Prenatal FGFR2 Signaling via PI3K/AKT Specifies the PDGFRA+ Myofibroblast.

It is well known that FGFR2 (fibroblast growth factor receptor 2) signaling is critical for proper lung development. Recent studies demonstrate that epithelial FGFR2 signaling during the saccular phase of lung development (sacculation) regulates alveolar type 1 (AT1) and AT2 cell differentiation. During sacculation, PDGFRA (platelet-derived growth factor receptor-α)-positive lung fibroblasts exist as three functional subtypes: contractile myofibroblasts, extracellular matrix-producing matrix fibroblasts, and lipofibroblasts. All three subtypes are required during alveolarization to establish a niche that supports AT2 epithelial cell self-renewal and AT1 epithelial cell differentiation. FGFR2 signaling directs myofibroblast differentiation in PDGFRA+ fibroblasts during alveolar reseptation after pneumonectomy. However, it remains unknown if FGFR2 signaling regulates PDGFRA+ myo-, matrix, or lipofibroblast differentiation during sacculation. In this study, FGFR2 signaling was inhibited by temporal expression of a secreted dominant-negative FGFR2b (dnFGFR2) by AT2 cells from embryonic day (E) 16.5 to E18.5. Fibroblast and epithelial differentiation were analyzed at E18.5 and postnatal days 7 and 21. At all time points, the number of myofibroblasts was reduced and the number of lipo-/matrix fibroblasts was increased. AT2 cells are increased and AT1 cells are reduced postnatally, but not at E18.5. Similarly, in organoids made with PDGFRA+ fibroblasts from dnFGFR2 lungs, increased AT2 cells and reduced AT1 cells were observed. In vitro treatment of primary wild-type E16.5 adherent saccular lung fibroblasts with recombinant dnFGFR2b/c resulted in reduced myofibroblast contraction. Treatment with the PI3K/AKT activator 740 Y-P rescued the lack of myofibroblast differentiation caused by dnFGFR2b/2c. Moreover, treatment with the PI3K/AKT activator 740 Y-P rescued myofibroblast differentiation in E18.5 fibroblasts isolated from dnFGFR2 lungs.

Maladaptive functional changes in alveolar fibroblasts due to perinatal hyperoxia impair epithelial differentiation.

Infants born prematurely worldwide have up to a 50% chance of developing bronchopulmonary dysplasia (BPD), a clinical morbidity characterized by dysregulated lung alveolarization and microvascular development. It is known that PDGFR alpha-positive (PDGFRA+) fibroblasts are critical for alveolarization and that PDGFRA+ fibroblasts are reduced in BPD. A better understanding of fibroblast heterogeneity and functional activation status during pathogenesis is required to develop mesenchymal population-targeted therapies for BPD. In this study, we utilized a neonatal hyperoxia mouse model (90% O2 postnatal days 0-7, PN0-PN7) and performed studies on sorted PDGFRA+ cells during injury and room air recovery. After hyperoxia injury, PDGFRA+ matrix and myofibroblasts decreased and PDGFRA+ lipofibroblasts increased by transcriptional signature and population size. PDGFRA+ matrix and myofibroblasts recovered during repair (PN10). After 7 days of in vivo hyperoxia, PDGFRA+ sorted fibroblasts had reduced contractility in vitro, reflecting loss of myofibroblast commitment. Organoids made with PN7 PDGFRA+ fibroblasts from hyperoxia in mice exhibited reduced alveolar type 1 cell differentiation, suggesting reduced alveolar niche-supporting PDGFRA+ matrix fibroblast function. Pathway analysis predicted reduced WNT signaling in hyperoxia fibroblasts. In alveolar organoids from hyperoxia-exposed fibroblasts, WNT activation by CHIR increased the size and number of alveolar organoids and enhanced alveolar type 2 cell differentiation.

mTOR-driven neural circuit changes initiate an epileptogenic cascade.

Mutations in genes regulating mTOR pathway signaling are now recognized as a significant cause of epilepsy. Interestingly, these mTORopathies are often caused by somatic mutations, affecting variable numbers of neurons. To better understand how this variability affects disease phenotype, we developed a mouse model in which the mTOR pathway inhibitor Pten can be deleted from 0 to 40 % of hippocampal granule cells. In vivo, low numbers of knockout cells caused focal seizures, while higher numbers led to generalized seizures. Generalized seizures coincided with the loss of local circuit interneurons. In hippocampal slices, low knockout cell loads produced abrupt reductions in population spike threshold, while spontaneous excitatory postsynaptic currents and circuit level recurrent activity increased gradually with rising knockout cell load. Findings demonstrate that knockout cells load is a critical variable regulating disease phenotype, progressing from subclinical circuit abnormalities to electrobehavioral seizures with secondary involvement of downstream neuronal populations.