Fig 2: Increase of intestinal Eomes+ T cells in the first year of life. a NK-T cell ratios of infant (white circles) and adult intestines (dark circles, A) in epithelium (EP, yellow circles) and lamina propria (LP, blue squares) (infant EP samples (N = 9) and LP samples (N = 10), adult EP and LP samples (N = 11). b Frequencies of epithelium and lamina propria-derived Eomes+ NK (turquoise circles) and Eomes+ T cells (violet triangles) within the total lymphocyte pool at different ages (infant EP and LP samples (N = 7) and adult EP and LP samples (N = 10)). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons. Asterisks represent the following p-values: **p < 0.01; ***p < 0.001; and ****p < 0.0001

Fig 3: High expression of Eomes in infant intestinal NK cells. a viSNE plots of combined flow cytometric data visualizing Eomes expression of infant and adult NK cells from epithelium (EP) and lamina propria (LP) intestinal tissues. Cell density of clusters is shown in first row. Expression of Eomes is shown by color coding in relative intensity in second row. viSNE plots have been calculated from concatenated FCS files gated on NK cells (infant samples N = 7, adult samples N = 5, iterations = 7500 perplexity = 100, KL divergence = 2.62). b Frequencies of EP and LP-derived infant (white circles) and adult (dark circles) Eomes+ NK cells. c Representative flow cytometric plots showing co-expression of Eomes and tissue-residency markers (CD103, CD49a, CD69) in NK cells from EP and LP of infant and adult intestines. d Frequencies of Eomes+ NK cells within CD103+, CD49a+, or CD69+ NK cell populations in EP and LP of infants (white circles) and adults (dark circles) (infant samples N = 8 (EP and LP), adult samples N = 8 (EP), and N = 10 (LP)). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons (b) and Wilcoxon matched-pairs signed rank tests (d). Asterisks represent the following p-values: *p < 0.05; **p < 0.01; and ***p < 0.001

Fig 4: Epithelial CD103+NKp44+lin- cells are the major ILC population in adults. a Frequencies as well as absolute cell numbers per cm2 of epithelial (EP) and lamina propria-derived (LP) NKp44+ NK cells in infant (white circles) and adult (dark circles) intestines (infant NK cell frequencies and absolute counts EP (N = 7) and LP (N = 5), adult NK cell frequencies (N = 9) and absolute counts (N = 8)). b Frequencies of NKp44+ cells within CD103+, CD49a+, or CD69+ NK cells in infants (white circles) and adults (dark circles). NKp44 expression by infant epithelial CD103+ (N = 7), CD49a+ (N = 7), and CD69+ (N = 6) NK cells. NKp44 expression by adult epithelial NK cell subsets (N = 9). Heatmap of median frequencies of NKG2A+, Eomes+, perforin+, granzyme B+, and NKp44+ cells within CD103+, CD49a+, or CD69+ NK cells as well as CD127+ ILCs in infant intestines. c SPADE tree of viable EP CD45+lin- lymphocytes. CD127+ ILCs (yellow outline), NK cells (red outline), including CD127-NKp44+CD103+lin- cells (blue outline) within NK cell population. Expression of NKp44 is shown by color coding in relative intensity. Node sizes represent the size of populations. SPADE analysis was computed by using the same signature parameters as in Fig. 1 (infant samples N = 4, adult samples N = 5, target number of nodes: 200, down sampled events target: 30%). Median frequencies indicated by red lines. Error bars define interquartile ranges between 75th and 25th percentiles. Statistical comparisons are Mann-Whitney U comparisons (a) and Wilcoxon matched-pairs signed rank tests (b). Asterisks represent the following p-values: *p < 0.05 and ***p < 0.001

Fig 5: Loss of FOXP1 results in decreased number of bRGCs.(A) Schematic showing the design of the “Ad-EGFP” virus infection experiment. (B) Representative images of immunostaining performed on the “Ad-EGFP”-infected organoids at week 6 showing detection of bRGCs by morphology including loss of ventricular contact. (C) Quantification of progenitors that are without apical attachment and are TBR2-. (D) Schematic showing the design of the BrdU assays. Incubation with 100 µM BrdU for 2 hours was followed by media change, 24-hour incubation, and harvesting the organoids for fixation. (E) Representative images of immunostaining performed on BrdU-treated organoids at week 6. (F) Schematic diagrams showing bRGC divisions. (G) Magnified images showing overlap between different markers. (H) Quantification of HOPX+ dividing bRGCs. (I) Quantification of HOPX+ basal progenitors going through neurogenesis via IPCs. For all quantifications, n = 3–8 organoids per sample per genotype were used. In each organoid, 3–8 cortical structures with clear lamination patterns were examined. Data are represented in bar graphs as mean ± STD with individual data as dots; n.s. means p > 0.05, *p < 0.05, p** < 0.01, and ***p <0.001, Kruskal–Wallis ANOVA test with Dunn’s multiple comparisons test as a post hoc was used. Scale bar = 100 µM. The numerical values that were used to generate the graphs can be found in S1 Data. Cartoon images were created with BioRender.com. Ad-EGFP, adenovirus-expressing GFP; BrdU, 5-bromo-2-deoxyuridine; bRGC, basal radial glial cell; FOXP1, Forkhead Box P1; IPC, intermediate progenitor cell; KO, knockout; WT, wild type.