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.

RNA-sequencing analysis excels in its ability to infer transcriptomic differences between discrete genetic states. This advantage is often leveraged to explore the consequences of single mutations causing disease, where prior knowledge of expected outcomes may not be established. Successful interpretation of such RNA-seq studies reveals the direct impacts of the mutation, as well as the homeostatic responses of the biological system. Recent studies have highlighted that, when homozygous mutations are studied in non-isogenic backgrounds, genes from the same chromosome as the mutation tend to be over-represented among differentially expressed (DE) genes. One hypothesis suggests that DE genes chromosomally linked to the mutation may not be true biological responses to the disruption of the mutation but, instead, result from differences in the representation of expression quantitative trait loci (eQTLs) that differ between the sample groups being compared. This can be problematic since including spurious DE genes in a functional enrichment study may result in incorrect inferences of mutation effect. Here we show thatchromosomally co-located differentially expressed genes(CC-DEGs) can also be observed in analyses of dominant mutations in heterozygotes. We define a method and a metric to quantify, in RNA-seq data, localised differential allelic representation (DAR) between groups of samples subject to DE analysis. We show how the DAR metric can predict regions prone to eQTL-driven differential expression, and how it can improve functional enrichment analyses through gene exclusion or weighting of gene-level rankings. Advantageously, this improved ability to identify probable eQTLs also reveals examples of CC-DEGs thatarelikely to be functionally related to a mutant phenotype. This situation was predicted by R.A. Fisher in 1930 as due to selection for advantageous linkage disequilibrium after chromosomal rearrangements. By comparing the genomes of zebrafish (Danio rerio) and medaka (Oryzias latipes), a teleost with a conserved ancestral karyotype, we find possible examples of chromosomal aggregation of CC-DEGs during evolution of the zebrafish lineage. The ability to identify and exclude eQTL artefacts from true transcriptomic responses to mutation provides exciting opportunities for developing new approaches for analysing RNA-seq data. Our DAR metric provides a solid foundation for addressing the eQTL issue in new and existing datasets because it relies solely on RNA-seq data, which will thus improve our understanding of the mechanisms of disease-causing mutations.

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Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.