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Several X-linked genes are involved in neuronal differentiation and may contribute to the generation of sex dimorphisms in the brain. Previous results showed that XX hypothalamic neurons grow faster, have longer axons, and exhibit higher expression of the neuritogenic gene neurogenin 3 (Ngn3) than XY before perinatal masculinization. Here we evaluated the participation of candidate X-linked genes in the development of these sex differences, focusing mainly on Kdm6a, a gene encoding for an H3K27 demethylase with functions controlling gene expression genome-wide. We established hypothalamic neuronal cultures from wild-type or transgenic Four Core Genotypes mice, a model that allows evaluating the effect of sex chromosomes independently of gonadal type. X-linked genes Kdm6a, Eif2s3x and Ddx3x showed higher expression in XX compared to XY neurons, regardless of gonadal sex. Moreover, Kdm6a expression pattern with higher mRNA levels in XX than XY did not change with age at E14, P0, and P60 in hypothalamus or under 17ß-estradiol treatment in culture. Kdm6a pharmacological blockade by GSK-J4 reduced axonal length only in female neurons and decreased the expression of neuritogenic genes Neurod1, Neurod2 and Cdk5r1 in both sexes equally, while a sex-specific effect was observed in Ngn3. Finally, Kdm6a downregulation using siRNA reduced axonal length and Ngn3 expression only in female neurons, abolishing the sex differences observed in control conditions. Altogether, these results point to Kdm6a as a key mediator of the higher axogenesis and Ngn3 expression observed in XX neurons before the critical period of brain masculinization., This study was supported by grants from Argentina: Consejo Nacional de Investigaciones Científcas y Técnicas (CONICET, PUE 2016 No. 22920160100135CO), Agencia Nacional de Promoción Científca y Tecnológica (ANPCyT, PICT 2015 No. 1333 and PICT 2019 No. 2176), and Secretaría de Ciencia y Tecnología de la Universidad Nacional de Córdoba (SECyT-UNC, 2018–2021) to MJC, from Spain: Agencia Estatal de Investigacion (AEI) co-funded by FEDER (BFU2017-82754-R and PID2020-115019RBI00) to MAA and LMGS and the Enhancing Mobility between Latin America, Caribbean and the European Union in Health & Environment (EMHE)-CSIC Program (MHE-200057) to LMGS and MJC, and from international organizations: International Brain Research Organization (IBRO) Return Home Fellowship and International Society for Neurochemistry (ISN) and Committee for Aid and Education in Neurochemistry (CAEN) Grant to CDC.

BackgroundThe assembly of neural circuits requires the concerted action of both genetically determined and activity-dependent mechanisms. Calcium-regulated transcription may link these processes, but the influence of specific transcription factors on the differentiation of synapse-specific properties is poorly understood. Here we characterize the influence of NeuroD2, a calcium-dependent transcription factor, in regulating the structural and functional maturation of the hippocampal mossy fiber (MF) synapse.ResultsUsing NeuroD2 null mice andin vivolentivirus-mediated gene knockdown, we demonstrate a critical role for NeuroD2 in the formation of CA3 dendritic spines receiving MF inputs. We also use electrophysiological recordings from CA3 neurons while stimulating MF axons to show that NeuroD2 regulates the differentiation of functional properties at the MF synapse. Finally, we find that NeuroD2 regulates PSD95 expression in hippocampal neurons and that PSD95 loss of functionin vivoreproduces CA3 neuron spine defects observed in NeuroD2 null mice.ConclusionThese experiments identify NeuroD2 as a key transcription factor that regulates the structural and functional differentiation of MF synapsesin vivo.