Molecular physiology of Phox2b-expressing RTN chemoreceptor neurons

A discrete group of intrinsically CO2/H+-sensitive brainstem neurons located in the retrotrapezoid nucleus (RTN) provide a critical state-dependent and CO2-modulated excitatory drive to respiratory and arousal circuits. RTN dysfunction is implicated in various syndromes associated with reduced arousal and ventilatory responses to high CO2 (e.g., sudden infant death, SIDS; congenital central hypoventilation, CCHS). RTN neurons express the transcription factor Phox2b throughout life. Phox2b expression is required for embryonic specification of RTN neurons, and RTN dysgenesis is the prominent neuropathological feature in mouse genetic models that incorpo- rate CCHS-relevant Phox2b mutations and phenocopy its cardinal signs (i.e., hypoventilation, blunted arousal and ventilatory responses to CO2). The broad transcriptional role of Phox2b has not been determined in RTN neurons, and it remains unclear whether continued postnatal expression of Phox2b is required to maintain the specific molecular, cellular, and network features of RTN neurons. This proposal examines gene regulatory func- tions of wild type and CCHS-relevant polyalanine expansion mutants of Phox2b in RTN neurons, and the rele- vance of Phox2b-regulated genes for RTN function (Aim 1) and connectivity (Aim 2). In Aim 1, we test the hypothesis that persistent expression of wild type Phox2b regulates genes critical for RTN neuron effects on breathing and arousal. Specifically, we use viral approaches to deplete or mutate Phox2b in RTN neurons and determine effects on: [1.1] CO2-regulated breathing and arousal, by combined EEG and whole animal plethys- mography; [1.2] the full RTN transcriptome, by single cell RNA sequencing; and [1.3] direct transcriptional regulation of GPR4 and TASK-2, two molecular CO2 detectors in RTN neurons. Exciting preliminary data reveals that Phox2b knockdown in RTN neurons results in basal hypoventilation, respiratory dysrhythmia, and blunted CO2-stimulated breathing and arousal. Moreover, our initial candidate gene analysis suggests that GPR4 and TASK-2 may be regulated in cis by Phox2b. In Aim 2, we test the hypothesis that RTN neurons target multiple, phenotypically distinct brainstem neuronal groups that are sustained by Phox2b expression. Specifically, we use anterograde and trans-synaptic genetic markers with single cell RNA sequencing to: [2.1] characterize the molecular phenotype of direct postsynaptic targets of RTN neurons in brainstem regions linked to CO2-regulated breathing and arousal; and [2.2] examine whether those specific cellular targets are disrupted in CCHS mouse models or after RTN-specific Phox2b depletion/mutation. Initial transcriptomic analysis of single RTN postsyn- aptic cells identified genetic markers suggestive of known neuronal subclusters, and exciting preliminary data indicates that Phox2b expression in RTN neurons is required to maintain appropriate network connectivity to these physiologically important brainstem target regions. Collectively, the proposed studies will provide novel information regarding the role of Phox2b in maintaining the molecular, cellular, and network properties for the critical respiratory and arousal functions of RTN neurons. Project Number: 1R01HL177563-01 | Fiscal Year: 2025 | NIH Institute/Center: National Heart Lung and Blood Institute (NHLBI) | Principal Investigator: Douglas Bayliss (+1 co-PI) | Institution: UNIVERSITY OF VIRGINIA, CHARLOTTESVILLE, VA | Award Amount: $744,305 | Activity Code: R01 | Study Section: Respiratory Integrative Biology and Translational Research Study Section[RIBT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01HL17756301