THE 7 STAGES OF NEURONAL DEVELOPMENT IN VITRO: MORPHOLOGICAL AND MOLECULAR SIGNATURES FROM BIRTH TO SENESCENCE

Neuronal development is a complex, multistep biological process that can be divided into distinct stages. The first in vitro description of rat hippocampal neuron (HPN) development was provided by Dotti (1988), who identified five stages between days in vitro (DIV) 0 and 7. Subsequently, Horton (2006) described an additional phase at DIV7–10. Subsequently, we proposed (Baj 2014) a model for mouse HPNs development, including a new sixth stage at DIV12–15. Although the cellular processes underlying these stages are well characterized, reliable biomarkers defining transitions between developmental stages are still lacking. In this study, we identify novel stage-specific biomarkers of each stage of neuronal development. We performed a bioinformatic analysis of the public RNA expression dataset GSE113680, reporting differentially expressed genes (DEGs) in cultured mouse HPNs at DIV0, 1, 3, 6, 12, and 15. We identified three DEGs with a unique expression pattern at stages 1–2, 4 specific to stage 3, one DEG for stage 4, and five DEGs associated with stage 6. The most promising candidate genes were validated by qPCR. Additionally, Beta-galactosidase assays and immunofluorescence analysis for gammaH2AX, a marker of DNA double-strand breaks, revealed a significant increase in senescence-associated signals at DIV18 and DIV21. Consistently, accumulation of the transcription factor GATA4, a key regulator of senescence-associated secretory phenotype (SASP) genes, increased from DIV18 onward. Based on these findings, we propose a new staging system of hippocampal neuron development including a novel stage 7 (DIV18–21) corresponding to neuronal senescence.