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Genetic and developmental models for the neural control of breathing in vertebrates
Respiration Physiology 122 (2000) 247 – 257 www.elsevier.com/locate/resphysiol
Genetic and developmental models for the neural control of breathing in vertebrates Gilles Fortin, Eduardo Domı´nguez del Toro, Ve´ronique Abadie, Laura Guimara˜es, Arthur S. Foutz, Monique Denavit-Saubie´, Franc¸ois Rouyer, Jean Champagnat * U.P.R. 2216, Neurobiologie Ge´ne´tique et Inte´grati6e, C.N.R.S., A6enue de la Terrasse, Baˆtiment 33, 91198 Gif-sur-Y6ette, France Accepted 17 May 2000
Abstract The present paper reviews some of the possible mechanisms that may link gene function in the brainstem and breathing patterns in vertebrates. On one hand, adaptation and acclimatisation of mature breathing to environmental constraints such as hypoxia, involves complex regulation of the gene expression in precise cardiorespiratory sites of the brainstem. On the other hand, targeted inactivation of different genes suggests that postnatal respiratory variables at rest depend on genes controlling the prenatal development of the brainstem. During embryogenesis, neurotrophins (gdnf, bdnf) regulate the survival of specific cellular populations composing the respiratory neuronal network. The expression of developmental genes such as Hox and Krox-20 initiates hindbrain segmentation, the earliest sign of regionalisation in the brainstem. As shown in the chick embryo, segmental specifications allow the establishment of an active embryonic rhythmic network and later insertion of specific neuronal circuits increasing the primordial rhythm frequency to near mature values. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Development, Embryo, Prenatal breathing; Control of breathing, Gene function, Gene expression, adaptation, environment
1. Introduction The existence of genetic diseases such as the congenital central hypoventilation syndrome (Shea, 1997; Gozal, 1998) points to the impact of * Corresponding author. Tel.: +33-1-69823404; fax: + 331-69070538. E-mail address: [email protected] (J. Champagnat).
genetic control on breathing. Genetic mechanisms controlling respiration can, otherwise, be postulated from the studies of breathing in human twins (Kobayashi et al., 1993) and inbred mice strains (Tankersley et al., 1998) or considering the diversity of respiratory patterns in mammalian species (Mortola and Noworaj, 1985). The present paper reviews some of the possible mechanisms that may link gene function and breathing patterns in vertebrates. It is suggested that brainstem
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respiratory rhythm generators and associated control systems should provide one of the best experimental models available so far to study how genetic specifications interact with integrative behaviours in vertebrates. General principles and strategies to help understanding the genetic control of behaviour result from studies on invertebrates, particularly the fruitfly Drosophila melanogaster. For example, chronobiological studies of locomotion and hatching in this species, have established the circadian function of an intracellular, cell-autonomous, endogenous clock (Dunlap et al., 1995). In this model, a link is established between gene expression in particular brain neurons and these highly integrated motor patterns. Within the 24-h period, the motor behaviour of the fly is time-linked with mRNA and protein level oscillations of clock genes like ‘period’ (per), ‘timeless’ (tim) (Hardin, 1998). Similar mechanisms are conserved in vertebrates, and a very similar picture of the clock components and mechanisms holds for mammals where per and tim homologs have been found (Whitmore et al., 1998). Investigation of the vertebrate brainstem control of breathing has started according to these strategies defined in invertebrates. First, modifications of mRNA and protein levels in the rodent brainstem have been time-linked with respiratory adaptive responses (Dumas et al., 1996). Second, experimental mutations, which in mammals, result mostly from targeted gene inactivation in mice, have altered selectively the brainstem control of respiration (Erickson et al., 1996; Jacquin et al., 1996; Poon et al., 2000). The breathing pattern is especially modified when certain genes operating during the prenatal development of the brainstem are inactivated (reviewed by Champagnat and Fortin, 1997; Katz and Balkowiec, 1997). Therefore, a major challenge in modelling the control of breathing would be to include genetic mechanisms influencing development. We will consider the role of neurotrophins, providing trophic support required for cell survival during restricted periods of maturation of the nervous system, and, finally, developmental control genes responsible for the segmentation of the neuroepithelium — a typical anatomical feature of the brainstem during the earliest stage of its development.
2. Gene expression is modified during hypoxia Physiologically realistic respiratory stimuli are known to affect expression of a variety of genes in the adult brainstem (Erickson and Millhorn, 1991). Acute hypoxia increases the expression of early response genes in precise cardiorespiratory sites of the brainstem, reflecting the increased electrical activity of neurons in these areas (see Larnicol et al., 1994; Belegu et al., 1999, and references herein). Regulation of gene expression during exposure to a hypoxic environment affects the respiratory control likely and contributes to the adaptive physiological responses. For example, acclimatisation to chronic hypoxia changes the expression of the gene encoding tyrosine hydroxylase in the nucleus tractus solitarius, the first order relay nucleus for sensory inputs to the respiratory network (Dumas et al., 1996). Tyrosine hydroxylase regulates the synthesis of catecholamine neuromodulators controlling the activity of brainstem respiratory neurons (Bianchi et al., 1995). Therefore, modifications of mRNA and protein levels time linked with responses to hypoxia are an integral part of the mechanisms underlying the adaptation of brainstem functions to environmental constraints. The finding of hypoxia-inducible factors (reviewed by Fandrey, 1995) such as HIF-1a (Wang et al., 1995; Gassman and Wenger, 1997; Guillemin and Krasnow, 1997) provides some information on how hypoxia can influence gene expression. The expression of HIF-1a is upregulated in the mammalian brainstem in response to a hypoxic stress compatible with normal respiratory physiology (Pequignot et al., 1998). Our knowledge on cellular mechanisms underlying this response derives from previous studies of the circadian clock genes per and tim. A time-controlled building of a PER–TIM heterodimer has been found in Drosophila. The dimer protects the PER protein against degradation and enters the nucleus to repress per and tim transcription. The PER–TIM heterodimerisation is mediated through a protein–protein interaction domain named PAS, that is found in PER and in other clock-, light- or oxygen-sensitive proteins (Taylor and Zhulin, 1999). HIF-1a has a PAS domain-containing
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partner for heterodimerisation, ARNT. In the presence of O2, HIF-1a is rapidly degraded (Huang et al., 1998). In hypoxic cells, HIF-1a is protected against degradation through heterodimerisation with ARNT, allowing the activation of its target genes (Jiang et al., 1997). Therefore, in the hypoxic response in mammals as well as in the Drosophila clock feedback loop, PAS domain protein that normally undergoes rapid turnover, can be stabilised by forming heterodimers that are responsible for the transcriptional regulation of the target genes. Whether HIF-1a/ARNT dimers could, in turn, be involved in the control of respiratory rhythm remains an open question.
3. Inactivation of genes encoding synaptic membrane receptors Inactivation of genes in transgenic mice provides a strategy to establish a link between genes expression and respiratory control. Genes of potential interest are essential encoding elements of the brainstem rhythm generator and controllers. We will consider the m-receptor involved in the opioid respiratory depression, and the NMDA-R1 receptor subunit involved in the excitatory glutamatergic transmission. These two synaptic membrane receptors are among the most effective in the brainstem respiratory neuronal network (Bianchi et al., 1995). Observations in knock-out animals show that defects observed are not simply understandable by considering only the deleted gene, others might be switched on and off, thereby causing deficits that are not seen after selective pharmacological blockade of the receptor protein function. Lack of the m-opioid receptor eliminates the morphine-induced respiratory depression. However, in these m-deficient mice, the d-opioid modulation of respiration is also absent (Matthes et al., 1998). The decrease in breathing frequency and minute volume induced in wildtype mice by the d-agonist analgesic deltorphin II, is not observed. These observations provide evidence for synergistic interactions between m- and d-receptors in respiratory pathways. Therefore, inactivation of the single gene encoding the m-opi-
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oid receptor modifies function of different protein products including the d-opioid receptor. Similarly, mutant mice lacking the glutamatergic receptor subunit NMDA-R1 exhibit novel, non-NMDA synaptic regulations. In wild-type mice, pharmacological blockade of NMDA receptor function has little effect on the breathing pattern of the intact awake neonates (Borday et al., 1998) and eliminates long-term depression of excitatory synaptic transmission in the nucleus tractus solitarius (Zhou et al., 1997). NMDAR1 − / − mice not only lack NMDA receptor function, they show a lethal depression of respiratory activities (Fig. 1) and a robust long term synaptic depression of synaptic transmission in the nucleus tractus solitarius (Poon et al., 2000). Of particular clinical significance is the fact that lethality takes place during the first postnatal day. This time window of the murine postnatal development is characterised in normal mice by irregular breathing with high incidence of apneas. Observations on NMDA-R1 − / − and other transgenic mice (Jacquin et al., 1996) show that the first postnatal day is also a period of increased risk of sudden death syndrome in mice. The acute pharmacological blockade of NMDA receptors has no effect on survival during this period. Therefore, disruption of the NMDA receptor function in utero and abnormalities appearing in mutants during prenatal development might explain discrepancies that are observed by comparing the effects of pharmacological and genetic blockade of synaptic receptor function.
4. The vital role of neurotrophins during development Most neuronal types are dependent on trophic support for survival during a restricted period of development. Survival is thought to be mediated by selective membrane receptors interacting with diffusible growth factors such as neurotrophins (Oppenheim, 1991). Both clinical evidence and observations on transgenic mice indicate that genetic disruption of the neurotrophic support during brainstem maturation may cause aberrant respiratory patterns after birth.
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4.1. Clinical obser6ations on the gdnf-RET pathway The glial cell line-derived neurotrophic factor (gdnf) is a ligand of the RET receptor tyrosine kinase. Mutations of the RET-gdnf signalling pathway have been identified in patients with congenital central hypoventilation syndrome (CCHS) (Bolk et al., 1996; Amiel et al., 1998), a rare congenital syndrome which is characterised by sleep-related hypopnea or apnea associated with a decreased sensitivity to hypercapnia. These clinical features occur in the absence of neuromuscular or lung disease, or an identifiable
brainstem lesion (Shea, 1997; Gozal, 1998). Inactivation of RET in mice also depresses the ventilatory response to inhaled CO2 (Burton et al., 1997). However, mutations of RET and the related defective function of vagal embryonic neural crest cells also contribute to approximately 50% of cases with aganglionic megacolon (Hirschsprung disease) in a much larger group of patients (Attie´ et al., 1995; Angrist et al., 1996). Hirschsprung disease is a congenital syndrome, which presents familial aggregation and genetic heterogeneity, and has been found in association with 16% of CCHS cases. Therefore, to what extent point mutations in the RET coding region account for CCHS remains an open issue.
Fig. 1. Effects of targeted inactivation of bdnf (Erickson et al., 1996), NMDA-R1 (Poon et al., 2000) and Krox-20 (Jacquin et al., 1996) on average (9 S.E.M.) respiratory variables, A, frequency; B, apneas (expirations lasting \ 3 sec); C, VT/M and suction; D, number of jaw openings elicited by peribuccal contact. Measurements (expressed in percent of wild-type values) compare wild-type control (black) and homozygous mutant (white) mice from the same litters, during the first day after birth. Although respiratory frequency is significantly decreased in all mutants (A), other behavioural traits define a functional phenotype that is typical of each mutation and related to the different patterns of lethality. Impairment of sensory input to the Medulla (in bdnf − / − and NMDA-R1 − / − ) and rhythm promoting circuits in the Pons (in NMDA-R1 − / − and Krox-20 − / − ) correlate with abnormal VT/M and suction-apnea, respectively. The time windows that are critical for surviving are the first 3 weeks in bdnf − / − (where VT/M is decreased and suction-apneas unaffected) and the first postnatal day in NMDA-R1 − / − and Krox-20 − / − (where breathing is apneic and suction impaired); lethality is 100% in NMDA-R1 − / − (where VT/M is small) and 2/3 in Krox-20 − / − (where a high VT/M alleviates hypoventilation).
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4.2. Obser6ations on bdnf in transgenic mice Another neurotrophin, the brain-derived neurotrophic factor (bdnf), signalling through the TrkB receptor tyrosine kinase, has been implicated in the chemoafferent control of breathing. In bdnf − / − mice, ventilatory responses to hyperoxia and hypoxia are depressed, but, in contrast to gdnf − / − mice, sensitivity to hypercapnia is intact (Erickson et al., 1996). Both respiratory frequency (fR) and tidal volume per body mass (VT/M) are smaller than normal (Fig. 1), leading to chronic hypoventilation that contributes to lethality during the first 3 weeks following birth (Erickson et al., 1996), a time windows clearly distinct from that observed in NMDA-R1 − / − mice. Lack of bdnf has been shown to influence survival of cranial sensory neurons (Brady et al., 1999) and function of central respiratory circuits (Balkowiec and Katz, 1998). Hence, bdnf − / − mice show a dramatic hypoplasia of cranial sensory ganglia while the brainstem anatomy appears unaffected (Conover et al., 1995). This trophic support of sensory ganglia by bdnf appears to be highly cell-specific. A sub-population of dopaminergic chemoafferent neurons that controls the respiratory network is selectively reduced by 50% in mice lacking bdnf; this effect is also found when bdnf expression is reduced, although not entirely blocked, in heterozygous mutants (Erickson et al., 1996). Neuronal growth factors are believed to regulate survival by modulating genetically programmed cell death and thereby quantitatively match neuronal populations to the size of their target field (Oppenheim, 1991). It appears that the expression of genes encoding neurotrophins may, therefore, control postnatal breathing patterns by modifying the size of specific neuronal populations within the respiratory network (Katz and Balkowiec, 1997).
5. From hindbrain segmentation in embryo to breathing after birth: the vital role of odd-rhombomeric specifications in the neuroepithelium The brainstem derives from the embryonic
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hindbrain (rhombencephalon), one of the vesicles that appears towards the anterior end of the neural tube. The hindbrain neuroepithelium is partitioned along the antero-posterior axis into an iterated series of eight cellular compartments called rhombomeres (Fig. 2A). A wealth of data have been accumulated on developmental control genes such as Hox and Krox-20, governing this segmentation process (Lumsden and Krumlauf, 1996). Their expression is transient and restricted to the earliest stages of the hindbrain embryonic development. Inactivation of these developmental control genes causes malformation of the brainstem organisation. Nevertheless, in many cases, it allows survival of the foetus and breathing at birth. Lethality may develop afterwards, during a critical time window of the postnatal development.
5.1. Obser6ations in transgenic mice Krox-20 is transiently expressed within the yet unsegmented hindbrain in two stripes with sharp edges corresponding to the future odd-numbered rhombomeres r3 and r5 (Wilkinson et al., 1989; Fig. 2A). The Krox-20 gene product acts as a direct transcription activator of other r3- and r5-related genes belonging to the Hox homeobox clusters (Nonchev et al., 1996). The inactivation of Krox-20 in transgenic mice results in the deletion of r3 and r5 (Fig. 2B) as demonstrated by the anatomical analysis of the hindbrain combined with the determination of the expression patterns of rhombomere-specific genes (SchneiderMaunoury et al., 1993; Swiatek and Gridley, 1993). Life-threatening respiratory deficiencies have been established in Krox-20 − / − mutant neonates during the first day after birth (Jacquin et al., 1996). During this precise time window, similar to that found in NMDA-R1 − / − mice, all these animals show apneas lasting ten-times longer than normal (Fig. 1B) and two thirds of them die. Krox-20 − / − mutant mice also exhibit abnormally slow respiratory and sucking rhythms (Fig. 1A, D), indicating that the elimination of r3 and r5 exerts irreversible influences on the control of frequency. The VT/M is higher than normal
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Fig. 2. Development of r3 and r5, abolished in Krox-20 − / − mice, is required for normal postnatal breathing (Jacquin et al., 1996), A, lateral view of the wild-type hindbrain showing rhombomeres (r1 to r8), trigeminal (5n), facial (7n) and glossopharyngeal (9n) exit points in even rhombomeres (r2, r4, r6) and expression of Krox-20 in odd rhombomeres r3 and r5; B, the homozygous inactivation of the Krox-20 gene leads to elimination of r3 and r5 (left) and life-threatening, low frequency respiration during the first postnatal day (P0.5, right; compare whole body plethysmographic recordings from Krox-20 − / − in B with wild-type in A); C, Postnatal temporal evolution of fR in Krox-20 − / − mice (average 9 S.E.M. expressed in percent of wild-type values, n =5) showing low values during the first postnatal day; untreated animals (not shown) in which fR is reduced to 20 – 40% of normal values usually die within a few hours; arrow, subcutaneous administration of naloxone, an antagonist of the enkephalinergic respiratory depression, stimulates respiration (see recording on the right) during 2 – 4 h, thereby allowing mutant mice to escape lethal processes, treated animals survive more than 5 days afterwards; note that fR remains lower than normal: r3 and r5 are, therefore, essential to properly specify respiratory rhythm frequency after birth.
(Fig. 1B), chronic partial de-afferentation and decrease of VT/M following the bdnf − / − mutation are not seen in Krox-20 − / − animals. Homeostatic regulation of VT/M, possibly reflex in origin, may therefore alleviate the apneic syndrome in some Krox-20 − / − mutants (Borday et al., 1997). Developmental control genes are believed to
specify neuronal fates by encoding positional information within the neuroepithelium (Lumsden and Krumlauf, 1996). Observations in Krox-20 − / − mutants suggest that the development of rhythmpromoting circuits in the brainstem requires such blueprints set in the embryo by specifications in the odd rhombomeres r3 and r5.
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Recordings performed in vitro have located one of these rhythm-promoting controls in the caudal pontine reticular nucleus (Jacquin et al., 1996). In the respiratory neuronal network, different types of rhythm promoting and depressing systems are spatially intermingled in the reticular formation. Immunochemical and pharmacological observations indicate that these systems are discriminated by the Krox-20 − / − mutation. For example, rhythm depressing enkephalinergic controls are spared and functionally predominant so that treatment with the selective antagonist naloxone greatly improves survival of the mutants during the critical time window of postnatal development (Fig. 2C). These observations suggest that the pattern of developmental control genes expressed in r3 and r5
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allows specific circuits to be inserted into the pontine neuronal network, thereby contributing to specify the respiratory frequency after birth.
5.2. Embryological obser6ations in chick Recordings have been performed in the embryo, mainly in chick, to further establish this link between the rhombomeric organisation and the development of circuits in the rhythmic neuronal network. By the end of the segmentation period (embryonic day E4 in chick), the hindbrain already exhibits a consistent and organised rhythmic respiration-like activity (Fortin et al., 1994, 1995; Abadie et al., 2000). This is a ‘low frequency’ rhythmic activity (Fig. 3B), one order of magnitude
Fig. 3. Development of the hindbrain circuit producing an episodic rhythm in the chick embryo (Fortin et al., 1999) requires specifications in odd-numbered rhombomeres r3 and r5 and a rostral-to-caudal oriented signalling (arrows). In vitro recordings (on the right) during the seventh embryonic day (E7), from facial (7n) or trigeminal (5n) roots, after isolation of segments of the embryonic hindbrain at E2 (on the left), A, High frequency episodes normally generated by intact hindbrain circuits are also recorded from the neuronal network derived from an r3r4 (or r5r6, not shown) segment isolated at E2; B, High frequency episodes fail to be produced when combinations of rhombomeres such as r2r3 (or r4r5, not shown) are isolated: an immature low frequency rhythmic pattern is maintained.
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slower than the rhythm produced by comparable postnatal brainstem preparations. A closer-to-mature activity with high frequency episodes (Fig. 3A) is turned on, at around E6 in chick, and increases in importance thereafter (Fortin et al., 1994). Segmental requirements for the generation of this high frequency pattern were investigated by isolating in ovo hindbrain segments at the time when they form and when Krox-20 is expressed (E2). Rhythmic patterns were recorded at E7 from brainstem segments deriving from isolated pairs of odd/even (e.g. r3r4) or even/odd (e.g. r4r5) rhombomeres (Fortin et al., 1999). Only networks deriving from the odd/even combinations, r3r4 or r5r6, developed a normal high frequency output (Fig. 3A). By contrast, neither isolated rhombomeres r4 or r6, nor isolated pairs r2r3 or r4r5 were able to generate typical high frequency, the immature low frequency pattern persisted in these segments (Fig. 3B). These results demonstrate that generation of a high frequency pattern requires specifications from r3 or r5 and inter-rhombomeric signalling with their caudal even-numbered neighbour r4 or r6 (arrows in Fig. 3). Furthermore, recording at the neuronal level has provided some information on the neural circuit that is eventually inserted into the rhythmic network according to previous rhombomeric specifications. The episodic pattern of high frequency activity was found in chick to correlate with the appearance of a new class of rhythmically inhibited neurons identified by their temporal pattern of membrane potentials. During motor bursts, these neurons show a strong synaptic inhibition followed by a post-inhibitory rebound discharge initiating another burst of motor activity. Hence, an inhibitory synapse, mediated by GABAA membrane receptors, is the essential component of the circuit responsible for the appearance of the high frequency pattern of rhythmic activity (Fortin et al., 1999). The chick embryo model, therefore, provides a first step towards the understanding of possible molecular mechanisms (discussed in Fortin et al., 1999) which link patterns of gene expression, network organisation and rhythmic activity in higher vertebrates.
5.3. Comparati6e obser6ations in 6ertebrates The segmented hindbrain is similar in all vertebrate embryos. Therefore, the segmentation stage is conserved and can be viewed as ‘phylotypic’ during the vertebrate hindbrain development, i.e. a stage at which all members of the phylum show the maximum degree of similarity, despite the large anatomical differences during earlier or later phases of development (Slack et al., 1993). The genetic mechanisms orchestrating segmentation are also highly conserved since developmental control gene homologues have been found in all vertebrates (Lumsden and Krumlauf, 1996). Basic principles that are emerging from studies in embryos are, therefore, worth confronting to phylogenetic data. The presence of ancestral rhythmic patterns would strengthen the view that the primordial brainstem neuronal network may stand as a genetically defined scaffold upon which further complexity and diversity can be built. Features of the hindbrain primordial rhythmic activities are indeed reminiscent of the primitive rhythmic motor control of respiration in lower vertebrates. In fact, central networks producing rhythmic motor patterns linked to the respiratory function exist in the brainstem reticular formation of all vertebrates (see Champagnat and Fortin, 1997 and references herein). Furthermore, the high frequency episodic pattern of rhythmic activity observed at E7 in chick (Fig. 2), has been recorded from the adult bullfrog brainstem under the same experimental conditions (Reid and Milsom, 1998). The same pattern has also been found recently in mice embryonic hindbrain at E13.5, shortly before the onset of foetal breathing (Abadie et al., 2000). Therefore, these observations in three evolutionary distant tetrapods indicate that the episodic pattern may identify a phylotypic stage during development of the brainstem rhythmic network. This pattern, which has a behavioural significance in an adult intermittent breather (amphibian), is transiently retained by the embryo of continuous breathers (birds and mammals). Inter-specific differences develop afterwards, with, for example, the mouse foetal pattern being different from the episodic rhythm that persists in chick (Abadie et al., 2000).
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More experiments are needed to identify in the different vertebrates, neuronal circuits that are specified by genes controlling segmentation. However, data obtained on transgenic mice and chick embryos, together with comparative physiological observations, support the existence of a basic functional organisation of the respiratory neuronal network that has been set by developmental control genes and conserved during the evolution of vertebrates.
6. Conclusion: how to build a vertebrate respiratory control system? Data accumulated during the last 10 years have shown that rhombomeres are units of gene expression. From the extensive analysis of the function and regulation of these genes, models can be proposed to understand ‘how to build a vertebrate hindbrain’ (Schneider-Maunoury et al., 1998). Data available so far from molecular, embryological and phylogenetical biology clearly show that in the near future, modelling of the respiratory control should integrate genetic processes specifying the neurobiological substrate upon which later complexity and diversity of the respiratory network can be built. However, assumption that important genetic events occur only at primordial stages of evolution or embryogenesis might lead to a rather stereotyped and rigid model of the central respiratory control that should also take environmental conditions into account. We believe, however, that it would provide the most realistic starting point to implement further the flexible and redundant controls of gene function that occur during mature breathing behaviour and acclimatisation to the environmental factors. Genetic tools for the analysis of the respiratory network are beginning to emerge, bridging the gap between embryonic pattern formation and functionality in the brain.
Acknowledgements This work was supported by Fondation pour la Recherche Me´dicale, a EEC training grant (BIO4-
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CT975-096) to E.D.T., FCT (BD/11299/97) and FLAD grants to L.G. and a Human Frontier Science Program RG0101/1997-B grant to J.C.
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Genetic and developmental models for the neural control of breathing in vertebrates Gilles Fortin, Eduardo Domı´nguez del Toro, Ve´ronique Abadie, Laura Guimara˜es, Arthur S. Foutz, Monique Denavit-Saubie´, Franc¸ois Rouyer, Jean Champagnat * U.P.R. 2216, Neurobiologie Ge´ne´tique et Inte´grati6e, C.N.R.S., A6enue de la Terrasse, Baˆtiment 33, 91198 Gif-sur-Y6ette, France Accepted 17 May 2000
Abstract The present paper reviews some of the possible mechanisms that may link gene function in the brainstem and breathing patterns in vertebrates. On one hand, adaptation and acclimatisation of mature breathing to environmental constraints such as hypoxia, involves complex regulation of the gene expression in precise cardiorespiratory sites of the brainstem. On the other hand, targeted inactivation of different genes suggests that postnatal respiratory variables at rest depend on genes controlling the prenatal development of the brainstem. During embryogenesis, neurotrophins (gdnf, bdnf) regulate the survival of specific cellular populations composing the respiratory neuronal network. The expression of developmental genes such as Hox and Krox-20 initiates hindbrain segmentation, the earliest sign of regionalisation in the brainstem. As shown in the chick embryo, segmental specifications allow the establishment of an active embryonic rhythmic network and later insertion of specific neuronal circuits increasing the primordial rhythm frequency to near mature values. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Development, Embryo, Prenatal breathing; Control of breathing, Gene function, Gene expression, adaptation, environment
1. Introduction The existence of genetic diseases such as the congenital central hypoventilation syndrome (Shea, 1997; Gozal, 1998) points to the impact of * Corresponding author. Tel.: +33-1-69823404; fax: + 331-69070538. E-mail address: [email protected] (J. Champagnat).
genetic control on breathing. Genetic mechanisms controlling respiration can, otherwise, be postulated from the studies of breathing in human twins (Kobayashi et al., 1993) and inbred mice strains (Tankersley et al., 1998) or considering the diversity of respiratory patterns in mammalian species (Mortola and Noworaj, 1985). The present paper reviews some of the possible mechanisms that may link gene function and breathing patterns in vertebrates. It is suggested that brainstem
0034-5687/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 3 4 - 5 6 8 7 ( 0 0 ) 0 0 1 6 3 - 8
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respiratory rhythm generators and associated control systems should provide one of the best experimental models available so far to study how genetic specifications interact with integrative behaviours in vertebrates. General principles and strategies to help understanding the genetic control of behaviour result from studies on invertebrates, particularly the fruitfly Drosophila melanogaster. For example, chronobiological studies of locomotion and hatching in this species, have established the circadian function of an intracellular, cell-autonomous, endogenous clock (Dunlap et al., 1995). In this model, a link is established between gene expression in particular brain neurons and these highly integrated motor patterns. Within the 24-h period, the motor behaviour of the fly is time-linked with mRNA and protein level oscillations of clock genes like ‘period’ (per), ‘timeless’ (tim) (Hardin, 1998). Similar mechanisms are conserved in vertebrates, and a very similar picture of the clock components and mechanisms holds for mammals where per and tim homologs have been found (Whitmore et al., 1998). Investigation of the vertebrate brainstem control of breathing has started according to these strategies defined in invertebrates. First, modifications of mRNA and protein levels in the rodent brainstem have been time-linked with respiratory adaptive responses (Dumas et al., 1996). Second, experimental mutations, which in mammals, result mostly from targeted gene inactivation in mice, have altered selectively the brainstem control of respiration (Erickson et al., 1996; Jacquin et al., 1996; Poon et al., 2000). The breathing pattern is especially modified when certain genes operating during the prenatal development of the brainstem are inactivated (reviewed by Champagnat and Fortin, 1997; Katz and Balkowiec, 1997). Therefore, a major challenge in modelling the control of breathing would be to include genetic mechanisms influencing development. We will consider the role of neurotrophins, providing trophic support required for cell survival during restricted periods of maturation of the nervous system, and, finally, developmental control genes responsible for the segmentation of the neuroepithelium — a typical anatomical feature of the brainstem during the earliest stage of its development.
2. Gene expression is modified during hypoxia Physiologically realistic respiratory stimuli are known to affect expression of a variety of genes in the adult brainstem (Erickson and Millhorn, 1991). Acute hypoxia increases the expression of early response genes in precise cardiorespiratory sites of the brainstem, reflecting the increased electrical activity of neurons in these areas (see Larnicol et al., 1994; Belegu et al., 1999, and references herein). Regulation of gene expression during exposure to a hypoxic environment affects the respiratory control likely and contributes to the adaptive physiological responses. For example, acclimatisation to chronic hypoxia changes the expression of the gene encoding tyrosine hydroxylase in the nucleus tractus solitarius, the first order relay nucleus for sensory inputs to the respiratory network (Dumas et al., 1996). Tyrosine hydroxylase regulates the synthesis of catecholamine neuromodulators controlling the activity of brainstem respiratory neurons (Bianchi et al., 1995). Therefore, modifications of mRNA and protein levels time linked with responses to hypoxia are an integral part of the mechanisms underlying the adaptation of brainstem functions to environmental constraints. The finding of hypoxia-inducible factors (reviewed by Fandrey, 1995) such as HIF-1a (Wang et al., 1995; Gassman and Wenger, 1997; Guillemin and Krasnow, 1997) provides some information on how hypoxia can influence gene expression. The expression of HIF-1a is upregulated in the mammalian brainstem in response to a hypoxic stress compatible with normal respiratory physiology (Pequignot et al., 1998). Our knowledge on cellular mechanisms underlying this response derives from previous studies of the circadian clock genes per and tim. A time-controlled building of a PER–TIM heterodimer has been found in Drosophila. The dimer protects the PER protein against degradation and enters the nucleus to repress per and tim transcription. The PER–TIM heterodimerisation is mediated through a protein–protein interaction domain named PAS, that is found in PER and in other clock-, light- or oxygen-sensitive proteins (Taylor and Zhulin, 1999). HIF-1a has a PAS domain-containing
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partner for heterodimerisation, ARNT. In the presence of O2, HIF-1a is rapidly degraded (Huang et al., 1998). In hypoxic cells, HIF-1a is protected against degradation through heterodimerisation with ARNT, allowing the activation of its target genes (Jiang et al., 1997). Therefore, in the hypoxic response in mammals as well as in the Drosophila clock feedback loop, PAS domain protein that normally undergoes rapid turnover, can be stabilised by forming heterodimers that are responsible for the transcriptional regulation of the target genes. Whether HIF-1a/ARNT dimers could, in turn, be involved in the control of respiratory rhythm remains an open question.
3. Inactivation of genes encoding synaptic membrane receptors Inactivation of genes in transgenic mice provides a strategy to establish a link between genes expression and respiratory control. Genes of potential interest are essential encoding elements of the brainstem rhythm generator and controllers. We will consider the m-receptor involved in the opioid respiratory depression, and the NMDA-R1 receptor subunit involved in the excitatory glutamatergic transmission. These two synaptic membrane receptors are among the most effective in the brainstem respiratory neuronal network (Bianchi et al., 1995). Observations in knock-out animals show that defects observed are not simply understandable by considering only the deleted gene, others might be switched on and off, thereby causing deficits that are not seen after selective pharmacological blockade of the receptor protein function. Lack of the m-opioid receptor eliminates the morphine-induced respiratory depression. However, in these m-deficient mice, the d-opioid modulation of respiration is also absent (Matthes et al., 1998). The decrease in breathing frequency and minute volume induced in wildtype mice by the d-agonist analgesic deltorphin II, is not observed. These observations provide evidence for synergistic interactions between m- and d-receptors in respiratory pathways. Therefore, inactivation of the single gene encoding the m-opi-
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oid receptor modifies function of different protein products including the d-opioid receptor. Similarly, mutant mice lacking the glutamatergic receptor subunit NMDA-R1 exhibit novel, non-NMDA synaptic regulations. In wild-type mice, pharmacological blockade of NMDA receptor function has little effect on the breathing pattern of the intact awake neonates (Borday et al., 1998) and eliminates long-term depression of excitatory synaptic transmission in the nucleus tractus solitarius (Zhou et al., 1997). NMDAR1 − / − mice not only lack NMDA receptor function, they show a lethal depression of respiratory activities (Fig. 1) and a robust long term synaptic depression of synaptic transmission in the nucleus tractus solitarius (Poon et al., 2000). Of particular clinical significance is the fact that lethality takes place during the first postnatal day. This time window of the murine postnatal development is characterised in normal mice by irregular breathing with high incidence of apneas. Observations on NMDA-R1 − / − and other transgenic mice (Jacquin et al., 1996) show that the first postnatal day is also a period of increased risk of sudden death syndrome in mice. The acute pharmacological blockade of NMDA receptors has no effect on survival during this period. Therefore, disruption of the NMDA receptor function in utero and abnormalities appearing in mutants during prenatal development might explain discrepancies that are observed by comparing the effects of pharmacological and genetic blockade of synaptic receptor function.
4. The vital role of neurotrophins during development Most neuronal types are dependent on trophic support for survival during a restricted period of development. Survival is thought to be mediated by selective membrane receptors interacting with diffusible growth factors such as neurotrophins (Oppenheim, 1991). Both clinical evidence and observations on transgenic mice indicate that genetic disruption of the neurotrophic support during brainstem maturation may cause aberrant respiratory patterns after birth.
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4.1. Clinical obser6ations on the gdnf-RET pathway The glial cell line-derived neurotrophic factor (gdnf) is a ligand of the RET receptor tyrosine kinase. Mutations of the RET-gdnf signalling pathway have been identified in patients with congenital central hypoventilation syndrome (CCHS) (Bolk et al., 1996; Amiel et al., 1998), a rare congenital syndrome which is characterised by sleep-related hypopnea or apnea associated with a decreased sensitivity to hypercapnia. These clinical features occur in the absence of neuromuscular or lung disease, or an identifiable
brainstem lesion (Shea, 1997; Gozal, 1998). Inactivation of RET in mice also depresses the ventilatory response to inhaled CO2 (Burton et al., 1997). However, mutations of RET and the related defective function of vagal embryonic neural crest cells also contribute to approximately 50% of cases with aganglionic megacolon (Hirschsprung disease) in a much larger group of patients (Attie´ et al., 1995; Angrist et al., 1996). Hirschsprung disease is a congenital syndrome, which presents familial aggregation and genetic heterogeneity, and has been found in association with 16% of CCHS cases. Therefore, to what extent point mutations in the RET coding region account for CCHS remains an open issue.
Fig. 1. Effects of targeted inactivation of bdnf (Erickson et al., 1996), NMDA-R1 (Poon et al., 2000) and Krox-20 (Jacquin et al., 1996) on average (9 S.E.M.) respiratory variables, A, frequency; B, apneas (expirations lasting \ 3 sec); C, VT/M and suction; D, number of jaw openings elicited by peribuccal contact. Measurements (expressed in percent of wild-type values) compare wild-type control (black) and homozygous mutant (white) mice from the same litters, during the first day after birth. Although respiratory frequency is significantly decreased in all mutants (A), other behavioural traits define a functional phenotype that is typical of each mutation and related to the different patterns of lethality. Impairment of sensory input to the Medulla (in bdnf − / − and NMDA-R1 − / − ) and rhythm promoting circuits in the Pons (in NMDA-R1 − / − and Krox-20 − / − ) correlate with abnormal VT/M and suction-apnea, respectively. The time windows that are critical for surviving are the first 3 weeks in bdnf − / − (where VT/M is decreased and suction-apneas unaffected) and the first postnatal day in NMDA-R1 − / − and Krox-20 − / − (where breathing is apneic and suction impaired); lethality is 100% in NMDA-R1 − / − (where VT/M is small) and 2/3 in Krox-20 − / − (where a high VT/M alleviates hypoventilation).
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4.2. Obser6ations on bdnf in transgenic mice Another neurotrophin, the brain-derived neurotrophic factor (bdnf), signalling through the TrkB receptor tyrosine kinase, has been implicated in the chemoafferent control of breathing. In bdnf − / − mice, ventilatory responses to hyperoxia and hypoxia are depressed, but, in contrast to gdnf − / − mice, sensitivity to hypercapnia is intact (Erickson et al., 1996). Both respiratory frequency (fR) and tidal volume per body mass (VT/M) are smaller than normal (Fig. 1), leading to chronic hypoventilation that contributes to lethality during the first 3 weeks following birth (Erickson et al., 1996), a time windows clearly distinct from that observed in NMDA-R1 − / − mice. Lack of bdnf has been shown to influence survival of cranial sensory neurons (Brady et al., 1999) and function of central respiratory circuits (Balkowiec and Katz, 1998). Hence, bdnf − / − mice show a dramatic hypoplasia of cranial sensory ganglia while the brainstem anatomy appears unaffected (Conover et al., 1995). This trophic support of sensory ganglia by bdnf appears to be highly cell-specific. A sub-population of dopaminergic chemoafferent neurons that controls the respiratory network is selectively reduced by 50% in mice lacking bdnf; this effect is also found when bdnf expression is reduced, although not entirely blocked, in heterozygous mutants (Erickson et al., 1996). Neuronal growth factors are believed to regulate survival by modulating genetically programmed cell death and thereby quantitatively match neuronal populations to the size of their target field (Oppenheim, 1991). It appears that the expression of genes encoding neurotrophins may, therefore, control postnatal breathing patterns by modifying the size of specific neuronal populations within the respiratory network (Katz and Balkowiec, 1997).
5. From hindbrain segmentation in embryo to breathing after birth: the vital role of odd-rhombomeric specifications in the neuroepithelium The brainstem derives from the embryonic
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hindbrain (rhombencephalon), one of the vesicles that appears towards the anterior end of the neural tube. The hindbrain neuroepithelium is partitioned along the antero-posterior axis into an iterated series of eight cellular compartments called rhombomeres (Fig. 2A). A wealth of data have been accumulated on developmental control genes such as Hox and Krox-20, governing this segmentation process (Lumsden and Krumlauf, 1996). Their expression is transient and restricted to the earliest stages of the hindbrain embryonic development. Inactivation of these developmental control genes causes malformation of the brainstem organisation. Nevertheless, in many cases, it allows survival of the foetus and breathing at birth. Lethality may develop afterwards, during a critical time window of the postnatal development.
5.1. Obser6ations in transgenic mice Krox-20 is transiently expressed within the yet unsegmented hindbrain in two stripes with sharp edges corresponding to the future odd-numbered rhombomeres r3 and r5 (Wilkinson et al., 1989; Fig. 2A). The Krox-20 gene product acts as a direct transcription activator of other r3- and r5-related genes belonging to the Hox homeobox clusters (Nonchev et al., 1996). The inactivation of Krox-20 in transgenic mice results in the deletion of r3 and r5 (Fig. 2B) as demonstrated by the anatomical analysis of the hindbrain combined with the determination of the expression patterns of rhombomere-specific genes (SchneiderMaunoury et al., 1993; Swiatek and Gridley, 1993). Life-threatening respiratory deficiencies have been established in Krox-20 − / − mutant neonates during the first day after birth (Jacquin et al., 1996). During this precise time window, similar to that found in NMDA-R1 − / − mice, all these animals show apneas lasting ten-times longer than normal (Fig. 1B) and two thirds of them die. Krox-20 − / − mutant mice also exhibit abnormally slow respiratory and sucking rhythms (Fig. 1A, D), indicating that the elimination of r3 and r5 exerts irreversible influences on the control of frequency. The VT/M is higher than normal
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Fig. 2. Development of r3 and r5, abolished in Krox-20 − / − mice, is required for normal postnatal breathing (Jacquin et al., 1996), A, lateral view of the wild-type hindbrain showing rhombomeres (r1 to r8), trigeminal (5n), facial (7n) and glossopharyngeal (9n) exit points in even rhombomeres (r2, r4, r6) and expression of Krox-20 in odd rhombomeres r3 and r5; B, the homozygous inactivation of the Krox-20 gene leads to elimination of r3 and r5 (left) and life-threatening, low frequency respiration during the first postnatal day (P0.5, right; compare whole body plethysmographic recordings from Krox-20 − / − in B with wild-type in A); C, Postnatal temporal evolution of fR in Krox-20 − / − mice (average 9 S.E.M. expressed in percent of wild-type values, n =5) showing low values during the first postnatal day; untreated animals (not shown) in which fR is reduced to 20 – 40% of normal values usually die within a few hours; arrow, subcutaneous administration of naloxone, an antagonist of the enkephalinergic respiratory depression, stimulates respiration (see recording on the right) during 2 – 4 h, thereby allowing mutant mice to escape lethal processes, treated animals survive more than 5 days afterwards; note that fR remains lower than normal: r3 and r5 are, therefore, essential to properly specify respiratory rhythm frequency after birth.
(Fig. 1B), chronic partial de-afferentation and decrease of VT/M following the bdnf − / − mutation are not seen in Krox-20 − / − animals. Homeostatic regulation of VT/M, possibly reflex in origin, may therefore alleviate the apneic syndrome in some Krox-20 − / − mutants (Borday et al., 1997). Developmental control genes are believed to
specify neuronal fates by encoding positional information within the neuroepithelium (Lumsden and Krumlauf, 1996). Observations in Krox-20 − / − mutants suggest that the development of rhythmpromoting circuits in the brainstem requires such blueprints set in the embryo by specifications in the odd rhombomeres r3 and r5.
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Recordings performed in vitro have located one of these rhythm-promoting controls in the caudal pontine reticular nucleus (Jacquin et al., 1996). In the respiratory neuronal network, different types of rhythm promoting and depressing systems are spatially intermingled in the reticular formation. Immunochemical and pharmacological observations indicate that these systems are discriminated by the Krox-20 − / − mutation. For example, rhythm depressing enkephalinergic controls are spared and functionally predominant so that treatment with the selective antagonist naloxone greatly improves survival of the mutants during the critical time window of postnatal development (Fig. 2C). These observations suggest that the pattern of developmental control genes expressed in r3 and r5
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allows specific circuits to be inserted into the pontine neuronal network, thereby contributing to specify the respiratory frequency after birth.
5.2. Embryological obser6ations in chick Recordings have been performed in the embryo, mainly in chick, to further establish this link between the rhombomeric organisation and the development of circuits in the rhythmic neuronal network. By the end of the segmentation period (embryonic day E4 in chick), the hindbrain already exhibits a consistent and organised rhythmic respiration-like activity (Fortin et al., 1994, 1995; Abadie et al., 2000). This is a ‘low frequency’ rhythmic activity (Fig. 3B), one order of magnitude
Fig. 3. Development of the hindbrain circuit producing an episodic rhythm in the chick embryo (Fortin et al., 1999) requires specifications in odd-numbered rhombomeres r3 and r5 and a rostral-to-caudal oriented signalling (arrows). In vitro recordings (on the right) during the seventh embryonic day (E7), from facial (7n) or trigeminal (5n) roots, after isolation of segments of the embryonic hindbrain at E2 (on the left), A, High frequency episodes normally generated by intact hindbrain circuits are also recorded from the neuronal network derived from an r3r4 (or r5r6, not shown) segment isolated at E2; B, High frequency episodes fail to be produced when combinations of rhombomeres such as r2r3 (or r4r5, not shown) are isolated: an immature low frequency rhythmic pattern is maintained.
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slower than the rhythm produced by comparable postnatal brainstem preparations. A closer-to-mature activity with high frequency episodes (Fig. 3A) is turned on, at around E6 in chick, and increases in importance thereafter (Fortin et al., 1994). Segmental requirements for the generation of this high frequency pattern were investigated by isolating in ovo hindbrain segments at the time when they form and when Krox-20 is expressed (E2). Rhythmic patterns were recorded at E7 from brainstem segments deriving from isolated pairs of odd/even (e.g. r3r4) or even/odd (e.g. r4r5) rhombomeres (Fortin et al., 1999). Only networks deriving from the odd/even combinations, r3r4 or r5r6, developed a normal high frequency output (Fig. 3A). By contrast, neither isolated rhombomeres r4 or r6, nor isolated pairs r2r3 or r4r5 were able to generate typical high frequency, the immature low frequency pattern persisted in these segments (Fig. 3B). These results demonstrate that generation of a high frequency pattern requires specifications from r3 or r5 and inter-rhombomeric signalling with their caudal even-numbered neighbour r4 or r6 (arrows in Fig. 3). Furthermore, recording at the neuronal level has provided some information on the neural circuit that is eventually inserted into the rhythmic network according to previous rhombomeric specifications. The episodic pattern of high frequency activity was found in chick to correlate with the appearance of a new class of rhythmically inhibited neurons identified by their temporal pattern of membrane potentials. During motor bursts, these neurons show a strong synaptic inhibition followed by a post-inhibitory rebound discharge initiating another burst of motor activity. Hence, an inhibitory synapse, mediated by GABAA membrane receptors, is the essential component of the circuit responsible for the appearance of the high frequency pattern of rhythmic activity (Fortin et al., 1999). The chick embryo model, therefore, provides a first step towards the understanding of possible molecular mechanisms (discussed in Fortin et al., 1999) which link patterns of gene expression, network organisation and rhythmic activity in higher vertebrates.
5.3. Comparati6e obser6ations in 6ertebrates The segmented hindbrain is similar in all vertebrate embryos. Therefore, the segmentation stage is conserved and can be viewed as ‘phylotypic’ during the vertebrate hindbrain development, i.e. a stage at which all members of the phylum show the maximum degree of similarity, despite the large anatomical differences during earlier or later phases of development (Slack et al., 1993). The genetic mechanisms orchestrating segmentation are also highly conserved since developmental control gene homologues have been found in all vertebrates (Lumsden and Krumlauf, 1996). Basic principles that are emerging from studies in embryos are, therefore, worth confronting to phylogenetic data. The presence of ancestral rhythmic patterns would strengthen the view that the primordial brainstem neuronal network may stand as a genetically defined scaffold upon which further complexity and diversity can be built. Features of the hindbrain primordial rhythmic activities are indeed reminiscent of the primitive rhythmic motor control of respiration in lower vertebrates. In fact, central networks producing rhythmic motor patterns linked to the respiratory function exist in the brainstem reticular formation of all vertebrates (see Champagnat and Fortin, 1997 and references herein). Furthermore, the high frequency episodic pattern of rhythmic activity observed at E7 in chick (Fig. 2), has been recorded from the adult bullfrog brainstem under the same experimental conditions (Reid and Milsom, 1998). The same pattern has also been found recently in mice embryonic hindbrain at E13.5, shortly before the onset of foetal breathing (Abadie et al., 2000). Therefore, these observations in three evolutionary distant tetrapods indicate that the episodic pattern may identify a phylotypic stage during development of the brainstem rhythmic network. This pattern, which has a behavioural significance in an adult intermittent breather (amphibian), is transiently retained by the embryo of continuous breathers (birds and mammals). Inter-specific differences develop afterwards, with, for example, the mouse foetal pattern being different from the episodic rhythm that persists in chick (Abadie et al., 2000).
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More experiments are needed to identify in the different vertebrates, neuronal circuits that are specified by genes controlling segmentation. However, data obtained on transgenic mice and chick embryos, together with comparative physiological observations, support the existence of a basic functional organisation of the respiratory neuronal network that has been set by developmental control genes and conserved during the evolution of vertebrates.
6. Conclusion: how to build a vertebrate respiratory control system? Data accumulated during the last 10 years have shown that rhombomeres are units of gene expression. From the extensive analysis of the function and regulation of these genes, models can be proposed to understand ‘how to build a vertebrate hindbrain’ (Schneider-Maunoury et al., 1998). Data available so far from molecular, embryological and phylogenetical biology clearly show that in the near future, modelling of the respiratory control should integrate genetic processes specifying the neurobiological substrate upon which later complexity and diversity of the respiratory network can be built. However, assumption that important genetic events occur only at primordial stages of evolution or embryogenesis might lead to a rather stereotyped and rigid model of the central respiratory control that should also take environmental conditions into account. We believe, however, that it would provide the most realistic starting point to implement further the flexible and redundant controls of gene function that occur during mature breathing behaviour and acclimatisation to the environmental factors. Genetic tools for the analysis of the respiratory network are beginning to emerge, bridging the gap between embryonic pattern formation and functionality in the brain.
Acknowledgements This work was supported by Fondation pour la Recherche Me´dicale, a EEC training grant (BIO4-
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CT975-096) to E.D.T., FCT (BD/11299/97) and FLAD grants to L.G. and a Human Frontier Science Program RG0101/1997-B grant to J.C.
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