Fabrice Chatonnet

Different respiratory control systems are affected in homozygous and heterozygous kreisler mutant mice

During embryonic development, restricted expression of the regulatory genes Krox20 and kreisler are involved in segmentation and antero‐posterior patterning of the hindbrain neural tube. The analysis of transgenic mice in which specific rhombomeres (r) are eliminated points to an important role of segmentation in the generation of neuronal networks controlling vital rhythmic behaviours such as respiration. Thus, elimination of r3 and r5 in Krox20–/– mice suppresses a pontine antiapneic system ( Jacquin et al., 1996 ). We now compare Krox20–/– to kreisler heterozygous (+/kr) and homozygous (kr/kr) mutant neonates. In +/kr mutant mice, we describe hyperactivity of the antiapneic system: analysis of rhythm generation in vitro revealed a pontine modification in keeping with abnormal cell specifications previously reported in r3 ( Manzanares et al., 1999b ). In kr/kr mice, elimination of r5 abolished all +/kr respiratory traits, suggesting that +/kr hyperactivity of the antiapneic system is mediated through r5‐derived territories. Furthermore, collateral chemosensory pathways that normally mediate delayed responses to hypoxia and hyperoxia were not functional in kr/kr mice. We conclude that the pontine antiapneic system originates from r3r4, but not r5. A different rhythm‐promoting system originates in r5 and kreisler controls the development of antiapneic and chemosensory signal transmission at this level.

Respiratory survival mechanisms in acetylcholinesterase knockout mouse

Cholinergic neurotransmission ensures muscle contraction and plays a role in the regulation of respiratory pattern in the brainstem. Inactivation of acetylcholinesterase (AChE) by organophosphates produces respiratory failure but AChE knockout mice survive to adulthood. Respiratory adaptation mechanisms which ensure survival of these mice were examined in vivo by whole body plethysmography and in vitro in the neonatal isolated brainstem preparation. AChE−/− mice presented no AChE activity but unaffected butyrylcholinesterase (BChE) activity. In vivo, bambuterol (50–500 µg/kg s.c.) decreased BChE activity peripherally but not in brain tissue and induced apnea and death in adult and neonate AChE−/− mice without affecting littermate AChE+/+ and +/− animals. In vitro, bath‐applied bambuterol (1–100 µm) and tetraisopropylpyrophosphoramide (10–100 µm) decreased BChE activity in the brainstem but did not perturb central respiratory activity recorded from spinal nerve rootlets. In vitro, the cholinergic agonists muscarine (50–100 µm) and nicotine (0.5–10 µm) induced tonic activity in respiratory motoneurons and increased the frequency of inspiratory bursts in AChE+/+ and +/− animals. These effects were greatly attenuated in AChE−/− animals. The results suggest that, in mice lacking AChE, (i) BChE becomes essential for survival peripherally but plays no critical role in central rhythm‐generating structures and (ii) a major adaptive mechanism for respiratory survival is the down‐regulated response of central respiratory‐related neurons and motoneurons to muscarinic and nicotinic agonists.

Early development of respiratory rhythm generation in mouse and chick

We are investigating neuronal circuits resulting from conservative developmental mechanisms orchestrating the segmentation of the vertebrates hindbrain into compartments called rhombomeres (r). Segmentation transcription factors Hoxa1, Krox20 and kreisler are expressed in the future rhombomeres r4-r5, r3 and r5, r5-r6, respectively. In mice, the in vivo and in vitro analysis of neuronal groups after inactivation of these three genes revealed distinct postnatal respiratory phenotypes associated with defects of central respiratory controls resulting from deletion, neoformation or reconfiguration of modular circuits. In chick and mice, we have found neuronal rhythm generators that conform to the rhombomeric anatomical pattern as early as at the end of the segmentation. By isolating chick hindbrain segments in vitro, we have also identified rhombomeric motifs allowing the formation or deletion of a specific (GABAergic) rhythm-promoting module. Therefore, primordial rhombomeric organization of the hindbrain seems to determine a modular organization of the rhythmogenic network, thereby influencing later function of brainstem respiratory control networks.

A Bimodal Influence of Thyroid Hormone on Cerebellum Oligodendrocyte Differentiation

Thyroid hormone (T(3)) can trigger a massive differentiation of cultured oligodendrocytes precursor cells (OPC) by binding the nuclear T(3) receptor α1 (TRα1). Whether this reflects a physiological function of TRα1 remains uncertain. Using a recently generated mouse model, in which CRE/loxP recombination is used to block its function, we show that TRα1 acts at two levels for the in vivo differentiation of OPC in mouse cerebellum. At the early postnatal stage, it promotes the secretion of several neurotrophic factors by acting in Purkinje neurons and astrocytes, defining an environment suitable for OPC differentiation. At later stages, TRα1 acts in a cell-autonomous manner to ensure the complete arrest of OPC proliferation. These data explain contradictory observations made on various models and outline the importance of T(3) signaling both for synchronizing postnatal neurodevelopment and restraining OPC proliferation in adult brain.

From hindbrain segmentation to breathing after birth: developmental patterning in rhombomeres 3 and 4.

Respiration is a rhythmic motor behavior that appears in the fetus and acquires a vital importance at birth. It is generated within central pattern-generating neuronal networks of the hindbrain. This region of the brain is of particular interest since it is the most understood part with respect to the cellular and molecular mechanisms that underlie its development. Hox paralogs and Hox-regulating genes kreisler/mafB and Krox20 are required for the normal formation of rhombomeres in vertebrate embryos. From studies of rhombomeres r3 and r4, the authors review mechanisms whereby these developmental genes may govern the early embryonic development of para-facial neuronal networks and specify patterns of motor activities operating throughout life. A model whereby the regional identity of progenitor cells can be abnormally specified in r3 and r4 after a mutation of these genes is proposed. Novel neuronal circuits may develop from some of these misspecified progenitors while others are eliminated, eventually affecting respiration and survival after birth.

Neural tube patterning by Krox20 and emergence of a respiratory control.

Recent data begin to bridge the gap between developmental events controlling hindbrain neural tube regional patterning and the emergence of breathing behaviour in the fetus and its vital adaptive function after birth. In vertebrates, Hox paralogs and Hox-regulating genes orchestrate, in a conserved manner, the transient formation of developmental compartments in the hindbrain, the rhombomeres, in which rhythmic neuronal networks of the brainstem develop. Genetic inactivation of some of these genes in mice leads to pathological breathing at birth pointing to the vital importance of rhombomere 3 and 4 derived territories for maintenance of the breathing frequency. In chick embryo at E7, we investigated neuronal activities generated in neural tube islands deriving from combinations of rhombomeres isolated at embryonic day E1.5. Using a gain of function approach, we reveal a role of the transcription factor Krox20, specifying rhombomeres 3 and 5, in inducing a rhythm generator at the parafacial level of the hindbrain. The developmental genes selecting and regionally coordinating the fate of CNS progenitors may hold further clues to conserved aspects of neuronal network formation and function. However, the most immediate concern is to take advantage of early generated rhythmic activities in the hindbrain to pursue their downstream cellular and molecular targets, for it seems likely that it will be here that rhythmogenic properties will eventually take on a vital role at birth.

Ontogeny of central rhythm generation in chicks and rodents

Recent studies help in understanding how the basic organization of brainstem neuronal circuits along the anterior-posterior (AP) axis is set by the Hox-dependent segmentation of the neural tube in vertebrate embryos. Neonatal respiratory abnormalities in Krox20(-/-), Hoxa1(-/-) and kreisler mutant mice indicate the vital role of a para-facial (Krox20-dependent, rhombomere 4-derived) respiratory group, that is distinct from the more caudal rhythm generator called Pre-Bötzinger complex. Embryological studies in the chick suggest homology and conservation of this Krox20-dependent induction of parafacial rhythms in birds and mammals. Calcium imaging in embryo indicate that rhythm generators may derive from different cell lineages within rhombomeres. In mice, the Pre-Bötzinger complex is found to be distinct from oscillators producing the earliest neuronal activity, a primordial low-frequency rhythm. In contrast, in chicks, maturation of the parafacial generator is tightly linked to the evolution of this primordial rhythm. It seems therefore that ontogeny of brainstem rhythm generation involves conserved processes specifying distinct AP domains in the neural tube, followed by diverse, lineage-specific regulations allowing the emergence of organized rhythm generators at a given AP level.

Distinct roles of Hoxa2 and Krox20 in the development of rhythmic neural networks controlling inspiratory depth, respiratory frequency, and jaw opening.

BackgroundLittle 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.ResultsWe 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.ConclusionThus, 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.

Developmental molecular switches regulating breathing patterns in CNS

The present paper presents some of the molecular switches that may operate at early embryonic stages to make development of the brainstem respiratory rhythm generator a robust and irreversible process. We concentrate on the role of transient Hox-related gene expression patterns in register with the regionalisation of the rhombencephalic neural tube along the antero-posterior axis. Using different recording and isolation procedures in chick embryos, we show that the hindbrain is subdivided at E1 into developmental units (rhombomeres) intrinsically able to produce rhythm generating neuronal circuits active at E5. At E6, intrinsic cues also allow a progressive maturation of episodic rhythm generators that persists after isolation of the hindbrain in vitro and requires odd/even rhombomeric interactions at E1. From these results and from respiratory pathologies observed in transgenic mice, we are beginning to understand that, despite diversity of breathing patterns and adaptations, there are links between developmental control genes and adult respiration.

Exposure to retinoic acid at the onset of hindbrain segmentation induces episodic breathing in mice.

Hyperpnoeic episodic breathing (HEB), a cyclic waxing and waning of breathing, has been widely reported in pre‐term neonates, patients with Joubert syndrome and adults (Cheyne‐Stokes respiration) with congestive heart failure and brainstem infarction. We now provide a developmental mouse model of neonatal HEB. We used retinoic acid (RA) (0.5–10 mg/kg of maternal weight) to alter embryonic development of the respiratory neuronal network at the onset of hindbrain segmentation (7.5 days post‐coitum). HEB was observed in vivo after RA treatment during post‐natal days 1–7 but not in control animals. HEB persisted after reduction of the chemoafferent input by hypocapnic hyperoxia (100% O2). A large increase and decrease of the rhythm resembling an HEB episode was induced in vitro by stimulating the parafacial respiratory oscillator in treated but not in control neonates. Post‐natal localization of the superior cerebellar peduncle and adjacent dorsal tegmentum was found to be abnormal in the pons of RA‐treated juvenile mice. Thus, early developmental specifications in the rostral hindbrain are required for the development of neurones that stabilize the function of the respiratory rhythm generator, thereby preventing HEB during post‐natal maturation.