Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

Abstract

Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells’ vulnerability. Gallus gallus embryos having 3–6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10−17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10−12, 60 genes), RNA Polymerase (P = 2.2 x 10−3, 15 genes) and Spliceosome (P = 2.6 x 10−2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work suggests ribosome biogenesis may be a novel target mediating alcohol’s damage to developing neural crest. Our findings are consistent with observations that gene-environment interactions contribute to vulnerability in FASD.

Gene-environment interactions characterized by single embryo transcriptomics

“…Our data demonstrates that the effect of this dose of ethanol on the early transcriptome is mild; but nonetheless caused detectable transcriptional responses using a single embryo assay. Our study is unique from other transcriptome profiling studies, which often use a higher dose of ethanol to induce craniofacial and neurological malformations, resulting in large-scale cellular and transcriptional changes (Berres et al, 2017;Green et al, 2007). Phenotypic dysmorphology results in a wild-type background with high enough concentrations of ethanol (Blader and Strahle, 1998;Joya et al, 2014), which can easily alter gene expression, but which also cause widespread changes in cellular composition, which obfuscate pathogenic gene expression changes.…”

Proteomic analysis of ametryn toxicity in zebrafish embryos

“…In addition, knockdown or mutation of rpl3 resulted in the impaired expansion of pancreatic progenitor cells in zebrafish . Berres et al reported that ribosome biogenesis may be a novel target mediating alcohol's damage to developing neural crest during embryonic development. We suggested that impairment of ribosomal proteins expression by AMT would cause the abnormal development in embryos.…”

The avian embryo as a model for fetal alcohol spectrum disorder

“…Using whole exome sequencing, we found that the greatest gene-level changes in response to alcohol were significant reductions in ribosome, oxidative phosphorylation, and spliceosome pathways (Berres et al 2017). Ribosome dysbiogenesis causes neural crest apoptosis and craniofacial deficits in humans and in animal models (Danilova and Gazda 2015; Yelick and Trainor, 2015), including Diamond-Blackfan anemia and Treacher-Collins syndrome.…”

“…To test the potential contribution of expression-level differences to alcohol vulnerability, we used a morpholino approach in zebrafish to create targeted reductions in RPL5A, RPL11, or RPS3A, but to intermediate levels that did not affect facial development. Addition of a moderate alcohol dose produces significant craniofacial deficits and neural crest apoptosis only in embryos that are also haploinsufficient for RPL5A, RPL11, or RPS3A (Berres et al 2017). This supports work in zebrafish and mouse showing that otherwise silent genetic alterations increase vulnerability to alcohol’s teratogenesis.…”