Ta AC et al. (2022), Temporal and spatial transcriptomic dynamics ac...

XB-ART-58609

G3 (Bethesda) 2022 Jan 04;121:. doi: 10.1093/g3journal/jkab387.

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Temporal and spatial transcriptomic dynamics across brain development in Xenopus laevis tadpoles.

Ta AC , Huang LC , McKeown CR , Bestman JE , Van Keuren-Jensen K , Cline HT .

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Amphibian metamorphosis is a transitional period that involves significant changes in the cell-type populations and biological processes occurring in the brain. Analysis of gene expression dynamics during this process may provide insight into the molecular events underlying these changes. We conducted differential gene expression analyses of the developing Xenopus laevis tadpole brain during this period in two ways: first, over stages of the development in the midbrain and, second, across regions of the brain at a single developmental stage. We found that genes pertaining to positive regulation of neural progenitor cell proliferation as well as known progenitor cell markers were upregulated in the midbrain prior to metamorphic climax; concurrently, expression of cell cycle timing regulators decreased across this period, supporting the notion that cell cycle lengthening contributes to a decrease in proliferation by the end of metamorphosis. We also found that at the start of metamorphosis, neural progenitor populations appeared to be similar across the fore-, mid-, and hindbrain regions. Genes pertaining to negative regulation of differentiation were upregulated in the spinal cord compared to the rest of the brain, however, suggesting that different programs may regulate neurogenesis there. Finally, we found that regulation of biological processes like cell fate commitment and synaptic signaling follow similar trajectories in the brain across early tadpole metamorphosis and mid- to late-embryonic mouse development. By comparing expression across both temporal and spatial conditions, we have been able to illuminate cell-type and biological pathway dynamics in the brain during metamorphosis.

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Species referenced: Xenopus laevis
Genes referenced: ache aif1l ano2 ascl1 c1qa cdh1 cmtm5 col2a1 dbx1 egr2 emp2 enpp6 eomes epcam epha8 esr1 fgf1 foxa2 gad1.2 gad2 gal3st1 glra1 glul gpr17 grin2b grp hepacam hes1 hes3 hes5 hes5.2 hgf klf9 lmx1b.2 mbp mpz nefm nes neurod1 nkx2-8 nodal1 notch1 nr4a2 ocln olig3 omg otx1 otx2 phyhip pik3ca pnmt podn rbp3 rgs9 slc18a3 slc30a2 slc4a4 slc6a4 sod3 sox10 stmn1 tc2n th thy1 tnfaip8l3 tnfrsf8 tph1 tph2 vim
GO keywords: metamorphosis [+]

???displayArticle.gses??? GSE183193: NCBI

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	Figure 1. Expression analysis in the Xenopus midbrain across developmental stages. A) X. laevis developmental stages sequenced based on Nieuwkoop and Faber, 1956 (hpf and dpf indicate hours/days post fertilization). B) Principal component analysis of samples collected from the X. laevis midbrain (MB) at developmental stages 44, 46, 55, and 61. Colors correspond to developmental stages in Figure 1A. C) Heat map of cell type marker expression over tadpole midbrain development. Cell types are categorized for genes listed as NPC, neural progenitor cell (including neuroepithelial cells, radial glia, intermediate progenitors, and immature neurons); G, glial cell (including mature oligodendrocytes, Schwann cells and Schwann cell precursors, astrocytes, and microglia); N, neuron; NPC+G, progenitor cell/glial marker; G+N, glial/neuron marker. Genes are clustered by similarity in expression across stages with complete linkage clustering, as indicated by the dendrogram to the left. Asterisks indicate significantly enriched cell types (p<0.001 for progenitor cell markers, p<0.005 for glial cell markers). Expression is normalized per-gene to the highest expression for that gene.
	Figure 2. Enriched biological pathways in the Xenopus midbrain across developmental stages. A) GO biological pathway analysis showing enriched sets of genes differentially expressed between stages of tadpole midbrain development. Dot size corresponds to the proportion of differentially expressed genes associated with the biological pathway, and dot color refers to the adjusted p-value. The numbers along the x-axis correspond to the total number of genes differentially expressed in each comparison. B) Clusters of genes differentially expressed during tadpole midbrain development that are associated with the “Positive Regulation of Cell Proliferation” GO category. The five clusters shown are Cyclin/Cdks (368 genes), PI3K-AKT/St55a-c, and PI3K/St61, numbers in parentheses indicates the number of genes in each cluster. The Cyclin/Cdk cluster is enriched in cell cycle-related genes at stages 44-46 (p<0.001) and the remaining clusters are enriched in PI3K-AKT pathway associated genes (St55a-c, p = 0.019; St61, p<0.001).
	Figure 3. Expression analysis of Xenopus stage 46 across different brain regions. A) Schematic depicting the four brain regions dissected and sequenced at Xenopus Stage 46: forebrain (pink), midbrain (blue), hindbrain (green), and spinal cord (purple). B) Principal component analysis of samples collected from the X. laevis stage 46 brain in the forebrain (FB ST46, pink), midbrain (MB ST46, blue), hindbrain (HB ST46, green), and spinal cord (SC ST46, purple). Colors correspond to brain regions in Figure 3A. C) Normalized expression of hox5, a hindbrain-spinal cord marker, and otx1 and otx2, forebrain-midbrain markers, across all brain regions. Colors correspond to brain regions in Figures 3A, B. D) Heat map of cell type marker expression over ST46 tadpole brain regions. General cell types are categorized for genes listed as NPC, neural progenitor cell (including neuroepithelial cells, radial glia, intermediate progenitors, and immature neurons); G, glial cell (including mature oligodendrocytes, Schwann cells and Schwann cell precursors, astrocytes, and microglia); N, neuron; NPC+G, progenitor cell/glial marker. E) Heat map of neuron subtype marker expression over brain region at stage 46. Specific neuronal subtypes are categorized for genes listed as CHO, cholinergic neuron; MN, mature neuron; DOP, dopaminergic neuron; SER, serotonergic neuron; GABA, GABAergic neuron; GLY, glycinergic neuron. Genes are clustered by similarity in expression across regions with complete linkage clustering, as indicated by the dendrogram to the left. Asterisks indicate significantly enriched cell types ((p<0.002 for PG, p<0.001 for N, p<0.001 for both DOP and CHO markers). Expression is normalized per-gene to the highest expression for that gene.
	Figure 4. Enriched biological pathways in ST46 Xenopus across brain regions. GO biological pathways enriched in the set of genes differentially expressed between two regions of the tadpole stage 46 brain. Brain regions, as shown in Figure 3A are FB, forebrain; MB, midbrain; HB, hindbrain; and SC, spinal cord. Dot size corresponds to the proportion of differentially expressed genes associated with the biological pathway, and dot color refers to the adjusted p-value. The numbers along the x-axis correspond to the total number of genes differentially expressed in each comparison.
	Figure 5. Expression analysis of mouse brain regions across development. A) Principal component analysis of mouse cerebral cortex (Ctx) and cerebellum (Cb) data, collected at timepoints E13.5-E18.5. B) Proportion of genes differentially expressed in the mouse cerebral cortex vs. cerebellum at each timepoint that was also differentially expressed in the tadpole stage 46 forebrain vs. hindbrain. The total number of differentially expressed genes in at this timepoint is indicated in parentheses next to the timepoint on the x-axis. All timepoints in the mouse model shared a similar proportion of genes with the tadpole timepoint. C) GO biological pathways enriched in the set of genes upregulated in both the mouse cerebral cortex and cerebellum at developmental timepoints from E13.5 to E18.5. Distinct pathways are upregulated at each developmental period. Dot size corresponds to the proportion of differentially expressed genes associated with the biological pathway, and dot color refers to the adjusted p-value. The numbers along the x-axis correspond to the total number of genes differentially expressed in each comparison.
	Figure 6. Comparison of gene expression between Xenopus and Mouse across development. A) Heat map showing expression of genes upregulated in the mouse cerebral cortex and cerebellum at E13.5/14.5 over the ST44-61 Xenopus midbrain. B) Heat map showing expression of genes upregulated in mouse cerebral cortex and cerebellum at E15.5/E16.5 over the ST44-61 Xenopus midbrain. C) Heat map showing expression of genes upregulated in the mouse cerebral cortex and cerebellum at E17.5/18.5 over the ST44-61 Xenopus midbrain. Genes are clustered by similarity in expression across Xenopus midbrain stages with complete linkage clustering, as indicated by the dendrograms to the left. E13.5/E14.5 genes were overrepresented in genes upregulated at Xenopus ST44 and ST46 (p<0.0001), while E17.5/E18.5 genes were overrepresented in genes upregulated at stage Xenopus ST61 (p<0.0001). Expression is normalized per-gene to the highest expression for gene.
	Schematic depicting the four brain regions dissected and sequenced at Xenopus Stage 46: forebrain (pink), midbrain (blue), hindbrain (green), and spinal cord (purple).

References [+] :

Akerman, Depolarizing GABAergic conductances regulate the balance of excitation to inhibition in the developing retinotectal circuit in vivo. 2006, Pubmed, Xenbase