ZFIN Image: Saraswathy et al., 2022, Fig. 6

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Figure Caption

Fig. 6

mstnb regulates neuronal differentiation after SCI via fgf1b

(A) mstnb^−/− and wild-type siblings were subjected to complete SC transections and collected at 7 dpi for RNA-seq. Control SC tissues were collected from uninjured fish. Sequencing was performed in independent duplicates from separate fish clutches. Principal-component analysis shows clustering of biological replicates. Principal components 1 and 2 (PC1 and PC2, respectively) show 56% and 38% variance, respectively.

(B and C) Volcano plot representation of genes that are significantly upregulated or downregulated in mstnb^−/− SCs relative to wild-type controls. Upregulated genes include genes with log₂(fold enrichment) > 1 and adjusted p < 0.01. Downregulated genes include transcripts with log₂(fold enrichment) < −1 and adjusted p < 0.01. Selected upregulated (green) and downregulated (magenta) neuronal genes are indicated. Unchanged genes are labeled in black.

(D) qRT-PCR for neuronal genes was performed on mstnb^−/− and wild-type SCs at 7 dpi. Uninjured mstnb^−/− and wild-type controls were used. For each time point, log₂(fold change) was normalized to eif1α and to gene expression levels in mstnb^+/+ controls.

(E) fgf1b qRT-PCR was performed on uninjured and injured mstnb^−/− and wild-type animals. For each time point, fgf1b expression was normalized to eif1α as a loading control and to fgf1b levels in mstnb^+/+ controls.

(F) Experimental timeline for human recombinant FGF1 treatment in mstnb^−/− animals.

(G) Neuronal gene expression was analyzed by qRT-PCR from FGF1- and vehicle-treated SC tissues at 7 dpi. For each gene, log₂(fold change) was normalized to eif1α as a loading control and to gene expression levels in uninjured mstnb^+/+ SCs.

(H) HuC/D and EdU staining was performed on vehicle- and FGF1-treated mstnb^−/− and wild-type animals. The numbers of regenerating HuC/D^>+ EdU^>+ neurons were quantified in SC cross sections 150 μm rostral to the lesion site. The proportions of HuC/D^>+ EdU^>+ neurons (%) were normalized to the numbers of HuC/D^>+ neurons for each section.

Dots represent individual animals from 2 independent clutches. Error bars depict SEM, and statistical significance was determined by one-way ANOVA. Multiple-comparisons p values are shown. ***p < 0.001; **p < 0.01; *p < 0.05; ns, p > 0.05.

Acknowledgments

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