Zebrafish Chromosome 14 Gene Differential Expression in the fmr1 h u 2787 Model of Fragile X Syndrome

Abstract

Zebrafish represent a valuable model for investigating the molecular and cellular basis of Fragile X syndrome (FXS). Reduced expression of the zebrafish FMR1 orthologous gene, fmr1, causes developmental and behavioural phenotypes related to FXS. Zebrafish homozygous for the hu2787 non-sense mutation allele of fmr1 are widely used to model FXS, although FXS-relevant phenotypes seen from morpholino antisense oligonucleotide (morpholino) suppression of fmr1 transcript translation were not observed when hu2787 was first described. The subsequent discovery of transcriptional adaptation (a form of genetic compensation), whereby mutations causing non-sense-mediated decay of transcripts can drive compensatory upregulation of homologous transcripts independent of protein feedback loops, suggested an explanation for the differences reported. We examined the whole-embryo transcriptome effects of homozygosity for fmr1 ^{h u2787} at 2 days post fertilisation. We observed statistically significant changes in expression of a number of gene transcripts, but none from genes showing sequence homology to fmr1. Enrichment testing of differentially expressed genes implied effects on lysosome function and glycosphingolipid biosynthesis. The majority of the differentially expressed genes are located, like fmr1, on Chromosome 14. Quantitative PCR tests did not support that this was artefactual due to changes in relative chromosome abundance. Enrichment testing of the "leading edge" differentially expressed genes from Chromosome 14 revealed that their co-location on this chromosome may be associated with roles in brain development and function. The differential expression of functionally related genes due to mutation of fmr1, and located on the same chromosome as fmr1, is consistent with R.A. Fisher's assertion that the selective advantage of co-segregation of particular combinations of alleles of genes will favour, during evolution, chromosomal rearrangements that place them in linkage disequilibrium on the same chromosome. However, we cannot exclude that the apparent differential expression of genes on Chromosome 14 genes was, (if only in part), caused by differences between the expression of alleles of genes unrelated to the effects of the fmr1 ^{h u2787} mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.

Keywords: FMR1; chromosome evolution; fragile X syndrome; homeostasis; linkage disequilibrium; transcriptional adaptation; transcriptome analysis; zebrafish.

FIGURE 1

Transcriptome analysis of fmr1^{hu2787/hu2787} vs. wild type entire embryos at 2 dpf. (A) Principal Component (PC) analysis of larval wild type (A, D, G, L) and fmr1^{hu2787/hu2787} (S2, S4, S5, S8) RNA-seq data. (B) Volcano plot of differential gene expression. (C) Mean-difference (MD) plot showing the average levels of expression of RNAs plotted against fold change differences in expression between mutant and wild type embryos. (D) Volcano plot of differential expression as in b, but indicating genes sharing some degree of sequence homology with fmr1 (indicated in blue). No significantly increased expression is seen.

FIGURE 2

Chromosome distribution of DE genes. (A) Manhattan plot of p-values for differential expression of genes on the chromosomes of zebrafish. An enrichment for DE genes on Chromosome 14 is apparent with possible enrichment also for genes on Chromosome 22 (see also Supplementary Data File 2). (B) A diagram of zebrafish Chromosome 14 taken from https://asia.ensembl.org (Yates et al., 2020) and including a representation of relative protein coding gene density along the chromosome. Loci for the Chromosome 14 genes found to be DE in fmr1^{hu2787/hu2787} 2 dpf embryos are indicated (see also Table 1).