Figures and data
fCpG barcode methylation
A) Barcode methylation was variable between cells with averages ∼ 50%
B) Most cells had different barcodes with average PWDs ∼ 0.5.
Brain data
Higher fCpG barcode methylation in earlier emerging cells
A) Average barcode methylation was higher in the brainstem and inhibitory neurons. Barcode methylation was lower for excitatory neurons, cerebellar, and glial cells. Notably, average methylation was lower for outer cortical (L2_3) compared to earlier appearing inner (L4_6) cortical excitatory neurons. Abbreviations are as in reference 3, with L2_3 all outer and L4_6 all inner cortical excitatory neurons, and NN are non-neuronal cells other than ASC, OPC and ODC.
B) Most fCpGs appear to start methylated in a progenitor because nearly all individual fCpGs are methylated in inhibitory neurons in the brain stem (PN, THM, MSN). Many fCpGs in inhibitory neurons (pvalb, sst) are still predominately methylated. Few fCpGs in excitatory neurons that differentiate later in development are fully methylated. Glial cells that also emerge late in development, and hippocampal cells that may divide postnatally had variable methylation with both highly methylated and unmethylated fCpGs.
C) Barcodes become fixed when their cells stop dividing and differentiate. Barcode methylation levels can indicate when neurons emerge and are correlated with a cartoon of physical caudal to rostral brain development. fCpGs infer that inhibitory neurons made in the ganglionic eminences appear before and reach their final adult content before most cortical excitatory neurons and glial cells. Notably, lower cortical layer neurons can be detected earlier in life relative to later appearing outer cortical neurons that reach adult levels late in development. Brain contents inferred by adult barcodes may differ from actual neonatal brains because neurons that die during development are not sampled.
Related cell pairs
A) Most cells had different barcodes with average PWDs between cell pairs of ∼ 0.5. Cell pairs of the same phenotype had different barcodes but were on average more related to each other.
B) Heatmap showing that cells of the same phenotype are more related.
C) Cells that emerge early in development are more related and more methylated. Closely related nearest neighbors (PWD <0.05) are numerically more common for more methylated cell types.
Brain lineage trees
A) Barcodes from 960 cells form lineage trees using IQtree (19) that are rooted by a fully methylated progenitor and generally follow caudal to rostral brain development, with sequential branching of brainstem neurons, inhibitory neurons, cerebellar neurons, and excitatory neurons, with hippocampal neurons furthest from the start. Trees are similar between the brains, with H04 inferring less distance between inhibitory and excitatory lineages. The degree of confidence was generally low with the data and numbers of bootstraps, with bootstrap branch support typically less than 15%.
B) H01 tree with labeled cell types. Neuron types generally clustered by phenotypes with closely branching excitatory and inhibitory neurons more common earlier in development.
C) H01 tree with labeled cell locations. Related inhibitory and excitatory neurons can be found in different parts of the brain (FC = frontal (red), TC = temporal, OC = occipital (blue), PC = parietal, HIP = hippocampus (orange), cere = cerebellum (yellow), BS = brainstem (black).
D) H01 tree with ∼2,853 cortical excitatory neurons has more evidence of localized radial migration because related neurons are more often found in the same cortical region. Excitatory neurons cluster by subtype, and closely related lower and upper excitatory neurons were still few.
E) H01 tree with ∼2,847 cortical inhibitory neurons still retains evidence of tangential migration with related neurons scattered throughout the cortex. Inhibitory neurons cluster by subtype with switching between some closely related pairs.
Lineage fidelity, migration, and differentiation
A) Inhibitory neurons have high lineage fidelity because nearest neighbor pairs (PWD <0.05) were nearly always both inhibitory neurons. Excitatory neurons had less lineage fidelity because a nearest neighbor was more often an inhibitory neuron. Data are for all three brains.
B) Nearest neighbor inhibitory neuron pairs often had subtypes differences. More lineage fidelity was generally present for brainstem and excitatory neurons. Numbers indicate percent lineage subtype fidelity.
C) Nearest neighbor inhibitory and excitatory neuron pairs showed evidence of tangential migration because they were found in different cortical regions. Data are for all three brains.
D) Nearest neighbor neurons were scattered in the cortex. Numbers indicate percent location fidelity. (NonC = non-cortical location, Paleo = paleocortex)
E) Phenotypic differences between inhibitory or excitatory neuron were measured by Euclidian distances between their annotated tsne coordinates (3). Although cells with more similar phenotypes (shaded yellow) were generally more related, cells with similar phenotypes could be distantly related, and related cells could have different phenotypes.
H02 data
A) Barcode methylation for different cell types
B) PWDs between cells of the same type
C) PWDs between cell types
D) Lineage cell type fidelity between nearest neighbor pairs (PWD<0.05)
E) Location fidelity between nearest neighbor pairs (PWD<0.05)
F) Barcode methylation versus final adult brain content indicates that inhibitory neurons appear first and reach their adult levels before excitatory or glial cells.
G) Ancestral tree with 1,001 cells rooted at a fully methylated progenitor shows sequential branching with excitatory, then brain stem, inhibitory and cerebellar neurons, then glial cells, and finally excitatory neurons with hippocampal neurons at the end.
H) Related cells colocalize for brain stem, cerebellar, and hippocampal neurons. Inhibitory neurons are more scattered. Excitatory neurons are also scattered with some localization within the cortex (see I & J for trees with more neurons)
I) Ancestral tree with more (2,806) excitatory neurons shows more localization between related neurons in the hippocampus, occipital and temporal cortex. Related neurons also tend to have similar phenotypes.
J) Ancestral tree with more (2,788) inhibitory neurons still shows scattering between related neurons. Related neurons tend to have similar phenotypes.
H04 data
A) Barcode methylation for different cell types
B) PWDs between cells of the same type
C) PWDs between cell types
D) Lineage cell type fidelity between nearest neighbor pairs (PWD<0.05)
E) Location fidelity between nearest neighbor pairs (PWD<0.05)
F) Barcode methylation versus final adult brain content indicates that inhibitory neurons appear first and reach their adult levels before excitatory or glial cells.
G) Ancestral tree with 1,033 cells rooted at a fully methylated progenitor shows sequential branching with excitatory, then brain stem, inhibitory and cerebellar neurons, then glial cells, and finally excitatory neurons with hippocampal neurons at the end.
H) Related cells colocalize for brain stem, cerebellar, and hippocampal neurons. Inhibitory neurons are more scattered. Excitatory neurons are also scattered with some localization within the cortex (see I & J for trees with more neurons)
I) Ancestral tree with more (2,797) excitatory neurons shows more localization between related neurons in the hippocampus, occipital and temporal cortex. Related neurons also tend to have similar phenotypes.
J) Ancestral tree with more (2,752) inhibitory neurons still shows scattering between related neurons. Related neurons tend to have similar phenotypes.
Brain tumor fCpG methylation:
fCpG sites were matched with brain tumor data from methylation arrays. Average methylation is displayed for each brain tumor sample. PA= pilocytic astrocytoma, GSE44684, 145 fCpGs covered; MB=medulloblastoma, GSE193646, 434f CpGs covered; MB=medulloblastoma, GSE63669, 883 fCpGs covered; GBM=glioblastoma multiforme, GSE109399, 1,138 fCpGs covered; Gliomas=various gliomas, GSE248471, 1,138 fCpGs covered. Includes both male and female tumors.
