hoxa1 mice), R. Krumlauf (BGZ40 plasmid and Hoxb1 probe), and J. F. Brunet (Phox2b
probe). The 4D5 antibody was obtained from the Developmental Studies
Hybridoma Bank under contract NO1-HD-7-3263.
E.D.T., V.B., and M.D. contributed equally to this work
Correspondence should be addressed to J. Champagnat,
Institut de Neurobiologie Alfred Fessard, Centre National de la
Recherche Scientifique, Unité Propre de Recherche 2216 (bât. 33), 91198 Gif-sur-Yvette, France. E-mail:
Jean.Champagnat{at}iaf.cnrs-gif.fr.
V. Borday's present address: Laboratoire de Biologie du
Développement, Université Paris 7, case 7077, 2 place
Jussieu, 75251 Paris, France.
M. Davenne's present address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724.
| |
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TOP
ABSTRACT
INTRODUCTION
