Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O 2 , CO 2 , and pH homeostasis. In mammals, it is generated by a neuronal network in the lower brainstem, the respiratory rhythm generator (Feldman et al., 2003). A century-old tenet in respiratory physiology posits that the respiratory chemoreflex, the stimulation of breathing by an increase in partial pressure of CO 2 in the blood, is indispensable for rhythmic breathing. Here we have revisited this postulate with the help of mouse genetics. We have engineered a conditional mouse mutant in which the toxic PHOX2B 27Ala mutation that causes congenital central hypoventilation syndrome in man is targeted to the retrotrapezoid nucleus, a site essential for central chemosensitivity. The mutants lack a retrotrapezoid nucleus and their breathing is not stimulated by elevated CO 2 at least up to postnatal day 9 and they barely respond as juveniles, but nevertheless survive, breathe normally beyond the first days after birth, and maintain blood PCO 2 within the normal range. Input from peripheral chemoreceptors that sense PO 2 in the blood appears to compensate for the missing CO 2 response since silencing them by high O 2 abolishes rhythmic breathing. CO 2 chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO 2 is dispensable for life-sustaining breathing and maintaining CO 2 homeostasis in the blood.

Phox2a is a vertebrate homeodomain protein expressed in subsets of differentiating neurons. Here, we show that it is essential for proper development of the locus coeruleus, a subset of sympathetic and parasympathetic ganglia and the VIIth, IXth, and Xth cranial sensory ganglia. In the sensory ganglia, we have identified two differentiation blocks in Phox2a-/- mice. First, the transient expression of dopamine-beta-hydroxylase in neuroblasts is abolished, providing evidence that Phox2a controls noradrenergic traits in vivo. Second, the expression of the GDNF receptor subunit Ret is dramatically reduced, and there is a massive increase in apoptosis of ganglion cells, which are known to depend on GDNF in vivo. Therefore, Phox2a appears to regulate conventional differentiation traits and the ability of neurons to respond to essential survival factors.

Many transcription factors, and most prominently among them, homeodomain proteins, are expressed in specific groups of cells in the developing nervous system in patterns that suggest their involvement in neural fate determination. How various aspects of neural identity are controlled by such transcription factors, or sets of them, is still mostly unknown. It has been shown previously that Phox2 is such a homeodomain protein, expressed exclusively in differentiated groups of neurons or their precursors, and that its expression correlated with that of the noradrenaline synthesis enzyme dopamine-␤-hydroxylase. Here we confirm this striking correlation at the single-cell level with the use of an anti-Phox2 antibody. Moreover, we uncover a second, nonmutually exclusive correlative clue to the Phox2 expression pattern: a high proportion of Phox2-expressing cells are involved in, or located in areas involved in, synaptic circuits, i.e., that of the medullary control reflexes of autonomic functions. This suggests that Phox2 could be involved in the establishment of these circuits.