S1.3 Phox2b and the autonomic control of breathing

Abstract

In all vertebrates, the Phox2b transcription factor is specifically expressed and required in a limited set of neuronal types, which match for the most part the neurons of the visceral reflex circuits that regulate cardio-vascular, respiratory and digestive functions. Until recently, the role of Phox2b in the circuits that control breathing in mammals seemed patchier than in those that control blood pressure and digestion. This changed with the discovery that dominant mutations in Phox2b cause congenital central hypoventilation syndrome (CCHS). CCHS is a rare, but severe congenital disease characterized by central hypoventilation in sleep and an absent response to hypercapnia, which have been attributed to a defect in the autonomic regulation of breathing. We have modeled the most frequent human mutation in the mouse. The heterozygous Phox2b27Ala/+ mice breathe irregularly, do not respond to hypercapnia and die in the first hours after birth from central apnoea. The sole anatomical defect we found was the lack of CO2-sensitive, Phox2b-expressing glutamatergic cells in the retrotrapezoid nucleus (RTN). The location and molecular phenotype of RTN neurons characterized by their chemosensitivity matches that of the parafacial respiratory group (pFRG) identified as intrinsically rhythmic pre-inspiratory neurons. We found that the respiratory-like spontaneous rhythmic activity of the pFRG was lost in Phox2b27Ala/+ embryos, while the pre-Bötzinger complex, the best studied respiratory rhythm generator, functioned normally when tested in isolation. We thus propose that, in addition to the unresponsiveness to CO2, the lack of the pFRG oscillator contributes to the severe phenotype of the Phox2b27Ala/+ pups.pFRG/RTN neurons not only succumb to the dominant Phox2b27Ala mutation, but are also depleted in the absence of Phox2b. Using genetic tracing to study the ontogeny of pFRG/RTN neurons, we found that they originate from a population of Lbx1+ dorsal interneurons in rhombomeres 3 or 5. This gave us the opportunity to generate conditional Phox2b mutants using cre recombinase expression in either rhombomere 3/5 or in Lbx1+ interneurons. Like the Phox2b27Ala/+ mutation, the two conditional Phox2b mutations resulted in fatal central apnoea and abolished the activity of the embryonic pFRG.