Zebrafish Chromosome 14 Gene Differential Expression in the fmr1hu2787 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 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.

Cyfip2 controls the acoustic startle threshold through FMRP, actin polymerization, and GABABreceptor function

“…These data indicate that FMRP, but not FXR1/2, regulates the startle threshold. The discrepancy between our hypersensitive fmr1 crispants and prior analysis of non-hypersensitive fmr1 hu2787 mutants could be due to genomic adaptation that has been reported in the fmr1 hu2787 mutant line that may partially compensate for the loss of FMRP [50]. In support of this, an independently created CRISPR-generated fmr1 mutant line displayed additional behavioral and developmental phenotypes not originally seen in the fmr1 hu2787 line [51].…”

“…Similarly, in Drosophila , which have only one Fragile X protein family member (dFMR1), re-expressing human FMRP (hFMR1), but not human FXR1 or FXR2, in dFMR1 mutants is sufficient to specifically rescue aberrant neuronal phenotypes [97]. As discussed above, the fact that fmr1 crispants but not fmr1 hu2787 mutants show startle hypersensitivity may be due to genomic adaptation in the ENU-induced fmr1 hu2787 line [50]. That we observed that fmr1 crispants show heightened startle sensitivity in the cyfip2 mutant background compared to cyfip2 mutants alone (Fig.…”

Differential allelic representation (DAR) identifies candidate eQTLs and improves transcriptome analysis

“…Our previous observation of CC-DEGs in our analysis of homozygous fmr1 mutant zebrafish embryos (8) led us to re-examine our numerous adult zebrafish brain transcriptome datasets where we had previously analysed the effects of dominant mutations in heterozygotes. Surprisingly, we saw CC-DEGs in a number of our analyses as illustrated by Manhattan plots of DE gene expression (Figure 2A and Supplementary Figure 1A-B).…”

“…If such selective pressure is important, then why have CC-DEGs not been observed more widely in transcriptome studies? It is notable that the only situations where our laboratory has observed distinct CC-DEG patterns are in well-controlled comparisons of single gene mutations in 2 dpf embryos (8), 7 dpf larvae, or in young (∼6-month-old zebrafish & ∼3-month-old mouse) whole brains/cortices. If genes are connected in complex, homeostatic networks, then disturbance of only one network node would cause great expression changes in only the most closely co-functional nodes, much as pulling on one point in a spider’s web causes greatest displacement for the most closely associated threads.…”

“…Indeed, obvious enrichments of DE genes on the same chromosome as a mutation-of-interest have previously been observed in analyses involving comparisons of homozygous mutant with homozygous wild-type individuals (6,7). We recently observed such CC-DEGs when comparing transcriptomes from pools of embryos either wild-type or homozygous for a loss-of-function mutation in the zebrafish gene fmr1 (orthologous to the human FMR1 gene mutated in Fragile X Syndrome) (8). However, that analysis also revealed enrichment in homozygous mutant embryos for a particular gene set previously seen as affected in Fmr1 knock-out mice (8,9), supporting the possibility that some of the CC-DEGs do participate in fmr1 functions, and highlighting challenges in this aspect of transcriptome analysis.…”