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 fmr1hu2787 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 fmr1hu2787 mutation and made manifest due to the limited, but non-zero, allelic diversity between the genotypes compared.
Highlights
Fragile X Syndrome (FXS) is an X-chromosome-linked form of human inherited intellectual disability affecting, on a panethnic basis, approximately 1 in every 2,400 male foetuses (Owens et al, 2018) (As females are diploid for the X-chromosome, the proportion of female foetuses affected is far lower)
We described changes in axonal branching that could be rescued by treatment with the metabotropic glutamate receptor (mGluR) antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP), similar to observations of mGluR effects in mouse and Drosophila analyses of FMRP translational regulator 1 (FMR1) activities
Differential Gene Expression in fmr1hu2787/hu2787 vs. + / + Embryos at 2 dpf Transcriptome (RNA-seq) analysis was conducted on clutches of wild type and fmr1hu2787 homozygous mutant embryos (n = 4 clutches for each genotype) that had been allowed to develop for 2 dpf at ∼28.5◦C
Summary
Fragile X Syndrome (FXS) is an X-chromosome-linked form of human inherited intellectual disability affecting, on a panethnic basis, approximately 1 in every 2,400 male foetuses (Owens et al, 2018) (As females are diploid for the X-chromosome, the proportion of female foetuses affected is far lower). FXS most commonly arises due to expansion of a CGG trinucleotide repeat sequence in the human FMR1 gene that codes for an RNA-binding protein involved in multiple aspects of RNA metabolism (Salcedo-Arellano et al, 2020). The trinucleotide repeat expansion promotes methylation and repression of FMR1 transcription (Pieretti et al, 1991). The syndrome is complicated by the existence of subtle cognitive effects due to limited expansion of the trinucleotide repeat known as a “premutation” (Salcedo-Arellano et al, 2020). The syndrome displays the phenomenon of anticipation whereby phenotype severity increases in successive generations due to expansion of the trinucleotide repeat beyond a premutation range (Sutherland et al, 1991)
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