Fig 1: Validation of neighbor‐dependent gene expression predictions upon co‐culture of cells derived from mouse hippocampus Model depicting the experimental methodology applied to analyze the expression of the predicted neighbor‐dependent genes in the mouse hippocampus. Briefly, we isolated Astrocyte and EnT cells from mouse hippocampus through cell sorting with specific cell membrane markers for these cell types. Then, we labeled Astrocyte with GFP and performed monocultures of Astrocyte and EnT as well as co‐culture of both cell types for 48 h, followed by sorting of Astrocyte and EnT cells based on their GFP expression. The sorted cells and the monocultures were used to perform RT–qPCR to determine the expression of the predicted neighbor‐dependent genes Trf, Fabp7, and Dbi.FACS of Astrocyte and EnT cells from mouse hippocampus. The top‐left gate shows the selection of CD11b negative cells, which were used to further select those with a positive expression of ACSA2 (top‐right gate), which represent the Astrocyte population. Additionally, we selected VEGFR1 negative cells (bottom‐left gate) to sort those that were double positive for VEGFR2 and CD34 as our EnT cell population.Sorting of Astrocyte and EnT cells based on GFP expression upon Astrocyte GFP positive and EnT GFP negative co‐cultures.The analysis of qPCR‐based mRNA expression of Astrocyte and EnT cells coculture shows that, upon co‐culture, the upregulation of Trf is specific for EnT cells (red bars, P = 8.13E‐11), while the expression of Fabp7 (P = 1.08E‐07) and Dbi (P = 4.6E‐06) is specific for Astrocyte (yellow bars). The expression of these genes in total mouse hippocampus represents the control (green bars). The expression of the predicted neighbor‐dependent genes was normalized for the expression of these genes in total mouse hippocampus (N = 5; bars represent averaged fold changes of gene expression relative to the control; error bars indicate mean ± SE). The P‐values shown in the bar plot were obtained by performing the two‐tailed Student's t‐test. The expression of all genes follows a normal distribution, which was calculated by the Shapiro–Wilk test and the F‐test was performed to study the equality of variances. The P‐value shown in the bar plot for the expression of Trf was obtained by performing the two‐tailed Student's t‐test, whereas the P‐values obtained for Fabp7 and Dbi were calculated by the two‐tailed Welch's t‐test with a confidence interval of 95%. Source data are available online for this figure.