Figure S4. Panel A shows immunostaining... | Rockefeller University Press

Figure S4.

$Characterization of the SMFCs in DS fetal brains (related to Fig. 8). (A) Whole view of a 14-gw DS fetal brain immunostained by IBA-1 and Ki67 antibodies. In the lower panel, the magnified rectangular region of the full-view image showed the entire middle section, and the other end displayed the shape and orientation of the SMFC in the DS brain. Scale bars, 2 mm (whole views) and 500 µm (below). (B) Stitched image of the immunostaining with IL-34, CSF-1R, and IBA-1 antibodies at one end of the SMFC of a DS fetal brain revealed that IL-34+ cells are preferentially enriched in the region of the SMFC characterized by a high CSF-1R level. Scale bar, 200 µm. (C) Immunostaining with L34, CSF-1R, and IBA-1 antibodies in a 14-gw DS fetal brain revealed that IL-34+ cells are almost absent in the CP. Scale bar, 200 µm. (D) Large SMFC region that contains a middle and an end stained by IBA-1, CSF-1R, and IL-34 antibodies in the DS fetal brain revealed that the SMFC region includes a large amount of IL-34+ cells. The inner insert on the right shows the IBA-1 signal, revealing IBA-1+ cell intensity in the SMFC. The inner insert on the left shows CSR-1R intensity in the SMFC and other regions. Scale bar, 100 µm. (E) Stitched image of immunostaining with IBA-1, NeuN, and IL-34 antibodies in the SMFC of the DS fetal brain (14 gw). Boxed, the magnified images (white arrowheads [bottom], NeuN+/IL-34+ cells). Scale bar, 500 µm. (F) Stitched image of immunostaining with IBA-1 and CSF-1 antibodies in the SMFC of the DS fetal brain. In the magnified image (below), the boxed area shows that CSF-1+ cells are rare in the SMFC. Scale bar, 500 µm. (G) SPP1, Ki67, and IBA-1 antibody immunostaining in the DS fetal brain. The magnified images: i, bipolar microglia with strong SPP1 at the end of the SMFC; ii, aggregate microglia with weak SPP1 in the middle fraction; iii, MV-interacting microglia with moderate SPP1; iv, a scattered ramified resident microglia with no SPP1. Scale bar, 200 µm. (H) SPP1, galectin-3, and IBA-1 antibody immunostaining in the DS fetal brain. The magnified images: i, aggregate microglia in the middle fraction with weak SPP1; ii, MV-interacting microglia with moderate SPP1; iii, ramified resident microglia with no SPP1; iv, bipolar microglia with strong SPP1. Scale bar, 200 µm. (I) Immunostaining of a 14-gw DS fetal brain with CTIP2, DARPP32, and IBA-1 antibodies revealed that the middle fraction of the SMFC does not overlap with the DARPP32+ region. Scale bar, 500 µm. (J) Immunostaining with CTIP2, DARPP32, and IBA-1 antibodies revealed that one end of the SMFC in the DS fetal brain overlapped with the cellular bridge of IC, whereas DARPP32+ did not overlap with the SMFC. AT, axon tract; CB, cellular bridge. Scale bar, 500 µm. (K) Super-resolution images of IBA-1, Ki67, and CD34 antibody immunostaining revealed that MV-interacting microglia in the DS fetal brain have more bulbous endings (white arrowheads, bulbous endings). Right, quantification of bulbous ending of MV-interacting microglia in DS and healthy fetal brains (DS, n = 3; healthy, n = 5; Data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (L) Super-resolution images of IBA-1, Ki-67, and CD34 antibody immunostaining revealed that RG-interacting microglia in DS fetal brains have more bulbous endings in the RG-interacting microglia (white arrowheads, indicated by bulbous endings). Right, bulbous ending count in the RG-interacting microglia of healthy and DS (DS, n = 4; healthy, n = 5; data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (M) Super-resolution images of IBA-1 and Ki67 antibody immunostaining in CP microglia in DS and healthy fetal brains. Right, bulbous ending count/microglia in the CP microglia of healthy and DS (DS, n = 4; healthy, n = 5; data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (N) Super-resolution images of IBA-1 and clathrin antibody immunostaining in the DS fetal brain. Right, measuring density of clathrin in bulbous endings and cellular processes (data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (O) Immunostaining with IBA-1, CD8, and CD177 antibodies in the healthy and DS fetal brains with the SMFC revealed that CD8+ T cells and CD177+ neutrophils are absent in the SMFCs (the inner insert in the LGE panel, intra-MV CD8+ T cell; white arrowheads, intra-MV CD8+ T cell). Scale bar, 100 µm (left) and 200 µm (right). (P) Immunostaining with iNOS, CD206, and IBA-1 antibodies revealed that iNOS+ microglia (M1) and CD206+ microglia (M2) are absent in the SMFC in healthy and DS fetal brains. Scale bar, 100 µm. MV, microvasculature. Refer to the image caption for details.$ Panel A shows immunostaining images whole-brain and magnified views of IBA-1 and Ki67 revealing overall structure, orientation, and expansion of SMFC regions in DS fetal brain. Panel B shows immunostaining images stitched views of IL34, CSF-1R, and IBA-1 demonstrating enrichment of IL34 positive cells in regions with high CSF-1R expression. Panel C shows immunostaining images indicating minimal IL34 expression within cortical plate regions. Panel D shows immunostaining images highlighting extensive SMFC regions with strong IL34 and CSF-1R signals and dense IBA-1 positive microglia. Panel E shows immunostaining images of IBA-1, NeuN, and IL34 identifying neuronal IL34 positive cells within SMFC regions. Panel F shows immunostaining images of IBA-1 and CSF-1 indicating low CSF-1 presence within SMFC areas. Panel G shows immunostaining images of SPP1, Ki67, and IBA-1 demonstrating heterogeneous microglia subtypes with distinct SPP1 expression patterns across SMFC regions. Panel H shows immunostaining images of SPP1, Galectin-3, and IBA-1 identifying subtype-specific marker expression among microglia populations. Panel I shows immunostaining images of CTIP2, DARPP32, and IBA-1 indicating lack of overlap between SMFC middle regions and DARPP32 positive areas. Panel J shows immunostaining images demonstrating spatial overlap of SMFC ends with internal capsule structures but not DARPP32 positive regions. Panel K shows immunostaining images super-resolution views with scatter bar plot quantification revealing increased bulbous endings in microvasculature-interacting microglia. Panel L shows immunostaining images super-resolution views with scatter bar plot quantification showing increased bulbous endings in radial glia-interacting microglia. Panel M shows immunostaining images super-resolution views with scatter bar plot quantification of increased bulbous endings in cortical plate microglia. Panel N shows immunostaining images super-resolution views with scatter bar plot quantification showing elevated clathrin density in microglial processes and endings. Panel O shows immunostaining images demonstrating absence of CD8 positive T cells and CD177 positive neutrophils within SMFC regions. Panel P shows immunostaining images indicating absence of M1 and M2 polarized microglia markers within SMFC regions.

Characterization of the SMFCs in DS fetal brains (related to Fig. 8). (A) Whole view of a 14-gw DS fetal brain immunostained by IBA-1 and Ki67 antibodies. In the lower panel, the magnified rectangular region of the full-view image showed the entire middle section, and the other end displayed the shape and orientation of the SMFC in the DS brain. Scale bars, 2 mm (whole views) and 500 µm (below). (B) Stitched image of the immunostaining with IL-34, CSF-1R, and IBA-1 antibodies at one end of the SMFC of a DS fetal brain revealed that IL-34⁺ cells are preferentially enriched in the region of the SMFC characterized by a high CSF-1R level. Scale bar, 200 µm. (C) Immunostaining with L34, CSF-1R, and IBA-1 antibodies in a 14-gw DS fetal brain revealed that IL-34⁺ cells are almost absent in the CP. Scale bar, 200 µm. (D) Large SMFC region that contains a middle and an end stained by IBA-1, CSF-1R, and IL-34 antibodies in the DS fetal brain revealed that the SMFC region includes a large amount of IL-34⁺ cells. The inner insert on the right shows the IBA-1 signal, revealing IBA-1⁺ cell intensity in the SMFC. The inner insert on the left shows CSR-1R intensity in the SMFC and other regions. Scale bar, 100 µm. (E) Stitched image of immunostaining with IBA-1, NeuN, and IL-34 antibodies in the SMFC of the DS fetal brain (14 gw). Boxed, the magnified images (white arrowheads [bottom], NeuN⁺/IL-34⁺ cells). Scale bar, 500 µm. (F) Stitched image of immunostaining with IBA-1 and CSF-1 antibodies in the SMFC of the DS fetal brain. In the magnified image (below), the boxed area shows that CSF-1⁺ cells are rare in the SMFC. Scale bar, 500 µm. (G) SPP1, Ki67, and IBA-1 antibody immunostaining in the DS fetal brain. The magnified images: i, bipolar microglia with strong SPP1 at the end of the SMFC; ii, aggregate microglia with weak SPP1 in the middle fraction; iii, MV-interacting microglia with moderate SPP1; iv, a scattered ramified resident microglia with no SPP1. Scale bar, 200 µm. (H) SPP1, galectin-3, and IBA-1 antibody immunostaining in the DS fetal brain. The magnified images: i, aggregate microglia in the middle fraction with weak SPP1; ii, MV-interacting microglia with moderate SPP1; iii, ramified resident microglia with no SPP1; iv, bipolar microglia with strong SPP1. Scale bar, 200 µm. (I) Immunostaining of a 14-gw DS fetal brain with CTIP2, DARPP32, and IBA-1 antibodies revealed that the middle fraction of the SMFC does not overlap with the DARPP32⁺ region. Scale bar, 500 µm. (J) Immunostaining with CTIP2, DARPP32, and IBA-1 antibodies revealed that one end of the SMFC in the DS fetal brain overlapped with the cellular bridge of IC, whereas DARPP32⁺ did not overlap with the SMFC. AT, axon tract; CB, cellular bridge. Scale bar, 500 µm. (K) Super-resolution images of IBA-1, Ki67, and CD34 antibody immunostaining revealed that MV-interacting microglia in the DS fetal brain have more bulbous endings (white arrowheads, bulbous endings). Right, quantification of bulbous ending of MV-interacting microglia in DS and healthy fetal brains (DS, n = 3; healthy, n = 5; Data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (L) Super-resolution images of IBA-1, Ki-67, and CD34 antibody immunostaining revealed that RG-interacting microglia in DS fetal brains have more bulbous endings in the RG-interacting microglia (white arrowheads, indicated by bulbous endings). Right, bulbous ending count in the RG-interacting microglia of healthy and DS (DS, n = 4; healthy, n = 5; data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (M) Super-resolution images of IBA-1 and Ki67 antibody immunostaining in CP microglia in DS and healthy fetal brains. Right, bulbous ending count/microglia in the CP microglia of healthy and DS (DS, n = 4; healthy, n = 5; data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (N) Super-resolution images of IBA-1 and clathrin antibody immunostaining in the DS fetal brain. Right, measuring density of clathrin in bulbous endings and cellular processes (data, mean ± SD; Mann–Whitney U test, **P < 0.001). Scale bar, 10 µm. (O) Immunostaining with IBA-1, CD8, and CD177 antibodies in the healthy and DS fetal brains with the SMFC revealed that CD8⁺ T cells and CD177⁺ neutrophils are absent in the SMFCs (the inner insert in the LGE panel, intra-MV CD8⁺ T cell; white arrowheads, intra-MV CD8⁺ T cell). Scale bar, 100 µm (left) and 200 µm (right). (P) Immunostaining with iNOS, CD206, and IBA-1 antibodies revealed that iNOS⁺ microglia (M1) and CD206⁺ microglia (M2) are absent in the SMFC in healthy and DS fetal brains. Scale bar, 100 µm. MV, microvasculature.