Fig 1: Transcriptome analysis (scRNA-seq) demonstrates an immediate increase in neurodifferentiation upon EPO application.a Experimental design of WT C57Bl6 mice (n = 3 per group), treated at P28 with a single i.p. injection of placebo or EPO (5000IU/kg), followed by isolation of the CA1 region after 6 h and processing for DropSeq analysis (processing of biological replicates performed separately, followed by pooling for graphical presentation and final analysis), as well as after 24 h for immunohistochemistry (figure created by Debia Wakhloo). b Visualization of hippocampal CA1 cell clusters using t-distributed stochastic neighbor embedding (tSNE). Each color represents a cluster of specific cells, characterized by a defined gene expression profile. c Individual cells derived from either EPO (red) or placebo (black)-treated mice are denoted. d Percentage of cells in the respective glutamatergic clusters per treatment condition (EPO: n = 583, placebo: n = 390); two-tailed Fisher’s exact test. e Representative images and quantification of Tbr1 and Tle4 staining (immature neuronal markers) in CA1 of mice at P29, i.e., 24 h after a single injection of EPO. Within bars, mouse n numbers indicated; mean ± SEM presented; two-tailed student’s t-test. f Trajectory analysis in Monocle2 of cells in the ‘Mature Glutamatergic1’ and the ‘Immature Glutamatergic’ cluster colored by pseudotime (the darker the more mature). g Trajectory colored by cell identity. Bar graph indicates average pseudotime of the respective clusters; mean ± SEM; two-tailed Mann–Whitney U test. h Trajectory colored and split by placebo (black; left) versus EPO treatment (red; right). Bar graph shows average pseudotime of cells in the respective treatment groups; two-tailed Mann–Whitney U test; mean ± SEM; see also Supplementary Figs. 1–3. Source data underlying graph e are provided as a Source Data File.
Fig 2: Brain developmental disorders with ventriculomegaly and cortical atrophy.(A) Viral loads in different organs. Viral genome copy number was assessed by qPCR using MCMV-DNA extracted from equal amounts of different organ tissues from 3 MCMV-infected mice at the indicated time points. Results are presented as mean ± SEM. (B) Dynamic changes in brain weight. Representative images of brains harvested at P7 using bright-field and fluorescence microscopies are shown. Hemorrhage is indicated by black arrow. Brain weights were measured at the indicated time points. Scale bar: 2 mm. (C) Lateral ventricle areas and thicknesses of cortical layers at P7. Coronal brain sections were stained for IE1 (green), DAPI (blue), and Tbr1 (red) or Ctip2 (red), respectively. Lateral ventricle areas of position-matched brain sections outlined by white dashed lines were measured. Thicknesses of cortical layers in the position-matched region (indicated in white dashed box) were measured. Data were collected from 3 mice/group in 3 independent experiments. Results are presented as mean ± SEM and analyzed by 2-tailed Student’s t test, respectively. *, P < 0.05; **, P < 0.01, ***, P < 0.001. Scale bar: 1 mm or 100 µm in the magnified images.
Fig 3: Grem1 is expressed in the glutamatergic excitatory neuron lineage cells. (A) Heatmap depicting unsupervised clustering of TdTomato+ and TdTomato- cells isolated from 14.5 dpc Grem1-reporter (red) mice induced with tamoxifen at 13.5 dpc based on expression of representative differentiation marker transcripts for neural stem cell (NSC), radial glia, intermediate progenitor, immature neuron and mature neuron. (B) GSEA for Glutamate Secretion genes between TdTomato+ and TdTomato- samples from A. NES=2.16, P=0.0049. (C) Representative images of immunofluorescence staining of 14.5 and 20.5 dpc telencephalon from Grem1-reporter (red) mice induced with tamoxifen at 13.5 dpc: Tbr1, green; DAPI, blue. (D) Quantification of C showing the percentage of TdTomato+ cells that were also Tbr1+ in three representative fields from three biological replicates. One way ANOVA with Tukey's multiple test. *P<0.05, ****P<0.0001. Data are mean±s.d. (E) tSNE plot of human scRNA-seq dataset. GREM1-expressing cells outlined in black. Dot size represents Grem1 expression value as indicated. NA, not applicable. Scale bars: 100 µm.
Fig 4: Transplanted Human PSC-Derived Cortical Neurons Integrate as Single Cells in the Mouse Cortex(A) Human ESC differentiation and transplantation protocol.(B) Left: confocal image of immunostained coronal section from the brain of a transplanted animal showing GFP+ transplanted human neurons (green) integrated in the mouse cortex, 14 days post-transplantation (DPT), with cell bodies stained with DAPI (blue). Right: high magnification of the boxed subregion on the left (dashed lines). Note the radial orientation and extended apical processes of the human cortical neurons.(C) Example images of cortical sections from transplanted animals immunostained for markers of deep (Tbr1, Ctip2, Foxp2) and upper (Satb2, Cux1) cortical layer identity at 2 months post-transplantation (2 MPT).(D) Fraction of GFP+ human neurons expressing markers in (C). N denotes the number of sampled human cells for each marker (n = 4 animals).(E) Monosynaptic rabies virus tracing shows that human neurons make synaptic connections with host mouse cortical neurons at 4 MPT. Left: confocal image of immunostained section of rabies-injected transplanted cortex with starter human cells (HSs) and rabies-labeled presynaptic mouse cortical neurons (1, 2). Right: high magnification of boxed areas on the left (dashed boxes).Scale bars: (B) left, 500 µm, right, 50 µm; (C) 200 µm; (E) low-magnification image, 200 µm, high-magnification enlargements, 10 µm.See also Figure S1.
Fig 5: Alterations in neural morphology after developmental exposure to Pb.A, illustration of the exposure, differentiation, and assessment scheme for this work. B, representative images of differentiating cortical neurons at Day 10, 18, and 35. TBR2 stains for intermediate progenitor cells, TBR1 stains for immature neurons, and MAP2 stains for mature neurons. Scale bar = 20 µm. Total neurite length (top) and branch number (bottom) were quantified at (C) Day 25 and (D) Day 45. N = 10 views from three independent differentiations. Data = mean ± SE. N.S., not significant; *p < 0.05 and ***p < 0.001.
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