Rubella virus exposure of brain organoids leads to interferon response. A. Single cell RNA sequencing analysis identified 13 clusters, including neurons and glial cells (Div.: dividing cells, RG: radial glia, Astros: astrocytes, IPC: intermediate progenitor cells). B. Dot plot depicting cluster marker genes for each cluster. C. UMAP plots of organoids colored by the presence or absence of microglia. D. UMAP plots of organoids colored by the presence or absence of RV treatment. E.

Contexts in source publication

Context 1

... transcripts and pseudogenes were excluded. Approximately 11,000 cells passed filtering criteria ( Figure 5A, Figure 5 -figure supplement 4A-C), revealing the expected major cell populations of the human developing brain , including radial glia cells, immature and mature neurons, and astrocytes ( Figure 5A-B). Cell cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). ...

Context 2

... transcripts and pseudogenes were excluded. Approximately 11,000 cells passed filtering criteria ( Figure 5A, Figure 5 -figure supplement 4A-C), revealing the expected major cell populations of the human developing brain , including radial glia cells, immature and mature neurons, and astrocytes ( Figure 5A-B). Cell cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). ...

Context 3

... transcripts and pseudogenes were excluded. Approximately 11,000 cells passed filtering criteria ( Figure 5A, Figure 5 -figure supplement 4A-C), revealing the expected major cell populations of the human developing brain , including radial glia cells, immature and mature neurons, and astrocytes ( Figure 5A-B). Cell cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). ...

Context 4

... 11,000 cells passed filtering criteria ( Figure 5A, Figure 5 -figure supplement 4A-C), revealing the expected major cell populations of the human developing brain , including radial glia cells, immature and mature neurons, and astrocytes ( Figure 5A-B). Cell cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). Cells derived from organoids with and without microglia were present in all clusters ( Figure 5C). ...

Context 5

... 11,000 cells passed filtering criteria ( Figure 5A, Figure 5 -figure supplement 4A-C), revealing the expected major cell populations of the human developing brain , including radial glia cells, immature and mature neurons, and astrocytes ( Figure 5A-B). Cell cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). Cells derived from organoids with and without microglia were present in all clusters ( Figure 5C). ...

Context 6

... 11,000 cells passed filtering criteria ( Figure 5A, Figure 5 -figure supplement 4A-C), revealing the expected major cell populations of the human developing brain , including radial glia cells, immature and mature neurons, and astrocytes ( Figure 5A-B). Cell cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). Cells derived from organoids with and without microglia were present in all clusters ( Figure 5C). ...

Context 7

... cluster annotations were assigned based on combinations of co-expressed cluster marker genes, such as FGFBP2 and SOX2 for radial glial cells (clusters 5 and 10), TAGLN3, HES6, NEUROD4 for neural progenitor cells (cluster 7), TUBB2A, TUBB2B, STMN2 for neurons (cluster 2), CLU, PTN and SPARCL1 for astrocytes (cluster 6), and MKI67, UBE2C and CENPF for dividing cells (clusters 3 and 4) ( Figure 5B, Figure 5 -figure supplement 4 D-E for individual cluster marker genes, Figure 5 -source data file 1 for the full list of markers). Cells derived from organoids with and without microglia were present in all clusters ( Figure 5C). A separate microglia cluster was not identified. ...

Context 8

... separate microglia cluster was not identified. Rare cells expressing the microglia marker AIF1 (encoding the Iba1 protein) were present, but such cells have been previously reported to develop spontaneously in organoids ( He et al., 2022) and the canonical microglia marker P2RY12 was not detected in those cells ( Figure 5 -figure supplement 4F). We attribute the apparent lack of microglia to both the small starting population and loss due to cell dissociation during processing for scRNAseq. ...

Context 9

... with the lack of microglia cells in our scRNAseq data, we did not recover appreciable numbers of the viral transcripts. However, exposure of organoids to RV resulted in significant transcriptomic differences including genes involved in the interferon signaling pathway and its response (IFI27, IFI6, IFITM3)(HLA-A (Campbell, Bizilj, Colman, Tuch, & Harrison, 1986;Keskinen, Ronni, Matikainen, Lehtonen, & Julkunen, 1997) andBST2 (Holmgren, Miller, Cavanaugh, & Rall, 2015)) (cluster 1, Figure 5E, Figure 5 -figure supplement 4 -source data file 1). The majority of cells in cluster 1 came from RV-exposed organoids ( Figure 5D). ...

Context 10

... with the lack of microglia cells in our scRNAseq data, we did not recover appreciable numbers of the viral transcripts. However, exposure of organoids to RV resulted in significant transcriptomic differences including genes involved in the interferon signaling pathway and its response (IFI27, IFI6, IFITM3)(HLA-A (Campbell, Bizilj, Colman, Tuch, & Harrison, 1986;Keskinen, Ronni, Matikainen, Lehtonen, & Julkunen, 1997) andBST2 (Holmgren, Miller, Cavanaugh, & Rall, 2015)) (cluster 1, Figure 5E, Figure 5 -figure supplement 4 -source data file 1). The majority of cells in cluster 1 came from RV-exposed organoids ( Figure 5D). ...

Context 11

... exposure of organoids to RV resulted in significant transcriptomic differences including genes involved in the interferon signaling pathway and its response (IFI27, IFI6, IFITM3)(HLA-A (Campbell, Bizilj, Colman, Tuch, & Harrison, 1986;Keskinen, Ronni, Matikainen, Lehtonen, & Julkunen, 1997) andBST2 (Holmgren, Miller, Cavanaugh, & Rall, 2015)) (cluster 1, Figure 5E, Figure 5 -figure supplement 4 -source data file 1). The majority of cells in cluster 1 came from RV-exposed organoids ( Figure 5D). While genes involved in the interferon response showed increased expression in organoids both with and without microglia, the magnitude of their upregulation was lower among cells in microglia-containing organoids ( Figure 5E-F). ...

Context 12

... majority of cells in cluster 1 came from RV-exposed organoids ( Figure 5D). While genes involved in the interferon response showed increased expression in organoids both with and without microglia, the magnitude of their upregulation was lower among cells in microglia-containing organoids ( Figure 5E-F). We confirmed higher expression levels of IFITM3 protein in organoids with microglia exposed to RV in comparison to organoids with microglia exposed to heat-inactivated-RV at 72 hours post-exposure ( Figure 5 G-H), while the overall cell numbers were not changed in either condition ( Figure 5I). ...

Context 13

... genes involved in the interferon response showed increased expression in organoids both with and without microglia, the magnitude of their upregulation was lower among cells in microglia-containing organoids ( Figure 5E-F). We confirmed higher expression levels of IFITM3 protein in organoids with microglia exposed to RV in comparison to organoids with microglia exposed to heat-inactivated-RV at 72 hours post-exposure ( Figure 5 G-H), while the overall cell numbers were not changed in either condition ( Figure 5I). immunofluorescence in brain organoids with microglia with wild type RV (bottom panel) or heat-inactivated control (top panel) 72 hours post-infection. ...

Context 14

... genes involved in the interferon response showed increased expression in organoids both with and without microglia, the magnitude of their upregulation was lower among cells in microglia-containing organoids ( Figure 5E-F). We confirmed higher expression levels of IFITM3 protein in organoids with microglia exposed to RV in comparison to organoids with microglia exposed to heat-inactivated-RV at 72 hours post-exposure ( Figure 5 G-H), while the overall cell numbers were not changed in either condition ( Figure 5I). immunofluorescence in brain organoids with microglia with wild type RV (bottom panel) or heat-inactivated control (top panel) 72 hours post-infection. ...

Context 15

... captured from microglia-containing organoids showed fewer differentially expressed genes in response to RV in each of the major cell classes compared to organoids that did not contain microglia ( Figure 6A, top vs bottom panel), with radial glia and neurons reaching statistically significant levels (p-values shown on the right side of the panel) and neural progenitor cells showing the overall trend without reaching statistical significance. One gene family that was specifically downregulated in the presence of RV in organoids without microglia included nuclear factor I -NFIB and NFIA ( Figure 6A, Figure 5 source data file 2) -two genes that form heterodimers in vivo and are associated with induction of gliogenesis ( Tchieu et al., 2019) in embryonic brain development. Early disruption in the function of either gene is associated with neurodevelopmental deficits and perinatal mortality in mice (das Neves et al., 1999;Steele-Perkins et al., 2005) and with intellectual disability in humans ( Schanze et al., 2018). ...

Context 16

... agree with the reviewer that further validation of the predicted molecular changes downstream of Rubella exposure would be valuable. We have opted to validate IFITM3 and NOVA1 expression differences using immunostaining, and the results are consistent with our predictions from scRNAseq, and the data is presented in revised Figure 5 and 6 of the current manuscript. ...