Christo Goridis

Phox2b is a transcription factor expressed in the central and peripheral neurons that control cardiovascular, respiratory, and digestive functions and essential for their development. Several populations known or suspected to regulate visceral functions express Phox2b in the developing hindbrain. Extensive cell migration and lack of suitable markers have greatly hampered studying their development. Reasoning that intersectional fate mapping may help to overcome these impediments, we have generated a BAC transgenic mouse line, P2b::FLPo, which expresses codon‐optimized FLP recombinase in Phox2b expressing cells. By partnering the P2b::FLPo with the FLP‐responsive RC::Fela allele, we show that FLP recombination switches on lineage tracers in the cells that express or have expressed Phox2b, permanently marking them for study across development. Taking advantage of the dual‐recombinase feature of RC::Fela, we further show that the P2b::FLPo transgene can be partnered with Lbx1^Cre as Cre driver to generate triple transgenics in which neurons having a history of both Phox2b and Lbx1 expression are specifically labeled. Hence, the P2b::FLPo line when partnered with a suitable Cre driver provides a tool for tracking and accessing genetically subsets of Phox2b‐expressing neuronal populations, which has not been possible by Cre‐mediated recombination alone. genesis 51:506–514.

Breathing is a spontaneous, rhythmic motor behavior critical for maintaining O₂, CO₂, 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 CO₂in 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 toxicPHOX2B^27Alamutation 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 CO₂at 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 PCO₂within the normal range. Input from peripheral chemoreceptors that sense PO₂in the blood appears to compensate for the missing CO₂response since silencing them by high O₂abolishes rhythmic breathing. CO₂chemosensitivity partially recovered in adulthood. Hence, during the early life of rodents, the excitatory input normally afforded by elevated CO₂is dispensable for life-sustaining breathing and maintaining CO₂homeostasis in the blood.

The retrotrapezoid nucleus (RTN) is a group of neurons in the rostral medulla, defined here as Phox2b-,Vglut2-, neurokinin1 receptor-, andAtoh1-expressing cells in the parafacial region, which have been proposed to function both as generators of respiratory rhythm and as central respiratory chemoreceptors. The present study was undertaken to assess these two putative functions using genetic tools. We generated two conditional Phox2b mutations, which target different subsets of Phox2b-expressing cells, but have in common a massive depletion of RTN neurons. In both conditional mutants as well as in the previously describedPhox2b^27Alamutants, in which the RTN is also compromised, the respiratory-like rhythmic activity normally seen in the parafacial region of fetal brainstem preparations was completely abrogated. Rhythmic motor bursts were recorded from the phrenic nerve roots in the mutants, but their frequency was markedly reduced. Both the rhythmic activity in the RTN region and the phrenic nerve discharges responded to a low pH challenge in control, but not in the mutant embryos. Together, our results provide genetic evidence for the essential role of the Phox2b-expressing RTN neurons both in establishing a normal respiratory rhythm before birth and in providing chemosensory drive.

In the last few years, elucidation of the architecture of breathing control centres has reached the cellular level. This has been facilitated by increasing knowledge of the molecular signatures of various classes of hindbrain neurons. Here, we review the advances achieved by studying the homeodomain factor Phox2b , a transcriptional determinant of neuronal identity in the central and peripheral nervous systems. Evidence from human genetics, neurophysiology and mouse reverse genetics converges to implicate a small population of Phox2b -dependent neurons, located in the retrotrapezoid nucleus, in the detection of CO ₂ , which is a paramount source of the ‘drive to breathe’. Moreover, the same and other studies suggest that an overlapping or identical neuronal population, the parafacial respiratory group, might contribute to the respiratory rhythm at least in some circumstances, such as for the initiation of breathing following birth. Together with the previously established Phox2b dependency of other respiratory neurons (which we review briefly here), our new data highlight a key role of this transcription factor in setting up the circuits for breathing automaticity.

Background: Branchiomotor neurons comprise an important class of cranial motor neurons that innervate the branchial-arch-derived muscles of the face, jaw and neck. They arise in the ventralmost progenitor domain of the rhombencephalon characterized by expression of the homeodomain transcription factors Nkx2.2 and Phox2b. Phox2b in particular plays a key role in the specification of branchiomotor neurons. In its absence, generic neuronal differentiation is defective in the progenitor domain and no branchiomotor neurons are produced. Conversely, ectopic expression of Phox2b in spinal regions of the neural tube promotes cell cycle exit and neuronal differentiation and, at the same time, induces genes and an axonal phenotype characteristic for branchiomotor neurons. How Phox2b exerts its pleiotropic functions, both as a proneural gene and a neuronal subtype determinant, has remained unknown. Results: To gain further insights into the genetic program downstream of Phox2b, we searched for novel Phox2b-regulated genes by cDNA microarray analysis of facial branchiomotor neuron precursors from heterozygous and homozygous Phox2b mutant embryos. We selected for functional studies the genes encoding the axonal growth promoter Gap43, the Wnt antagonist Sfrp1 and the transcriptional regulator Sox13, which were not previously suspected to play roles downstream of Phox2b and whose expression was affected by Phox2b misexpression in the spinal cord. While Gap43 did not produce an obvious phenotype when overexpressed in the neural tube, Sfrp1 induced the interneuron marker Lhx1,5 and Sox13 inhibited neuronal differentiation. We then tested whether Sfrp1 and Sox13, which are down-regulated by Phox2b in the facial neuron precursors, would antagonize some aspects of Phox2b activity. Co-expression of Sfrp1 prevented Phox2b from repressing Lhx1,5 and alleviated the commissural axonal phenotype. When expressed together with Sox13, Phox2b was still able to promote cell cycle exit and neuronal differentiation, but the cells failed to relocate to the mantle layer and to extinguish the neural stem cell marker Sox2. Conclusion: Our results suggest novel roles for Sfrp1 and Sox13 in neuronal subtype specification and generic neuronal differentiation, respectively, and indicate that down-regulation of Sfrp1 and Sox13 are essential aspects of the genetic program controlled by Phox2b in cranial motoneurons.

Breathing is maintained and controlled by a network of neurons in the brainstem that generate respiratory rhythm and provide regulatory input. Central chemoreception, the mechanism for CO ₂ detection that provides an essential stimulatory input, is thought to involve neurons located near the medullary surface, whose nature is controversial. Good candidates are serotonergic medullary neurons and glutamatergic neurons in the parafacial region. Here, we show that mice bearing a mutation in Phox2b that causes congenital central hypoventilation syndrome in humans breathe irregularly, do not respond to an increase in CO ₂ , and die soon after birth from central apnea. They specifically lack Phox2b-expressing glutamatergic neurons located in the parafacial region, whereas other sites known or supposed to be involved in the control of breathing are anatomically normal. These data provide genetic evidence for the essential role of a specific population of medullary interneurons in driving proper breathing at birth and will be instrumental in understanding the etiopathology of congenital central hypoventilation syndrome.

Within the developing vertebrate nervous system, specific subclasses of neurons are produced in vastly different numbers at defined times and locations. This implies the concomitant activation of a program that controls pan-neuronal differentiation and of a program that specifies neuronal subtype identity, but how these programs are coordinated in time and space is not well understood. Our previous loss- and gain-of-function studies have defined Phox2b as a homeodomain transcription factor that coordinately regulates generic and type-specific neuronal properties. It is necessary and sufficient to impose differentiation towards a branchio- and viscero-motoneuronal phenotype and at the same time promotes generic neuronal differentiation. We have examined the underlying genetic interactions. We show thatPhox2b has a dual action on pan-neuronal differentiation. It upregulates the expression of proneural genes (Ngn2) when expressed alone and upregulates the expression of Mash1 when expressed in combination with Nkx2.2. By a separate pathway, Phox2brepresses expression of the inhibitors of neurogenesis Hes5 andId2. The role of Phox2b in the specification of neuronal subtype identity appears to depend in part on its capacity to act as a patterning gene in the progenitor domain. Phox2b misexpression represses the Pax6 and Olig2 genes, which should inhibit a branchiomotor fate, and induces Nkx6.1 and Nkx6.2, which are expressed in branchiomotor progenitors. We further show that Phox2b behaves like a transcriptional activator in the promotion of both, generic neuronal differentiation and expression of the motoneuronal marker Islet1. These results provide insights into the mechanisms by which a homeodomain transcription factor through interaction with other factors controls both generic and type-specific features of neuronal differentiation.

The specification of neurotransmitter phenotype is an important aspect of neuronal fate determination. Substantial progress has been made in uncovering key extracellular signals and transcriptional regulators that control the mode of neurotransmission in several model systems, among which catecholaminergic and serotonergic neurons feature prominently. Here, we review our current knowledge of the regulatory circuits that direct neurotransmitter choice, and discuss the development of well-studied types of catecholaminergic and serotonergic neurons. One emerging concept is that different types of neuron use a similar core programme to control shared modes of neurotransmission, but recruit different factors that are specific for each neuronal type. Another is that most factors that specify neurotransmitter identity also control other features of the neuronal phenotype.