PHOX2B-PARMs perturb hindbrain patterning in human pluripotent stem cell-derived brainstem organoids of congenital central hypoventilation syndrome [Poster presentation]

Abstract

<p>Congenital central hypoventilation syndrome (CCHS) is caused by the loss of retrotrapezoid nucleus (RTN) neurons in the brainstem, leading to an impaired ventilatory response to hypercarbia. PHOX2B<em>-</em>polyalanine repeat mutations (PHOX2B<em>-</em>PARMs) are associated with CCHS and it often occurs with Hirschsprung disease (HSCR) which affects the enteric nervous system (ENS) of the colon. However, it is still unclear how PHOX2B-PARMs contribute to the pathogenesis of CCHS and HSCR and the underlying disease mechanisms. In this study, using human pluripotent stem cells (hPSCs), we generated brainstem (HBSO) and cerebral (HCO) organoids, which contain hypercapnia-responsive neurons and resemble the RTN-respiratory center. HCOs and HBSOs derived from PHOX2B-PARM mutant hPSCs lacked PHOX2B⁺VGLUT2⁺ RTN neurons, recapitulating the CCHS phenotypes. Single-cell transcriptomic analyses of the organoids further revealed that PHOX2B+7Ala PARM interrupts the progenitor-to-neuronal transition of <em>PHOX2B</em>-expressing neurons and also the differentiation of other hindbrain neurons. Dysregulations of Hedgehog pathway and <em>HOX</em> genes were observed in the neurons expressing <em>PHOX2B-7Ala</em>, suggesting that PHOX2B-PARMs disrupt the pattern specification process. A CCHS-HSCR hPSC line carrying a PHOX2B+7Ala PARM displayed severe defects in the production of PHOX2B⁺ glutamatergic CNS neurons and dopaminergic ENS neurons. The correction of PARM could rescue most of the CCHS-associated CNS phenotypes, but only some of the HSCR-associated ENS defects. This implies the clinical phenotypes associated with CCHS-HSCR are likely contributed by the sensitizing genetic background of the patients. In sum, using the hPSC-derived brain organoid models, we defined the molecular mechanisms underlying the detrimental effects of PHOX2B-PARMs in hindbrain development and demonstrated different vulnerability of the CNS and ENS neurons to the PHOX2B-PARMs.</p>