The Microbiota Shapes Central Nervous System Myelination in Early Life

Abstract

Maturation of the gut microbiota coincides with neurodevelopmental processes such as myelination, essential for efficient neural signal transmission. While a role for the microbiome in regulating adult prefrontal cortex (PFC) myelination is known, its effects on early-life myelin formation, growth, and integrity remain unclear. Using a cross-species approach in germ-free (GF) mice and zebrafish, we examined how the microbiota influences early myelination and neural development. Multi-system, multi-level analyses showed that the microbiota impacts glial maturation and myelination across species. In GF mice, we observed sex- and age-dependent alterations in pathways linked to neuronal activity and myelination, with myelin-related transcriptomic changes correlating with functional shifts in neurotransmission- and metabolism-related metabolites over time. Myelin growth and integrity were also affected in a sex- and time-dependent manner. As microglia regulate neuronal activity and engulf myelin, we examined microbiota-microglia interactions and found altered expression of genes involved in microglia maturation and synaptic pruning in both species. In zebrafish larvae, the microbiota influenced the spatial distribution of microglia and oligodendrocytes within the brain and spinal cord. These findings reveal conserved microbiota-mediated modulation of neuronal activity, myelination, and glial maturation in early life, providing a foundation for future studies into these mechanisms.

Keywords: development; germ‐free; microbiota‐gut‐brain axis; microglia; myelination; neurodevelopment; neuronal activity; zebrafish.

FIGURE 1

The microbiota alters the developmental trajectory of PFC transcriptome associated with oligodendrocyte maturation and myelin‐related pathways. (A) Schematic representation of the experimental timeline for RNASeq of PFC. (B) Volcano plot illustrating the most differentially expressed genes (DEGs) in germ‐free (GF) male mice in young adulthood (p < 0.05). Genes (dots) to the left of “0” are downregulated, while genes to the right are upregulated. (C) Targeted enrichment analysis of genes associated with myelin‐related pathways in young adult GF mice relative to controls. Enrichment analysis was conducted by hypergeometric testing, where a solid grey circle indicates p < 0.05. (D) Heatmap showing the expression fold change (logFC) and significance of differentially expressed genes associated with myelin maturation, activity‐induced, myelin regulation, and nodal physiology of young adult GF versus control mice. Colour indicates logFC, with purple indicating increased expression and blue indicating decreased expression in GF mice compared to controls. (E) Volcano plot illustrating the most differentially expressed genes (DEGs) in GF mice during each of the four early developmental timepoints (p < 0.05). Genes (dots) to the left of “0” are downregulated, while genes to the right are upregulated. (F) Targeted enrichment analysis of genes associated with myelin‐related pathways in early development of GF mice relative to controls. Enrichment analysis was conducted by hypergeometric testing, where a solid grey circle indicates p < 0.05. (G–I) Heatmap showing the expression fold change (logFC) and significance of differentially expressed genes associated with (G) myelin maturation, (H) activity‐induced, myelin regulation, and (I) nodal physiology of GF versus control mice during all four early developmental timepoints. Colour indicates logFC, with purple indicating increased expression and blue indicating decreased expression in GF mice compared to controls. (J) Venn diagrams illustrating the overlap of significantly different genes (p < 0.05) between GF and CON groups across different developmental timepoints and sexes. Sample size: Young Adulthood: n = 8 mice/group, Early Development: n = 8–10 mice/group. Microbiota; *p < 0.05, **p < 0.01, ***p < 0.001, Sex; #p < 0.05, ##p < 0.01. Abbreviations: P: postnatal day, F: females, M: males, GF: germ‐free. Detailed statistical analysis can be found in Supplementary Table S1.

FIGURE 2

The microbiota influences the developing PFC metabolome, which correlates with transcriptomic signatures. (A) Schematic representation of the experimental timeline for metabolomics of PFC. (B) Principal Component Analysis (PCA) displaying differences in the PFC metabolome of germ‐free (GF) and conventionally‐raised (CON) in each of the early developmental timepoints assessed. Data analysed using PERMANOVA analysis. (C) Heatmap demonstrating the concentration (z‐scored) of metabolites that are differentially abundant in GF versus CON mice in at least one of the assessed timepoints (p < 0.05). Each square represents one mouse. (D) Untargeted enrichment analysis of metabolites that were significantly altered in GF compared to CON mice during early developmental timepoints. (E) Heatmap demonstrating the concentration (z‐score) of metabolites that are differentially abundant in young adult GF versus CON mice (p < 0.05). Each square represents one mouse. (F,G) Venn diagrams illustrating the overlap of significantly different metabolites (p < 0.05) between GF and CON groups across different developmental timepoints and sexes. (H) Schematic illustrating the procedure involved in multi‐omics analysis. (I) Representative schematic of glutamine metabolism in the tripartite synapse. Data presented as Mean ± SEM. Sample size: Young Adulthood: n = 8 mice/group, Early Development: n = 8‐10 mice/group. Microbiota; *p < 0.05, **p < 0.01, ***p < 0.001, Sex; #p < 0.05, ##p < 0.01. Detailed statistical analysis can be found in Supplementary Table S1.

FIGURE 3

The microbiota influences myelination. (A) Schematic representation of PFC region of interest. Olig2+/PDGFR⍺+ analysis was performed on the mPFC: anterior cingulate (AC), prelimbic (PL), and infralimbic (IL) (green outline). CNPase staining was performed across prefrontal cortex, including grey matter, white matter (corpus callosum), and the striatum (caudate and putamen), as well as other regions of interest, outlined in magenta. (B–D) Total number of (B) Olig2+ (C) PDGFR⍺+ and (D) Olig2+/PDGFR⍺+ cells per mm² within the mPFC of germ‐free (GF) and conventionally‐raised (CON) at P21. (E) Representative images of pan oligodendrocytes (Olig2⁺; green) and oligodendrocyte progenitor cells (PDGFR⍺⁺; magenta) immunofluorescence in the mPFC of GF and CON mice at P21. Scale bar, 100 µm. (F) Representative images of mature oligodendrocytes (CNPase⁺; white) immunofluorescence across GF and CON brain sections from rostral to caudal at P21. Magenta lines illustrate areas of interest. Scale bar, 200 µm. (G) Percentage of CNPase per mm² from rostral to caudal brains in GF and CON mice at P21. (H) Total percentage of CNPase per mm² in the PFC of GF and CON mice at P21. Data presented as Mean ± SEM. Sample size: Early Development P21: n = 6 mice/group. Microbiota; *p < 0.05, **p < 0.01, ***p < 0.001, Sex; #p < 0.05, ##p < 0.01. Detailed statistical analysis can be found in Supplementary Table S1.

FIGURE 4

The microbiota induces temporal changes in myelin growth and integrity. (A) Electron micrographs of axons in the mPFC of germ‐free (GF) and conventionally‐raised (CON) mice. Scale bar, 1 µm. (B) Total number of myelinated axons in the mPFC of GF and CON mice throughout the early life developmental timepoints. (C) Average axon diameter of myelinated axons in the mPFC of GF and CON mice at P21. (D) Representative images of myelin abnormalities (arrowheads) in GF male mice at P21. Scale bar, 1 µm. (E) Proportion of axons with and without outfolding and unravellings per mm² in the mPFC of GF and CON mice during the early developmental timepoints. (F) Average inner tongue thickness in GF and CON mice at P21. (G) Scatter plot of mean inner tongue thickness per axon diameter in the PFC of GF and CON mice at P21. (H) Representative electron micrographs of GF and CON mice at P21. (Green; inner tongue region). Scale bar, 0.2 µm. (I)Average number of laminae per myelinated axon in the mPFC of GF and CON mice during the early developmental timepoints. (J) Average myelin thickness in GF and CON mice at P21. (K‐L) Myelin thickness versus axon diameter in the PFC of GF and CON mice at P21. Data presented as Mean ± SEM. Sample size: Early Development: n = 3 mice/group, >50 axons/per mouse. Microbiota; *p < 0.05, **p < 0.01, ***p < 0.001, Sex; #p < 0.05, ##p < 0.01. Detailed statistical analysis can be found in Supplementary Table S1.

FIGURE 5

The microbiota modulates oligodendrocyte maturation and myelination across species. (A) Relative gene expression (RT‐qPCR) of glia, myelin, and neurotransmitter markers in GF and conventionally‐raised (CON) whole larvae at 5 dpf. (B) Schematic of the lateral larval zebrafish showing the region of interest and the method for calculating the g‐ratio. Imaging was conducted at the urogenital opening level, around somite 15. (C) Representative images of whole‐body myelin (white—Tg(mbp:memScarlet); nacre) in GF and CON larvae at 5 dpf. Scale bar, 300 µm. (D) Quantification of the total area of myelin (µm²) proportional to larval length at 5 dpf. (E) Super‐resolution confocal live‐imaging depicting the diameter of the Mauthner axon (magenta—Tg(hspGFF62A:Gal4)); Tg(UAS:mem‐Scarlet)) with myelination (green—Tg(mbp:eGFP‐CAAX)) at somite 15 at 5 dpf. The white boxed region depicts the area where analysis was performed (somite 15). Scale bar, 10 µm. (F,G) Quantification of Mauthner (F) axon diameter growth and (G) number of synaptic boutons at somite 15 at 5 dpf. (H) Representative images of the Mauthner axon tracing (region outlined with magenta) along with myelin sheath tracing (region outlined with green) for both GF and CON larvae at 5 dpf. Scale bar, 10 µm. (I–K) Quantification of (I) myelin diameter (J) myelin thickness, and (K) (g‐ratio) in GF larvae compared to controls at 5 dpf. Data presented as Mean ± SEM. Sample size: RT‐qPCR; n = 10–13, Whole body myelin; n = 21–26 larvae, confocal Imaging; n = 16–18 axons from individual larvae. Microbiota; *p < 0.05, **p < 0.01, ***p < 0.001. Detailed statistical analysis can be found in Supplementary Table S1.

FIGURE 6

The microbiota modulates microglial homeostasis and localization across species. (A‐B) Heatmap showing the expression fold change (logFC) and significance of differentially expressed genes associated with microglia homeostasis, activation and priming, and synaptic pruning in germ‐free (GF) versus conventionally‐raised (CON) mice during (A) early development and (B) adulthood. Colour indicates logFC, with purple indicating increased expression and blue indicating decreased expression in GF mice compared to controls. (C) Representative images of microglia/macrophages (magenta; Tg(mpeg1:eGFP) in the head of GF and CON larvae at 5 dpf. Scale bar 50 µm. (D,E) Quantification of the total number of (D) mepg1⁺ cells and the (E) total area and total area occupied per mepg1⁺ cell within the head of GF and CON larvae at 5 dpf. (F) Representative images of mepg1⁺ cells (magenta; Tg(mpeg1:eGFP) and oligodendrocytes (green; Tg(mbp:mScarlet)) in the dorsal and ventral spinal cord of GF and CON larvae at 5 dpf. Scale bar 300 µm. (G) Quantification of the total number of oligodendrocytes and microglia within the dorsal and ventral spinal cord of GF and CON larvae at 5 dpf. (H) Quantification of the total volume of oligodendrocyte and microglia within the dorsal and ventral spinal cord of GF and CON larvae at 5 dpf. (I) Schematic illustrating the region of interest, with emphasis on the lateral and dorsal spinal tracts as well as the peripheral tract. (J) Representative images of microglia/macrophages (magenta; Tg(mpeg1:eGFP) and oligodendrocytes (green; Tg(mbp:mScarlet)) in the spinal cord of GF and CON larvae at 5 dpf. Scale bar 50 µm. (K) Quantification of the total number of microglia‐oligodendrocyte contacts within the dorsal and ventral spinal cord of GF and CON larvae at 5 dpf. Data presented as Mean ± SEM. Sample size: Adulthood: n = 8 mice/group, Early Development: n = 8–10 mice/group, Zebrafish Head: n = 33–47 larvae. Zebrafish whole spinal cord: n = 21–23 larvae, Zebrafish microglia‐oligodendrocyte contacts: n = 30–45 larvae, Microbiota; *p < 0.05, **p < 0.01, ***p < 0.001. Abbreviations: P: postnatal day, F: females, M: males, GF: germ‐free. Detailed statistical analysis can be found in Supplementary Table S1.