Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a) Schematic illustration of a coronally sectioned embryonic brain hemisphere, depicting the lateral and dorsal localization of the two bin selections, used to quantify the density and distribution of EOMES + – and TBR1 + -cells in E14.5 and E16.5 brain sections. (b-e) TBR1 and EOMES immunostaining in coronally sectioned (50 µm) brains of E14.5 Sst-Cre/tdTomato (control) and Sst-Cre/tdTomato/Dnmt1 loxP (KO) embryos. (b and d) Exemplary microphotographs of ( b ) TBR1 immunostaining (green) and (d) EOMES immunostaining (green); scale bars: 50 µm (DAPI is shown in blue). (c and e) Quantitative analysis of the TBR1 + postmitotic neuron numbers (c) and EOMES + intermediate progenitor cell (IPC) numbers (e) per bin in the E14.5 cortex of control and KO embryos, analyzed in 50 µm coronal cryosections (two-way ANOVA with subsequent Bonferroni correction , n = 13 control and n = 9 KO sections from N = 3 embryos for both genotypes). (f) TBR1 immunostaining (green) in coronally sectioned (50 µm) brains of E16.5 control and KO embryos, scale bars: 50 µm. (g) Quantitative analysis of the TBR1-positive postmitotic neuron numbers per bin in the E16.5 cortex of control and KO embryos, analyzed in 50 µm coronal cryosections (two-way ANOVA with subsequent Bonferroni correction , n = 8 for control and KO from N = 3 embryos for both genotypes). (h and j) Exemplary microphotographs of ( h ) EOMES immunostaining (green) and (j) TBR1 immunostaining (green) in coronal sections of E18.5 control and KO brains; scale bars: 50 µm (DAPI is shown in blue). (i and k) Quantitative analysis of the density of EOMES-positive IPCs (i) and TBR1-positive postmitotic neurons (k) in the E18.5 cortex of control and KO embryos, analyzed in 50 µm coronal cryosections(Nested t -test , n = 11 sections for both genotypes from N = 4 embryos per genotype). (l) Schematic illustration of the cortex of a coronally sectioned embryonic brain (E18.5), depicting the parameters analyzed for the quantification shown in ( m ). Three regions (lateral (1), dorsolateral (2), and dorsal (3)) were averaged to obtain the mean thickness respectively for each layer (nested t -test ; n = 12 control and n = 9 KO sections . N = 4 brains for both genotypes). (n and o) Panels depict significant ligand-receptor pairs involved in communication from SST + cINs to cortical progenitors, filtered by differentially expressed genes (DEG) from single-cell RNA-sequencing data. Communication probability is represented by dot color, and p -value by dot size, with p -values being computed using a one-sided permutation test. Panel ( n ) shows the communication with different apical progenitor (AP) populations at E14.5. Panel (o) illustrates communication with IPCs at E14.5. Panels (p-r) present microphotographs of cell pair combinations formed from cortical neurons isolated from E14.5 C57BL6/J embryos, plated at clonal density, after 24 hours. Cell clones were either treated with control-or recombinant efnB2-FC for 24 h, prior to immunocytochemical staining for nestin (magenta), β-III-tubulin (TUBB3, green), and DAPI (blue). Scale bars: 5 µm. (s) Quantification of the proportion of nestin/nestin, nestin/β-III-tubulin and β-III-tubulin/β-III-tubulin-positive cell pairs normalized to the overall number of these pairs is shown in (p-r) . Student’s t -test (control-Fc: 66 cell pairs, efnB2-Fc: 58 cell pairs, N = 3). p < 0.05 *, p < 0.01 ** , p < 0.001 ***. MZ: marginal zone, CP: cortical plate, IZ-VZ: intermediate zone-ventricular zone, n.s.: not significant, ctrl: control.

Article Snippet: Then a final washing step with 1x PBS for 10 min and a DAPI staining (1:10000/1x PBS; Carl Roth, Germany) for 5 min were performed and coverslips were mounted in Mowiol.

Techniques: Immunostaining, Control, RNA Sequencing Assay, Isolation, Clone Assay, Recombinant, Staining