Distinct roles of Hoxa2 and Krox20in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening
© Chatonnet et al.; licensee BioMed Central Ltd. 2007
Received: 10 April 2007
Accepted: 26 September 2007
Published: 26 September 2007
Little is known about the involvement of molecular determinants of segmental patterning of rhombomeres (r) in the development of rhythmic neural networks in the mouse hindbrain. Here, we compare the phenotypes of mice carrying targeted inactivations of Hoxa2, the only Hox gene expressed up to r2, and of Krox20, expressed in r3 and r5. We investigated the impact of such mutations on the neural circuits controlling jaw opening and breathing in newborn mice, compatible with Hoxa2-dependent trigeminal defects and direct regulation of Hoxa2 by Krox20 in r3.
We found that Hoxa2 mutants displayed an impaired oro-buccal reflex, similarly to Krox20 mutants. In contrast, while Krox20 is required for the development of the rhythm-promoting parafacial respiratory group (pFRG) modulating respiratory frequency, Hoxa2 inactivation did not affect neonatal breathing frequency. Instead, we found that Hoxa2-/- but not Krox20-/- mutation leads to the elimination of a transient control of the inspiratory amplitude normally occurring during the first hours following birth. Tracing of r2-specific progenies of Hoxa2 expressing cells indicated that the control of inspiratory activity resides in rostral pontine areas and required an intact r2-derived territory.
Thus, inspiratory shaping and respiratory frequency are under the control of distinct Hox-dependent segmental cues in the mammalian brain. Moreover, these data point to the importance of rhombomere-specific genetic control in the development of modular neural networks in the mammalian hindbrain.
The role of hindbrain segmentation  in the organization and function of neural networks has been investigated using mutant mouse models for key regulatory genes, of which members of the Hox gene family are important. These genes display partially overlapping expression domains with rostral limits matching rhombomere (r) boundaries, providing a specific expression code for each segment along the anterior-posterior (AP) axis (reviewed in [1, 2]). Segment-specific Hox expression is regulated by transcription factors exhibiting rhombomere-restricted expression patterns, such as Krox20 expressed in r3 and r5 [3–5], and by cross- and auto-regulatory activity of Hox proteins themselves [6–8]. Defining the biological significance of these rhombomere-specific gene regulatory networks is essential for understanding the development and functional organization of neuronal circuits in the vertebrate hindbrain. Hoxa2 is particularly interesting as it is the most anteriorly expressed Hox gene up to the r1/r2 border, and because it participates in complex rhombomere-specific regulatory pathways [6, 9]. Targeted inactivation in the mouse revealed that Hoxa2 is indeed required for normal patterning of the rostralmost rhombomeres, as well as for the development of topographic brainstem circuitry [10–13]. However, the behavioral implication of Hoxa2 control has not yet been addressed.
A very sensitive method to evaluate behavioral significance of disturbed rhombomere development is to identify uncompensated abnormalities of vital postnatal behavior, for example, alimentary and breathing behaviors, in vivo in transgenic animals. The oro-facial control in particular is tightly linked with trigeminal sensory and motor pathways, as well as surrounding rhythmogenic and pre-motor reticular neurons [14–16]. Interestingly, mutations affecting rostral hindbrain segmentation differentially affect the control of jaw opening in neonates, which requires Krox20  but not Hoxa1 expression . A recent study  showed that Hoxa2 controls the connectivity pattern of the trigeminal sensory afferents to the rostral pons governing the formation of the whisker-to-barrel somatosensory circuit in the mouse. The implication of the Hoxa2 mutation on oro-buccal behavior remains to be investigated in these mice.
Breathing in rodents is thought to be governed by a rhythm generator named the pre-Bötzinger Complex (pre-BötC) [19, 20], which acts as an oscillator. Previous studies from our group showed that it arises from post-otic rhombomeres [21, 22]. The most recent findings support an additional parafacial respiratory group (pFRG) , which also shows an oscillating rhythmic activity. A dual origin of respiratory rhythm generation in newborn rodents involving a coupling between the pre-BötC and the pFRG  has been hypothesized and the roles of each oscillator in respiratory rhythm generation are still under discussion .
We have used the above-mentioned developmental approach to investigate the origin and the architecture of the respiratory rhythm generator. We previously described life-threatening anomalies of respiratory frequency that can be alleviated by naloxone in Krox20-/- and Hoxa1-/- mice. In these mice an anti-apneic system that exerts a rhythm-promoting function during the first postnatal days, likely the pFRG, is eliminated [17, 18, 26]. Krox20-dependent signaling and the r3-r4 segment are required in chicken and mice for the development of the pFRG .
In addition to the pre-BötC and the pFRG, the rostral pons has a role in control of breathing but its function in the intact animal remains questionable (see ). Distinct pontine inspiratory control in vivo has been recently proposed, the development of which can be altered by retinoic acid at embryonic day (E) 7.5 without affecting the respiratory frequency . The present study investigates in vivo whether anti-apneic and inspiratory controls result from different AP specifications (Krox20-dependent, para-facial , and rostral , Hoxa2-dependent, respectively) caudal to the r2/r3 boundary. Alternatively, the present study also considers that abnormal inspiratory control in vivo may result from behavioral adaptation to r3-r5 defects in which Krox20 expression is altered but not entirely eliminated. These two hypothesis are investigated comparing Krox20 null mutant mice with null Hoxa2  and hypomorphic Krox20  mutant mice. We found that inactivation of either Hoxa2 or Krox20 impairs the rhythmic control of the jaw opening in agreement with HOXA2-dependent trigeminal defects and direct regulation of Hoxa2 by KROX20 in r3. However, an inspiratory pontine activity residing in the rostral pons and requiring an intact r2 is selectively abolished in Hoxa2-/-, but not in Krox20, mutants. These results indicate that pontine inspiratory and para-facial anti-apneic control systems are embryologically and functionally distinct and are under the control of distinct Hox-dependent segmental cues in the mammalian brain.
Impairment of oro-buccal behavior in Hoxa2-/-mice at birth
Respiratory parameters and anatomical measures of wild-type, heterozygous and homozygous Hoxa2 mutant mice at birth
Respiratory and behavioral parameters at P0.1
Birth weight (g)
1.45 ± 0.006
1.46 ± 0.022
1.40 ± 0.024
Weight gain at P0.75
0.24 ± 0.01
0.22 ± 0.01
-0.09 ± 0.008*
16.5 ± 1.0
15.1 ± 0.9
-6.6 ± 0.6*
Jaw openings (in 30 s)
24.0 ± 0.41
23.9 ± 1.22
13.6 ± 1.63*
107 ± 1.3
107 ± 4.0
110 ± 5.7
Tidal volume (μl/g)
4.6 ± 0.06
5.3 ± 0.41
10.6 ± 0.87*
0.52 ± 0.01
0.58 ± 0.06
1.21 ± 0.14*
Apneas (% of recording duration)
11 ± 0.8
9.6 ± 3.0
8.1 ± 3.7
AP length of locus coeruleus (μm)
455 ± 18
420 ± 60
717 ± 23*
DV height of locus coeruleus (μm)
625 ± 60
700 ± 20
1,017 ± 73†
Distance between LC ventral limit (μm)
1,050 ± 25
607 ± 79*
Compared with the Krox20 null mutation, the Hoxa2-/-mutation selectively affects the inspiratory amplitude without affecting respiratory frequency during the first postnatal hours
In human infants, the first inspiratory efforts are the deepest breaths of the whole neonatal period [31–33]. The VT is later reduced and increases afterwards progressively during several hours (see page 26 of ). The same was observed in the wild-type (Figure 2a, b and inset) and Krox20-/- (Figure 2b) mice, in which values of VT were reduced to about 5 μl/g at P0.1 and increased to about 10 μl/g at P0.5, which is the normal value maintained during the postnatal week (Figure 2a, b). At P0.5, the absolute tidal volume values of all genotypes were very similar, ranging from 8–11 μl/g. The tidal volume of Hoxa2-/- mutant mice was the same as at birth, indicating that the decrease in VT observed in wild-type mice after the first breaths was impaired by the mutation (Figure 2a). Thus, the Hoxa2 invalidation perturbs a developmentally regulated mechanism involved in the control of the tidal volume during the first 12 postnatal hours.
Thus, the respiratory phenotype of the Krox20 null mutation was dramatically distinct from that observed in Hoxa2 null mutants. Hoxa2 null mutation specifically affected the inspiratory control at birth, without modifying the respiratory frequency, whereas Krox20 null mutation had the exact opposite effect, deeply affecting the respiratory frequency without changing the respiratory amplitude.
Partial impairment of Krox20 function does not mimic the Hoxa2-/-respiratory phenotype and preserves breathing rhythm at birth
Krox20 is required for the development of r3, where Hoxa2 is expressed under Krox20 control ; nevertheless, the respiratory phenotype of the Krox20 null mutation was quite distinct from that observed in Hoxa2 null mutants (see above), suggesting that it was not induced by the loss of Hoxa2 expression in r3. However, as the Krox20 mutation results in the complete elimination of r3, it cannot be compared to the changes induced in the Hoxa2 null mutants, in which r3 is still present but displays patterning defects [10, 11]. The abnormal inspiratory control in Hoxa2 mutants might, therefore, originate from an adaptive respiratory behavior following compensation for the loss of Hoxa2 in r3, while Krox20 is still functional.
Because development of rhythmic circuits in r3r4 has been ascribed to r3-related control of neurogenesis in r4 , we analyzed the early pattern or neuronal differentiation in Krox20Cre/flox mutants (Figure 4c, d). We found that r3, although dramatically reduced in size, preserves its ability to delay neurogenesis and axonal invasion . We conclude that function of the anti-apneic control does not depend on the ability to maintain quantitatively the size of embryonic territories, but rather as previously suggested , on odd-numbered rhombomere properties required to qualitatively control neuronal circuit formation.
Taken together, these results establish that Hoxa2 does not mediate the Krox20 null respiratory phenotype in r3 since Hoxa2 null mutation does not reproduce Krox20 null mutation phenotypic traits. In addition, they suggest that the Hoxa2 mutant respiratory phenotype may be contributed by abnormalities in pontine areas more rostral than the Krox20 phenotype, since Hoxa2 has greater effects in its rostral domain of expression [10, 36].
Apneas are not responsible for the lethality of the Hoxa2-/-mutation
At P0, the respiratory pattern was very irregular in Hoxa2-/- mutant mice and the time spent in apneas (that is, respiratory pauses lasting more than 2 s) was about 10% of the total time of observation, similar to heterozygous and wild-type animals (Table 1). At P0.5, the time spent in apneas in wild-type animals normally decreased to 3.2 ± 2.0% of the recording time. In contrast, no decrease was observed in Hoxa2-/- mutants (time spent in apneas at P0.5, 17.5 ± 5.5%) and at P0.5 these animals spent significantly more time in apneas than controls. However, apneas did not greatly influence the average volume inhaled by Hoxa2-/- mutants. At P0.5, shortly before the death of the animals, the minute ventilation in homozygous mutants (1.36 ± 0.42 ml/g/minute) was not significantly different from that at birth (1.21 ± 0.14 ml/g/minute) or from that in the wild-type (1.71 ± 0.11 ml/g/minute) or heterozygous (1.43 ± 0.09 ml/g/minute) littermates.
Anatomical defects in rostral pontine areas in Hoxa2-/-mutants
Using heterozygous Hoxa2EGFP(lox-neo-lox)/+;r2::Cre animals at E18.5, we showed that the estimated lateral parabrachial medial and Kölliker-Fuse nuclei (Figure 6b, e), as well as the more medial noradrenergic locus coeruleus, identified by PHOX2A or tyrosine hydroxylase (Figure 6g), were located outside, though adjacent to, the trigeminal r2-derived domain. Therefore, the putative inspiratory related domain is derived from r1, about two rhombomeres distant from the r3-r4 region, the most anterior site known to induce formation of a respiratory frequency controller [17, 26].
Analysis of EGFP expression in E18.5 Hoxa2EGFP(lox-neo-lox)/-;r2::Cre homozygous mutant mice (Figure 6c, f, h) indicated that the r2-derived peri-trigeminal nuclei were reduced while a ventral hyperplasia of the estimated Kölliker-Fuse (KF in Figure 6a–c) and parabrachial medial (PB in Figure 6d–f) nuclei was observed (arrowheads in Figures 5f and 6c). Other r1-derived structures, such as the locus coeruleus (LC in Figure 6c and Table 1) and the pediculo-pontine tegmental nuclei also expanded ventrally.
The anatomical structures modified in Krox20-/-, Hoxa1-/- and kreisler mutants [17, 18, 29, 37] have also been investigated in Hoxa2-/- neonates. Specifically, we found a normal morphology of the anterior fourth ventricle, a normal AP length of the dorsal Pons, and a normal position of the ambiguus and facial branchial motor nuclei (data not shown). The parvocellular reticular nucleus, a dorsal pontine structure originating in r3 and extending between the trigeminal motor nucleus and the facial nerve [17, 18, 29] was also normal in Hoxa2-/- mutants.
In conclusion, the brainstem defects likely responsible for the observed abnormalities of inspiratory control are restricted to the rostral pons of Hoxa2-/- mutants and include the r1-r2 derived territory.
The Hoxa2mutation affects the development of the rostral pons
Consistent with the original observations of the Hoxa2-/- defects being restricted to the anteriormost domain of Hoxa2 expression [10, 36], the function of respiratory rhythm controllers located in the caudal pons and medulla was normal in Hoxa2-/- animals. The phenotypic defects of the Hoxa2-/- mice that were not observed in Krox20 mutants are likely to be related to the reorganization of neural structures derived from the alar plate at the r1-r2 level of the brainstem . The distribution of cells expressing EGFP in the pons of Hoxa2EGFP(lox-neo-lox)/-);r2::Cre homozygous mutants pons suggests that the territories derived from r2 are reduced along the AP axis or have lost r2-specific characteristics. Lack of an r1/r2 boundary [10–12] explains that territories derived from r1 and adjacent to r2 are also modified, with, for example, an ectopic projection of the ascending branch of the sensory trigeminal nerve to the cerebellum . The abnormal location of locus coeruleus neurons is also consistent with an altered positioning of neurons within the r1-derived territory . Thus, anatomical observations indicate that a large part of the rostral pontine territory deriving from r1-r2 is reorganized in Hoxa2-/- mutants.
The r1-derived territory adjacent to r2 includes the estimated location of medial parabrachial and Kölliker-Fuse nuclei where a pontine respiratory group regulates adult mammalian inspirations [38–41] (for review see [28, 42]). Therefore, Hoxa2 might be required for normal development of the pontine respiratory group. Our data further suggest that the pons contributes in shaping inspirations during the first postnatal hours in intact mice in vivo.
In addition, catecholaminergic  and cholinergic  control of breathing are probably affected. Interestingly, a large VT with normal respiratory frequency in vivo has been observed after the inactivation of the gene encoding acetylcholinesterase, a procedure found to greatly reduce the muscarinic and nicotinic control of the respiratory generator in vitro . It is possible, therefore, that abnormalities of the pediculo-pontine tegmental nucleus (PPTg), the major source of ponto-bulbar cholinergic neurons, contribute to the control of inspirations in Hoxa2 mutants.
Selective inspiratory control by the rostral pons has been postulated to explain postnatal respiratory deficits following the exposure to sub-teratogenic doses of retinoic acid at the onset of hindbrain segmentation (E7.5) . We presently show that development of inspiratory control requires rhombomere-related expression of the most rostral Hox gene in the neural tube. These results further suggest that the genetic network controlling hindbrain segmentation is involved in the formation of neuronal circuits controlling breathing, thereby giving the modular organization of the ponto-bulbar respiratory network .
Control of the oro-buccal reflex requires both Hoxa2 and Krox20
As shown recently, Hoxa2 is required in r2-r3 for trigeminal nerve pathfinding at early stages and axonal arborization of afferents at later stages . Here we show that the anomaly of facial circuits in Hoxa2-/- mutant embryos is associated with an impairment of the reflex-induced rhythmic oro-buccal behavior at birth. Because the Hoxa2-/- oro-buccal behavior resembles that induced by the elimination of r3 in Krox20-/- , trigeminal behavioral deficits may involve both r2- and r3-derived processes, including the Hoxa2-dependent arborization of sensory axons in the rostral principal trigeminal nucleus . In addition, Hoxa2-/- and Krox20-/- deficits may involve rhythmic pre-motor reticular neurons in the vicinity of the trigeminal motor nucleus controlling rhythmic opening of the jaw during alimentary behaviors [14–16, 18]http://www.jneurosci.org/cgi/external_ref?access_num=000077737200006&link_type=ISI. This deficit in suction might participate in the early death of Hoxa2 null mutant mice, impeding the correct feeding behavior together with extensive transformation of the first branchial arch-derived facial skeleton [2, 36].
Robustness of Krox20-induced formation of anti-apneic circuits in the mouse
The most rostral rhombomere that has been shown to be required for the development of normal respiratory rate and to prevent apneas is r3. Elimination of Krox20 expression in r3 reduces quiet breathing frequency by 50% and multiplies by 10 the time spent in apnea, eventually leading to opioid-sensitive lethality [17, 26]. Because, in contrast, hypomorphic Krox20 neonates breathe at a normal rate without apneas, Krox20 expression must be entirely eliminated to alter respiration. Our results, therefore, suggest that the transient expression of Krox20 and the subsequent reduction of r3 size observed in hypomorphic Krox20 mutants do not prevent the development of the para-facial anti-apneic system. This result extends to mice previous observations in chick embryos using loss- and gain-of function strategies . Krox20 expression in r3 was found necessary for the non-cell autonomous induction of a neuronal rhythm controller from r4 . In contrast, it was found to be unlikely that this induction requires the generation of a specific population of r3 neurons because KROX20 inhibits (rather than stimulates) neurogenesis and neuronal differentiation . Furthermore, ectopic Krox20-expression in a limited territory (for example, obtained by unilateral electroporation in a rhombomere) was sufficient to induce a fully functional rhythm generator, although the population of neurons specified by Krox20 was certainly smaller than normal r3 in these preparations . We presently show that, in mice as in chicks, induction of the anti-apneic parafacial function by Krox20 is a robust process that persists despite quantitative alterations of pontine cell populations. Our working hypothesis is, therefore, that Krox20 may act by initiating a cell non-autonomous control of specific neuronal fates rather than by generating a respiratory-related neuronal population in a cell autonomous manner. In addition, rhythm generators may compensate for minor abnormalities during fetal development to restore normal function at birth [29, 44]. To further explore the parafacial development in mouse embryos, experiments are in progress in our laboratories to identify cell lineages that could be targets of the induction initiated by Krox20 expression.
Hoxa2 is not a crucial target of Krox20for the formation of the para-facial neuronal group controlling respiratory frequency
High levels of Hoxa2 are expressed in r3 and are required at late stages (E13) in trigeminal principal sensory neurons to induce arborization of whisker-related maxillary primary afferents . In contrast, we show that Hoxa2 is not necessary in r3 for the development of the Krox20-dependent anti-apneic respiratory frequency controller; because in Hoxa2-/- neonates respiratory frequency was normal, apneas were not life-threatening and treatment with naloxone had no effect on survival. In r3, Hoxa2 function downstream of Krox20 may be partially redundant with that of its paralogue Hoxb2 , also a direct target of Krox20 . Functional redundancy of Hox2 paralogs can be expected at early developmental stages (end-segmental stages, about E9.5), during which Krox20 expression initiates formation of rhythm generators. Indeed, synergistic genetic interaction of Hoxa2 and Hoxb2 has been shown for the early patterning of r3 . Redundancy might be less at later stages (about E13), when Hoxa2 exerts cell type-specific functions . Previous observations  have shown that the anti-apneic activity abolished in Krox20-/- and Hoxa1-/- mutants is preserved in kreisler mutants lacking r5. Altogether, the available data support the location of the anti-apneic activity to be within the para-facial r3r4-derived territories and support Krox20, but not Hoxa2, as a major player in this process.
We present evidence that distinct circuits regulate respiratory frequency and inspiration depth in vivo and involve different patterning mechanisms and progenitor populations during development. Hoxa2 inactivation, affecting the r1-r2 region and respiratory amplitude, did not severely perturb respiratory rhythm that requires normal Krox20 and Hoxa1 expression in the r3-r5 region . In contrast, inactivation of either Hoxa2 or Krox20 impairs the rhythmic control of jaw opening, consistent with Hoxa2 being required for normal development of the trigeminal function  and Krox20 being a direct regulator of Hoxa2 in r3 [4, 5].
Materials and methods
All experiments were carried out following the ethical guidelines of the European Union Council (86/609/EU), the French Agriculture Ministry regulations for the care and use of laboratory animals in acute and chronic experiments. These experiments were also approved by the respective Institution Committees for animal care and handling.
Mouse lines and genotype analysis
A total of 38 wild-type, 58 Hoxa2+/- and 29 Hoxa2-/- littermates resulting from crosses between heterozygous animals [10–12] were used in the present study. These numbers are in accordance with Mendelian repartition of genotypes (χ2 test, P = 0.469). The Hoxa2EGFP(lox-neo-lox) mouse knock-in allele allows selective activation of EGFP expression from the Hoxa2 locus only upon Cre recombinase mediated recombination, as described in . The r2::Cre transgenic line allows selective expression of Cre in r2 and its derivatives, as described in . The original Hoxa2 null mutation is described in . The phenotypes of Hoxa2-/- and Hoxa2EGFP(lox-neo-lox)/EGFP(lox-neo-lox) homozygous mutants are indistinguishable. The analysis of Hoxa2EGFP(lox-neo-lox)/+ and Hoxa2EGFP(lox-neo-lox)/- specimens allowed, therefore, the comparison of heterozygous and homozygous Hoxa2 mutants carrying only one dose of EGFP in both genotypes. DNA was extracted from the tail of the neonate mouse and the genotype was subsequently determined by a PCR assay using specific sets of oligonucleotide primers, as described in . All homozygous Hoxa2-/- animals did not feed, lost 6.6% of their birth weight in their first 18 hours of life and died within 12–20 hours after birth, whereas their heterozygous or wild-type littermates fed, gained about 15% of their birth weight during the same period, survived (Table 1; see also [36, 45]) and were, therefore, studied during the first week after birth.
We also analyzed 7 hypomorphic Krox20 mutants at P0.5 and P3-4 and we reinvestigated the breathing behavior of 17 Krox20-/- animals  during the first hours following birth. The hypomorphic Krox20 mutant was obtained by combining two previously developed Krox20 alleles: the Krox20 Cre allele consists of an insertion of the gene for the Cre recombinase into the Krox20 locus, resulting in Krox20 inactivation and expression of the Cre gene with a pattern that faithfully recapitulates the normal Krox20 pattern ; in the Krox20 flox allele, the second Krox20 exon is flanked by loxP sites – this allele behaves like the wild-type until excision of the floxed exon by the Cre recombinase results in inactivation of Krox20 [30, 46]. The compound heterozygous animals, Krox20Cre/flox, express Krox20 only transiently, due to subsequent elimination of the second exon. In the hindbrain, this combination behaves as a hypomorphic mutation, resulting in a severe reduction in, but not elimination of, r3, and a slight reduction of r5 (Figure 4).
Respiratory activity was measured using a modified barometric method, previously employed in neonates, called whole-body plethysmography . The plethysmograph chamber (20 ml) equipped with a temperature sensor was connected to a reference chamber of the same volume. The pressure difference between the two chambers was measured with a differential pressure transducer connected to a sine wave carrier demodulator. The spirogram was stored on a computer using a Labmaster interface at a sampling frequency of 1 kHz. Calibrations were performed at the end of each recording session by injecting 2.5–5 μl of air in the chamber with a Hamilton syringe.
Measurements started 0.5–2 hours after birth and were repeated every 4–6 hours. 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 (165 s). In each sample, periods of quiet breathing were identified by the absence of limb or body movements. Periods of limb, body and head movements were measured to determine neonatal activity during recordings. During quiet breathing, a computer-assisted method was used to measure the duration of inspiration and expiration from which respiratory frequency (fR) is derived and the tidal volume (VT, μl/g) from which minute ventilation (VE = f × VT/1,000, ml/g/minute) is derived. Total duration of apneas per recording session (given in percentages) provided an estimation of the apneic breathing. Apneas were included in the calculation of the minute ventilation, giving a mean value of this physiological parameter. In the case of naloxone treatment, separate calculations of respiratory frequency with and without inclusion of apneas were performed. Naloxone (1 mg/kg in saline) was administered subcutaneously using a Hamilton syringe, and the next recording carried out one hour after injection.
Before plethysmographic recordings, the number of jaw openings within 30 s was counted by introducing a catheter into the mouth of the newborns, therefore testing oro-buccal reflexes . In order to obtain comparable data, counting was done by the same person for the whole study. Both plethysmographic recordings and jaw opening counting were done blindly, without knowing the genotype. Values are given as mean ± standard error of the mean. Comparisons between two sets of data were performed by paired or unpaired Student's T-tests.
Anatomical observation and immunochemistry
E18.5 dpc (days post coitum) Hoxa2EGFP(lox-neo-lox)/+;r2-Cre and Hoxa2EGFP(lox-neo-lox)/EGFP(lox-neo-lox);r2-Cre foetuses were obtained by caesarean section. Brains were dissected out, fixed in 4% paraformaldehyde, 1 × phosphate buffer saline (PBS) at 4°C overnight, cryoprotected, and sagittally sectioned. Adjacent sections were processed for immunohistochemistry using anti-EGFP (1:2000; Molecular Probes (Carlsbad, California, USA)) or anti-Phox2a (1:1,000; a gift from Jean-François Brunet, ENS, Paris, France) polyclonal antibodies. Sections were then incubated for 2 hours with biotinylated secondary antibodies (1:200, pH 7.4; Vector (Burlingame, California, USA)). Signal amplification was obtained by using the Vectastain ABC kit (Vector). Peroxidase was subsequently revealed in a staining mixture containing 0.05% 3,3'-diaminobenzidine hydrochloride (DAB, Sigma (Lyon, France)) and 0.03% H2O2 in 0.05 M TrisHCl pH 7.6.
We thank L McKay for useful comments on the manuscript. We appreciate the kind gift of PHOX2A antibody by Drs JF Brunet and C Goridis. Work in JC's laboratory was funded by CNRS, Ministère de la Recherche et de la Technologie. Work in FMR's laboratory is supported by the Agence Nationale pour la Recherche (ANR), the Fondation pour la Recherche Medicale (FRM, 'Equipe Labelisée'), the Association pour la Recherche contre le Cancer (ARC), the Association Française contre les Myopathies (AFM), the Ministère pour le Recherche (ACI program), and by institutional funds from CNRS and INSERM. Work in PC's laboratory was supported by the ARC, ARCEP, AFM and by institutional grants from INSERM and MENRT; ET was supported by a fellowship from the LNFCC.
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