The clustered gamma protocadherin PcdhγC4 isoform regulates cortical interneuron programmed cell death in the mouse cortex.

Cortical inhibitory interneurons (cINs) are born in the ventral forebrain and migrate into the cortex where they make connections with locally produced excitatory glutamatergic neurons. Cortical function critically depends on the number of cINs, which is also key to establishing the appropriate inhibitory/excitatory balance. The final number of cINs is determined during a postnatal period of programmed cell death (PCD) when ~40% of the young cINs are eliminated. Previous work shows that the loss of clustered gamma protocadherins (Pcdhgs), but not of genes in the Pcdha or Pcdhb clusters, dramatically increased BAX-dependent cIN PCD. Here, we show that PcdhγC4 is highly expressed in cINs of the mouse cortex and that this expression increases during PCD. The sole deletion of the PcdhγC4 isoform, but not of the other 21 isoforms in the Pcdhg gene cluster, increased cIN PCD. Viral expression of the PcdhγC4, in cIN lacking the function of the entire Pcdhg cluster, rescued most of these cells from cell death. We conclude that PcdhγC4 plays a critical role in regulating the survival of cINs during their normal period of PCD. This highlights how a single isoform of the Pcdhg cluster, which has been linked to human neurodevelopmental disorders, is essential to adjust cIN cell numbers during cortical development.

Loss of Ezh2 in the medial ganglionic eminence alters interneuron fate, cell morphology and gene expression profiles.

Introduction: Enhancer of zeste homolog 2 (Ezh2) is responsible for trimethylation of histone 3 at lysine 27 (H3K27me3), resulting in repression of gene expression. Here, we explore the role of Ezh2 in forebrain GABAergic interneuron development. Methods: We removed Ezh2 in the MGE by generating Nkx2-1Cre;Ezh2 conditional knockout mice. We then characterized changes in MGE-derived interneuron fate and electrophysiological properties in juvenile mice, as well as alterations in gene expression, chromatin accessibility and histone modifications in the MGE. Results: Loss of Ezh2 increases somatostatin-expressing (SST+) and decreases parvalbumin-expressing (PV+) interneurons in the forebrain. We observe fewer MGE-derived interneurons in the first postnatal week, indicating reduced interneuron production. Intrinsic electrophysiological properties in SST+ and PV+ interneurons are normal, but PV+ interneurons display increased axonal complexity in Ezh2 mutant mice. Single nuclei multiome analysis revealed differential gene expression patterns in the embryonic MGE that are predictive of these cell fate changes. Lastly, CUT&Tag analysis revealed that some genomic loci are particularly resistant or susceptible to shifts in H3K27me3 levels in the absence of Ezh2, indicating differential selectivity to epigenetic perturbation. Discussion: Thus, loss of Ezh2 in the MGE alters interneuron fate, morphology, and gene expression and regulation. These findings have important implications for both normal development and potentially in disease etiologies.

Cultivation and identification of neural GABAergic precursor cells derived from medial ganglionic eminence of embryonic mice / 中华神经医学杂志

Objective To explore a method for isolation,cultivation and identification of GABAergic precursor cells derived from medial ganglionic eminence (MGE-NPCs) of embryonic mice.Methods The MGE brain tissues of pregnant mice of 14.5 d were isolated under stereomicroscope;and the cells from these tissues were cultured to third passage or above in serum-free medium with SHH signal path stimulator.(1) Fluorescence immunocytochemistry was used to detect the expressions of neural stem cells (NSCs) markers nestin,sex determining region Y-box protein 2 (SOX2),MGE transcription factor NK2 homeobox 1 (NKX2-1) and intemeuron progenitor marker LIM homeobox 6 (LHX6) to identify the NSCs maintenance ability.(2) Proliferation potential of MGE-NPCs was detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay and bromodeoxyuridine (BrdU) labeling one and 3 d after culture in vitro.(3) The expressions of neuronal marker neuronal nuclear antigen (NeuN),and interneuron markers gamma-aminobutyric acid (GABA),glutamic acid decarboxylase(GAD67),and parvalbumin (PV) were examined by immunocytofluorescent 5 d after induced culture with GABAergic induced medium.(4) The differentiations of MGE-NPCs into GABA-,GAD67-and PV-positive cells in vivo one month after transplantation into the mice were detected by frozen section immunofluorescence staining.Results The neurospheres with self-renewal and proliferation capacity were obtained from the MGE of embryonic mice.Immunofluorescent staining showed that nestin,SOX2,Nkx2.1 and LHX6 positively expressed in the MGE-NPCs.The results of MTT assay revealed that the optical density (OD) one d after culture was significantly less than that 3 d after culture (0.392±0.032 vs.0.811±0.017,P＜0.05).BrdU labeling indicated that the ratio of proliferated MGE-NPCs one d after culture was significantly less than that 3 d after culture (45.086±7.122 vs.61.786±10.540,P＜0.05).The MGE-NPCs could differentiate into NeuN-,GABA-,GAD67-and PV-positive inhibitory interneurons 5 d after differentiation culture and one month after transplantion into mice.Conclusion The MGE-NPCs cultured in vitro still have NSCs characteristics of neuronal precursors and remain the capacity of differentiated intemeuron.