Transcription Factor Phox2b Research Articles

Molecular Ontology of the Nucleus of Solitary Tract.

The nucleus of the solitary tract (NTS) receives visceral information and regulates appetitive, digestive, and cardiorespiratory systems. Within the NTS, diverse processes operate in parallel to sustain life, but our understanding of their cellular composition is incomplete. Here, we integrate histologic and transcriptomic analysis to identify and compare molecular features that distinguish neurons in this brain region. Most glutamatergic neurons in the NTS and area postrema co-express the transcription factors Lmx1b and Phox2b, except for a ventral band of neurons in the far-caudal NTS, which include the Gcg-expressing neurons that produce glucagon-like peptide 1 (GLP-1). GABAergic interneurons intermingle through the Lmx1b+Phox2b macropopulation, and dense clusters of GABAergic neurons surround the NTS. The Lmx1b+Phox2b macropopulation includes subpopulations with distinct distributions expressing Grp, Hsd11b2, Npff, Pdyn, Pou3f1, Sctr, Th, and other markers. These findings highlight Lmx1b-Phox2b co-expression as a common feature of glutamatergic neurons in the NTS and improve our understanding of the organization and distribution of neurons in this critical brain region.

6421 Imaging Phenotype May Predict Genotype In Human Pheochromocytoma: Relationships Between Genetic Mutation Clusters, Metabolite Transporter Expression, And Radiotracer Uptake

Abstract Disclosure: I. Sakuma: None. M. Fujimoto: None. D.F. Vatner: None. K. Yokote: None. T. Tanaka: None. Context [1]23I-metaiodobenzylguanidine scintigraphy (MIBG) and [1]8F-fluoro-2-doxy-D-glucose positron emission tomography (FDG-PET) are helpful for the localization of pheochromocytoma. The norepinephrine transporter (NET) and the glucose transporter 1 (GLUT1) are thought to be critical for the uptake of these radioisotopes. NET expression is activated by Paired Like Homeobox (PHOX2) and glucocorticoid receptor (GR) in neuroblastoma cells. Around 70% of pheochromocytomas carry well-known mutations. Pheochromocytoma-associated genes are divided into two clusters: the pseudohypoxia-related cluster 1 and the kinase signaling-related cluster 2. Cluster 1 mutation are associated with noradrenergic pheochromocytomas with an elevated metastatic risk. Cluster 2 tumors demonstrate an adrenergic phenotype with a less aggressive course. How mutations of cluster 1 and cluster 2 impact the expression of NET/GLUT1 and on radiotracer uptake remains unclear. Hypothesis Cluster 1 and cluster 2 will have different effects on the expression of transcription factors associated with chromaffin cell differentiation with resultant differential effects on NET/GLUT1 expression. Methods 42 patients with pheochromocytoma were evaluated by MIBG and FDG-PET. Tumor gene expression was evaluated by real-time qPCR, and mutation analyses were performed by Sanger or next-generation sequencing. Results 88% of tumors were MIBG-avid, and 67% were FDG-avid. The expression of NET in MIBG-negative tumors was 70% lower than in MIBG-positive tumors. The expression of GLUT1 in FDG-PET-negative tumors was 75% lower than in FDG-PET-positive tumors. The expression of NET positively correlated with transcription factors involved in chromaffin cell maturation, including PHOX2A, PHOX2B, GATA binding protein 3 (GATA3), GR, catecholamine synthases, and insulinoma associated 1 (INSM1). The JASPAR database for promoter analysis revealed putative responsive regions of the transcription factors PHOX2A, PHOX2B, GATA3, GR, and INSM1 on the NET gene. Seven patients (16.7%) had mutations in cluster 1, and fourteen patients (33.3%) had mutations in cluster 2. Cluster 2 exhibited elevated NET, PHOX2A, PHOX2B, GATA3, GR, and INSM1 expression levels compared to cluster 1. Cluster 1 showed a higher expression level of GLUT1 and BMP4 that is associated with early chromaffin cell differentiation. Conclusions In pheochromocytomas, MIBG and FDG uptake correlated with the expression levels of NET and GLUT1, respectively. NET expression is associated with chromaffin cell differentiation transcription factor expression. We propose that pheochromocytomas that carry Cluster 1 mutations are more likely to be missed in MIBG due to low NET expression, while pheochromocytomas that carry Cluster 2 mutations are more likely to be missed on FDG-PET due to low GLUT1 expression with a more mature chromaffin cell phenotype. Presentation: 6/2/2024

Control of breathing by orexinergic signaling in the nucleus tractus solitarii

Orexin signaling plays a facilitatory role in respiration. Abnormalities in orexin levels correlate with disordered breathing patterns and impaired central respiratory chemoreception. Nucleus tractus solitarii (NTS) neurons expressing the transcription factor Phox2b contribute to the chemoreceptive regulation of respiration. However, the extent to which orexinergic signaling modulates respiratory activity in these Phox2b-expressing NTS neurons remains unclear. In the present study, the injection of orexin A into the NTS significantly increased the firing rate of the phrenic nerve. Further analysis using fluorescence in situ hybridization and immunohistochemistry revealed that orexin 1 receptors (OX1Rs) were primarily located in the ventrolateral subdivision of the NTS and expressed in 25% of Phox2b-expressing neurons. Additionally, electrophysiological recordings showed that exposure to orexin A increased the spontaneous firing rate of Phox2b-expressing neurons. Immunostaining experiments with cFos revealed that the OX1R-residing Phox2b-expressing neurons were activated by an 8% CO2 stimulus. Crucially, OX1R knockdown in these NTS neurons notably blunted the ventilatory response to 8% CO2, alongside an increase in sigh-related apneas. In conclusion, orexinergic signaling in the NTS facilitates breathing through the activation of OX1Rs, which induces the depolarization of Phox2b-expressing neurons. OX1Rs are essential for the involvement of Phox2b-expressing NTS neurons in the hypercapnic ventilatory response.

Structural characterization of PHOX2B and its DNA interaction shed light on the molecular basis of the +7Ala variant pathogenicity in CCHS.

An expansion of poly-alanine up to +13 residues in the C-terminus of the transcription factor PHOX2B underlies the onset of congenital central hypoventilation syndrome (CCHS). Recent studies demonstrated that the alanine tract expansion influences PHOX2B folding and activity. Therefore, structural information on PHOX2B is an important target for obtaining clues to elucidate the insurgence of the alanine expansion-related syndrome and also for defining a viable therapy. Here we report by NMR spectroscopy the structural characterization of the homeodomain (HD) of PHOX2B and HD + C-terminus PHOX2B protein, free and in the presence of the target DNA. The obtained structural data are then exploited to obtain a structural model of the PHOX2B-DNA interaction. In addition, the variant +7Ala, responsible for one of the most frequent forms of the syndrome, was analysed, showing different conformational proprieties in solution and a strong propensity to aggregation. Our data suggest that the elongated poly-alanine tract would be related to disease onset through a loss-of-function mechanism. Overall, this study paves the way for the future rational design of therapeutic drugs, suggesting as a possible therapeutic route the use of specific anti-aggregating molecules capable of preventing variant aggregation and possibly restoring the DNA-binding activity of PHOX2B.

Phox2b-expressing neurons contribute to breathing problems in Kcnq2 loss- and gain-of-function encephalopathy models

Loss- and gain-of-function variants in the gene encoding KCNQ2 channels are a common cause of developmental and epileptic encephalopathy, a condition characterized by seizures, developmental delays, breathing problems, and early mortality. To understand how KCNQ2 dysfunction impacts behavior in a mouse model, we focus on the control of breathing by neurons expressing the transcription factor Phox2b which includes respiratory neurons in the ventral parafacial region. We find Phox2b-expressing ventral parafacial neurons express Kcnq2 in the absence of other Kcnq isoforms, thus clarifying why disruption of Kcnq2 but not other channel isoforms results in breathing problems. We also find that Kcnq2 deletion or expression of a recurrent gain-of-function variant R201C in Phox2b-expressing neurons increases baseline breathing or decreases the central chemoreflex, respectively, in mice during the light/inactive state. These results uncover mechanisms underlying breathing abnormalities in KCNQ2 encephalopathy and highlight an unappreciated vulnerability of Phox2b-expressing ventral parafacial neurons to KCNQ2 pathogenic variants.

Phox2b mutation mediated by Atoh1 expression impaired respiratory rhythm and ventilatory responses to hypoxia and hypercapnia.

Mutations in the transcription factor Phox2b cause congenital central hypoventilation syndrome (CCHS). The syndrome is characterized by hypoventilation and inability to regulate breathing to maintain adequate O2 and CO2 levels. The mechanism by which CCHS impact respiratory control is incompletely understood, and even less is known about the impact of the non-polyalanine repeat expansion mutations (NPARM) form. Our goal was to investigate the extent by which NPARM Phox2b mutation affect (a) respiratory rhythm; (b) ventilatory responses to hypercapnia (HCVR) and hypoxia (HVR); and (c) number of chemosensitive neurons in mice. We used a transgenic mouse line carrying a conditional Phox2bΔ8 mutation (same found in humans with NPARM CCHS). We crossed them with Atoh1cre mice to introduce mutation in regions involved with respiratory function and central chemoreflex control. Ventilation was measured by plethysmograph during neonatal and adult life. In room air, mutation in neonates and adult did not greatly impact basal ventilation. However, Phox2bΔ8, Atoh1cre increased breath irregularity in adults. The HVR and HCVR were impaired in neonates. The HVR, but not HCVR, was still partially compromised in adults. The mutation reduced the number of Phox2b+/TH--expressing neurons as well as the number of fos-activated cells within the ventral parafacial region (also named retrotrapezoid nucleus [RTN] region) induced by hypercapnia. Our data indicates that Phox2bΔ8 mutation in Atoh1-expressing cells impaired RTN neurons, as well as chemoreflex under hypoxia and hypercapnia specially early in life. This study provided new evidence for mechanisms related to NPARM form of CCHS neuropathology.

Etonogestrel Administration Reduces the Expression of PHOX2B and Its Target Genes in the Solitary Tract Nucleus.

Heterozygous mutations of the transcription factor PHOX2B are responsible for Congenital Central Hypoventilation Syndrome, a neurological disorder characterized by inadequate respiratory response to hypercapnia and life-threatening hypoventilation during sleep. Although no cure is currently available, it was suggested that a potent progestin drug provides partial recovery of chemoreflex response. Previous in vitro data show a direct molecular link between progestins and PHOX2B expression. However, the mechanism through which these drugs ameliorate breathing in vivo remains unknown. Here, we investigated the effects of chronic administration of the potent progestin drug Etonogestrel (ETO) on respiratory function and transcriptional activity in adult female rats. We assessed respiratory function with whole-body plethysmography and measured genomic changes in brain regions important for respiratory control. Our results show that ETO reduced metabolic activity, leading to an enhanced chemoreflex response and concurrent increased breathing cycle variability at rest. Furthermore, ETO-treated brains showed reduced mRNA and protein expression of PHOX2B and its target genes selectively in the dorsal vagal complex, while other areas were unaffected. Histological analysis suggests that changes occurred in the solitary tract nucleus (NTS). Thus, we propose that the NTS, rich in both progesterone receptors and PHOX2B, is a good candidate for ETO-induced respiratory modulation.

Neural Stem Cell-Derived Exosomal Netrin1 Contributes to Neuron Differentiation of Mesenchymal Stem Cells in Therapy of Spinal Bifida Aperta.

Spinal bifida aperta (SBA) is a congenital malformation with a high incidence. Bone marrow mesenchymal stem cell (BMSC) transplantation has the potential to repair the structure of damaged tissues and restore their functions. This is an optional treatment that can be used as a supplement to surgery in the treatment of SBA. However, the application of BMSCs is limited, as the neuronal differentiation rate of BMSCs is not satisfactory when used in treating severe SBA. Thus, we aimed to assess the effect of neural stem cell (NSC)-derived exosomes on BMSC neuronal differentiation and observe the therapeutic effect in an ex vivo rat SBA embryo model. We found that NSC-derived exosomes increased the neuronal differentiation rate of BMSCs in vitro and in the SBA embryo model ex vivo. Proteomic analysis showed that NSC-derived exosomes were enriched in Netrin1, which positively regulated neuronal differentiation. Netrin1 increased the neuronal differentiation rate of BMSCs and NSCs and upregulated the expression of the neuronal markers, microtubule-associated protein (Map2), neurofilament, and β3-tubulin. Bioinformatic analysis revealed that Netrin1 treatment increased the expression of the transcription factors Hand2 and Phox2b, related to neuronal differentiation. Furthermore, the Netrin1-induced NSC neuronal differentiation was significantly blocked by Phox2b knockdown. We suggest that NSC-derived exosomal Netrin1 induces neuronal differentiation via the Hand2/Phox2b axis by upregulating the expression of Hand2 and Phox2b. Therefore, NSC-derived exosomes are a critical inducer of BMSC neuronal differentiation and represent a potential treatment agent that can benefit BMSC treatment in SBA.

Chapter 2 - Central respiratory chemoreception

Brain PCO2 is sensed primarily via changes in [H+]. Small pH changes are detected in the medulla oblongata and trigger breathing adjustments that help maintain arterial PCO2 constant. Larger perturbations of brain CO2/H+, possibly also sensed elsewhere in the CNS, elicit arousal, dyspnea, and stress, and cause additional breathing modifications. The retrotrapezoid nucleus (RTN), a rostral medullary cluster of glutamatergic neurons identified by coexpression of Phoxb and Nmb transcripts, is the lynchpin of the central respiratory chemoreflex. RTN regulates breathing frequency, inspiratory amplitude, and active expiration. It is exquisitely responsive to acidosis in vivo and maintains breathing autorhythmicity during quiet waking, slow-wave sleep, and anesthesia. The RTN response to [H+] is partly an intrinsic neuronal property mediated by proton sensors TASK-2 and GPR4 and partly a paracrine effect mediated by astrocytes and the vasculature. The RTN also receives myriad excitatory or inhibitory synaptic inputs including from [H+]-responsive neurons (e.g., serotonergic). RTN is silenced by moderate hypoxia. RTN inactivity (periodic or sustained) contributes to periodic breathing and, likely, to central sleep apnea. RTN development relies on transcription factors Egr2, Phox2b, Lbx1, and Atoh1. PHOX2B mutations cause congenital central hypoventilation syndrome; they impair RTN development and consequently the central respiratory chemoreflex.

A Focus on Regulatory Networks Linking MicroRNAs, Transcription Factors and Target Genes in Neuroblastoma.

Simple SummaryNeuroblastoma is a tumor of the sympathetic nervous system that substantially contributes to childhood cancer mortality. Neuroblastoma originates from the neural crest cells that are subjected to genetic alterations during embryonic development. These impairments hit key genes, whose expression is activated/repressed by transcription factors and inhibited by negative regulators, named microRNAs, thereby promoting tumorigenesis. Here, we have focused on the interactions between the transcription factors MYCN and PHOX2B with their target genes ALK and LIN28B and the miRNAs let-7, miR-34 and miR-204, which should act as negative regulators of their expression. In neuroblastoma, the physiologic regulatory networks among these genes and microRNAs are disrupted, leading to a complex and aberrant oncogene expression that underlies the development of the tumor. We also looked into the role of these genetic determinants of neuroblastoma starting from their physiological role in neural crest development and ending with their pathogenic dysregulation that leads to neuroblastoma oncogenesis.Neuroblastoma (NB) is a tumor of the peripheral sympathetic nervous system that substantially contributes to childhood cancer mortality. NB originates from neural crest cells (NCCs) undergoing a defective sympathetic neuronal differentiation and although the starting events leading to the development of NB remain to be fully elucidated, the master role of genetic alterations in key oncogenes has been ascertained: (1) amplification and/or over-expression of MYCN, which is strongly associated with tumor progression and invasion; (2) activating mutations, amplification and/or over-expression of ALK, which is involved in tumor initiation, angiogenesis and invasion; (3) amplification and/or over-expression of LIN28B, promoting proliferation and suppression of neuroblast differentiation; (4) mutations and/or over-expression of PHOX2B, which is involved in the regulation of NB differentiation, stemness maintenance, migration and metastasis. Moreover, altered microRNA (miRNA) expression takes part in generating pathogenetic networks, in which the regulatory loops among transcription factors, miRNAs and target genes lead to complex and aberrant oncogene expression that underlies the development of a tumor. In this review, we have focused on the circuitry linking the oncogenic transcription factors MYCN and PHOX2B with their transcriptional targets ALK and LIN28B and the tumor suppressor microRNAs let-7, miR-34 and miR-204, which should act as down-regulators of their expression. We have also looked at the physiologic role of these genetic and epigenetic determinants in NC development, as well as in terminal differentiation, with their pathogenic dysregulation leading to NB oncogenesis.

Pituitary hormones are specifically expressed in trigeminal sensory neurons and contribute to pain responses in the trigeminal system

Trigeminal (TG), dorsal root (DRG), and nodose/jugular (NG/JG) ganglia each possess specialized and distinct functions. We used RNA sequencing of two-cycle sorted Pirt-positive neurons to identify genes exclusively expressing in L3–L5 DRG, T10-L1 DRG, NG/JG, and TG mouse ganglion neurons. Transcription factor Phox2b and Efcab6 are specifically expressed in NG/JG while Hoxa7 is exclusively present in both T10-L1 and L3–L5 DRG neurons. Cyp2f2, Krt18, and Ptgds, along with pituitary hormone prolactin (Prl), growth hormone (Gh), and proopiomelanocortin (Pomc) encoding genes are almost exclusively in TG neurons. Immunohistochemistry confirmed selective expression of these hormones in TG neurons and dural nerves; and showed GH expression in subsets of TRPV1+ and CGRP+ TG neurons. We next examined GH roles in hypersensitivity in the spinal versus trigeminal systems. Exogenous GH produced mechanical hypersensitivity when injected intrathecally, but not intraplantarly. GH-induced thermal hypersensitivity was not detected in the spinal system. GH dose-dependently generated orofacial and headache-like periorbital mechanical hypersensitivity after administration into masseter muscle and dura, respectively. Periorbital mechanical hypersensitivity was reversed by a GH receptor antagonist, pegvisomant. Overall, pituitary hormone genes are selective for TG versus other ganglia somatotypes; and GH has distinctive functional significance in the trigeminal versus spinal systems.

Neonatal apneic phenotype in a murine congenital central hypoventilation syndrome model is induced through non-cell autonomous developmental mechanisms.

Congenital central hypoventilation syndrome (CCHS) represents a rare genetic disorder usually caused by mutations in the homeodomain transcription factor PHOX2B. Some CCHS patients suffer mainly from deficiencies in CO2 and/or O2 respiratory chemoreflex, whereas other patients present with full apnea shortly after birth. Our goal was to identify the neuropathological mechanisms of apneic presentations in CCHS. In the developing murine neuroepithelium, Phox2b is expressed in three discrete progenitor domains across the dorsal‐ventral axis, with different domains responsible for producing unique autonomic or visceral motor neurons. Restricting the expression of mutant Phox2b to the ventral visceral motor neuron domain induces marked newborn apnea together with a significant loss of visceral motor neurons, RTN ablation, and preBötzinger complex dysfunction. This finding suggests that the observed apnea develops through non‐cell autonomous developmental mechanisms. Mutant Phox2b expression in dorsal rhombencephalic neurons did not generate significant respiratory dysfunction, but did result in subtle metabolic thermoregulatory deficiencies. We confirm the expression of a novel murine Phox2b splice variant which shares exons 1 and 2 with the more widely studied Phox2b splice variant, but which differs in exon 3 where most CCHS mutations occur. We also show that mutant Phox2b expression in the visceral motor neuron progenitor domain increases cell proliferation at the expense of visceral motor neuron development. We propose that visceral motor neurons may function as organizers of brainstem respiratory neuron development, and that disruptions in their development result in secondary/non‐cell autonomous maldevelopment of key brainstem respiratory neurons.

ASCL1 is a MYCN- and LMO1-dependent member of the adrenergic neuroblastoma core regulatory circuitry

A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1—all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC.

Conversion of Astrocytes and Fibroblasts into Functional Noradrenergic Neurons

Dysfunction of noradrenergic (NA) neurons is associated with a number of neuronal disorders. Diverse neuronal subtypes can be generated by direct reprogramming. However, it is still unknown how to convert non-neuronal cells into NA neurons. Here, we show that seven transcription factors (TFs) (Ascl1, Phox2b, AP-2α, Gata3, Hand2, Nurr1, and Phox2a) are able to convert astrocytes and fibroblasts into induced NA (iNA) neurons. These iNA neurons express the genes required for the biosynthesis, release, and re-uptake of noradrenaline. Moreover, iNA neurons fire action potentials, receive synaptic inputs, and control the beating rate of co-cultured ventricular myocytes. Furthermore, iNA neurons survive and integrate into neural circuits after transplantation. Last, human fibroblasts can be converted into functional iNA neurons as well. Together, iNA neurons are generated by direct reprogramming, and they could be potentially useful for disease modeling and cell-based therapies.

Molecular insights into the role of the polyalanine region in mediating PHOX2B aggregation.

About 90% of congenital central hypoventilation syndrome (CCHS) patients show polyalanine triplet expansions in the coding region of transcription factor PHOX2B, which renders this protein an intriguing target to understand the insurgence of this syndrome and for the design of a novel therapeutical approach. Consistently with the role of PHOX2B as a transcriptional regulator, it is reasonable that a general transcriptional dysregulation caused by the polyalanine expansion might represent an important mechanism underlying CCHS pathogenesis. Therefore, this study focused on the biochemical characterization of different PHOX2B variants, such as a variant containing the correct C-terminal (20 alanines) stretch, one of the most frequent polyalanine expansions (+7 alanines), and a variant lacking the complete alanine stretch (0 alanines). Comparison of the different variants by a multidisciplinary approach based on different methodologies (including circular dichroism, spectrofluorimetry, light scattering, and Atomic Force Microscopy studies) highlighted the propensity to aggregate for the PHOX2B variant containing the polyalanine expansion (+7-alanines), especially in the presence of DNA, while the 0-alanines variant resembled the protein with the correct polyalanine length. Moreover, and unexpectedly, the formation of fibrils was revealed only for the pathological variant, suggesting a plausible role of such fibrils in the insurgence of CCHS.

Ablation of PHOX2B‐Derived Astrocytes Results in Neuronal Dystrophy‐Like Neuropathology in the RTN

The transcription factor Phox2b is a regulator of the autonomic nervous system development and is especially expressed by respiratory circuits involved in chemosensory integration, as neurons of solitary tract nucleus (NTS) that relay information from peripheral chemoreceptors to the ventral respiratory column, and by central chemoreceptors located in the retrotrapezoid nucleus (RTN). In addition to neurons, recent studies show that astrocytes from ventral medullary surface also detect PCO2 increase, and contribute to respiratory drive. Since PHOX2B biased progenitor cells generate astrocytes that have a developmental connection with RTN neurons, the aim of this study is determine the extent to which selective ablation of these astrocytes affect synapse integrity in the retrotrapezoid nucleus. We utilized the astrocyte ablation technique described by Tsai et al., 2012. In this model, the astrocyte‐specific ALDH1L1 promoter induces GFP expression in the absence of cre recombinase, and in the presence of cre recombinase expresses diphtheria toxin A (DTA). We interbred ALDH1L1loxp‐GFP‐STOP‐loxp‐DTA mice to PHOX2Bcre mice. Transmission electron photomicrographs were captured from the RTN/VLM area and morphometric analysis was performed using FIJI. We noted that axon terminals in the mutants (PHOX2Bcre, ALDH1L1loxp‐GFP‐STOP‐loxp‐DTA) were on average larger in area and perimeter. Furthermore, we noted an increase in the percentage of axon terminals showing autophagic vacuoles/phagosomes. The proportion of axon terminals with other organelles, including mitochondria and dense core neurosecretory vesicles, was not different between groups. In summary, PHOX2B‐derived, astrocyte ablated mice show synaptic pathology characterized by dysmorphic neuroaxonal morphology, suggesting an underlying defect in the afferent input into the RTN. These data suggest that PHOX2B‐derived astrocytes may play a role in regulating the synaptic integrity of neuronal inputs into RTN neurons. Other experiments are being performed in order to elucidate if the cell body of these neurons is located in the commissural NTS, and also, if PHOX2b derived astrocytes depletion can imply local regulation of presynaptic autophagy, which can lead to neurodegeneration and breathing impair.Support or Funding InformationFAPESP and NIHThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Altered enteric expression of the homeobox transcription factor Phox2b in patients with diverticular disease.

Diverticular disease, a major gastrointestinal disorder, is associated with modifications of the enteric nervous system, encompassing alterations of neurochemical coding and of the tyrosine receptor kinase Ret/GDNF pathway. However, molecular factors underlying these changes remain to be determined. We aimed to characterise the expression of Phox2b, an essential regulator of Ret and of neuronal subtype development, in the adult human enteric nervous system, and to evaluate its potential involvement in acute diverticulitis. Site-specific gene expression of Phox2b in the adult colon was analysed by quantitative polymerase chain reaction. Colonic specimens of adult controls and patients with diverticulitis were subjected to quantitative polymerase chain reaction for Phox2b and dual-label immunochemistry for Phox2b and the neuronal markers RET and tyrosine hydroxylase or the glial marker S100β. The results indicate that Phox2b is physiologically expressed in myenteric neuronal and glial subpopulations in the adult enteric nervous system. Messenger RNA expression of Phox2b was increased in patients with diverticulitis and both neuronal, and glial protein expression of Phox2b were altered in these patients. Alterations of Phox2b expression may contribute to the enteric neuropathy observed in diverticular disease. Future studies are required to characterise the functions of Phox2b in the adult enteric nervous system and to determine its potential as a therapeutic target in gastrointestinal disorders.

Dissecting the Contribution of Specific Hindbrain Cell Populations in the Phenotypic Characterization of a Mouse Model of Central Congenital Hypoventilation Syndrome

Hindbrain respiratory neuron networks undergo critical developmental maturation during the late embryonic, perinatal and post‐natal periods in mammals. Thus, abnormalities in neuron network development may contribute to autonomic respiratory dysfunction. Central Congenital Hypoventilation Syndrome (CCHS) is a rare genetic disorder characterized by an inability to sense and respond to altered levels of CO2 or O2 (Chemosensation). CCHS patients present with a spectrum of central respiratory control pathophysiologies, ranging from deficiency in chemosensation, to most severe manifestations of neonatal apnea that requires life‐sustaining mechanical ventilation. Heterozygous Mutations of the transcription factor PHOX2B, classified as either poly‐alanine repeat (PARM) and non‐poly‐alanine repeat (NPARM), have been linked to the development of CCHS. In order to explore CCHS pathophysiology, a novel transgenic mouse expressing an inducible NPARM Phox2b mutation (Phox2bΔ8) was developed and characterized by Nobuta and collaborators (Nabuta et al. 2015). Following this study and aiming to reveal the contribution of specific hindbrain populations to CCHS, we have generated two additional mouse models in which of Phox2bΔ8 expression is restricted to cell populations derived from either NKX2.2+ or OLIG3+ progenitors. Expression of Phox2bΔ8 on NKX2.2+ derived cells resulted in a strong apneic phenotype on newborn pups. Detail examination of this mouse model hindbrains showed deficiencies in a spectrum of rhombomeric nuclei not developmentally derived from NKX2.2+‐expressing progenitors, indicating that the developmental neuropathologies identified result through non‐cell‐autonomous mechanisms. Additionally, we find that these pathologies result from early rhombomeric patterning defects in embryonic ventral neural progenitor zones, which showed reduced endogenous PHOX2B expression. On the other hand, preliminary results indicate that expression of Phox2bΔ8 on OLIG3+ derived cells result in a subtle deficit in chemosensation, but we have been unable to find any neuropathological change in the hindbrain. Together, our results help to dissect the contributions of different cell populations in the hindbrain and to further clarify the different manifestations and severity of chemosensation and respiratory deficits seen in human CCHS patientsSupport or Funding InformationNIH R01HL132355This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Analysis of the neuronal network of the medullary respiratory center in transgenic rats expressing archaerhodopsin-3 in Phox2b-expressing cells

Preinspiratory (Pre-I) neurons in the parafacial respiratory group (pFRG) comprise one of the respiratory rhythm generators in the medulla of the neonatal rat. A subgroup of pFRG/Pre-I neurons expresses the transcription factor Phox2b. To further analyze detailed neuronal mechanisms of respiratory rhythm generation in the neonatal rat, we developed a transgenic (Tg) rat line in which Phox2b-positive cells expressed archaerhodopsin-3 (Arch). Brainstem-spinal cord preparations were isolated from 0–2-day-old Tg newborn rats and were superfused with artificial cerebrospinal fluid equilibrated with 95% O2 and 5% CO2, pH 7.4, at 25–26 °C. Inspiratory fourth cervical ventral root (C4) activity was monitored, and membrane potentials of neurons in the pFRG including Pre-I and inspiratory neurons were recorded. Phox2b-positive cells in the Tg rats were essentially positive for enhanced green fluorescent protein (EGFP) signals (reporter for Arch) in the pFRG. Continuous photo-stimulation of the rostral ventral medulla for up to 90 s by covering the pFRG with green laser light (532 nm) induced a decrease of respiratory rate measured at C4 accompanied by membrane hyperpolarization of Phox2b-positive pFRG/Pre-I neurons. In contrast, Phox2b-negative inspiratory neurons were not hyperpolarized during the photo-stimulation. Our findings showed that Phox2b-expressing pFRG/Pre-I neurons are involved in the maintenance of the basic respiratory rhythm in neonatal rat.

Striking parallels between carotid body glomus cell and adrenal chromaffin cell development

Carotid body glomus cells mediate essential reflex responses to arterial blood hypoxia. They are dopaminergic and secrete growth factors that support dopaminergic neurons, making the carotid body a potential source of patient-specific cells for Parkinson's disease therapy. Like adrenal chromaffin cells, which are also hypoxia-sensitive, glomus cells are neural crest-derived and require the transcription factors Ascl1 and Phox2b; otherwise, their development is little understood at the molecular level. Here, analysis in chicken and mouse reveals further striking molecular parallels, though also some differences, between glomus and adrenal chromaffin cell development. Moreover, histology has long suggested that glomus cell precursors are ‘émigrés’ from neighbouring ganglia/nerves, while multipotent nerve-associated glial cells are now known to make a significant contribution to the adrenal chromaffin cell population in the mouse. We present conditional genetic lineage-tracing data from mice supporting the hypothesis that progenitors expressing the glial marker proteolipid protein 1, presumably located in adjacent ganglia/nerves, also contribute to glomus cells. Finally, we resolve a paradox for the ‘émigré’ hypothesis in the chicken - where the nearest ganglion to the carotid body is the nodose, in which the satellite glia are neural crest-derived, but the neurons are almost entirely placode-derived - by fate-mapping putative nodose neuronal 'émigrés' to the neural crest.