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.

Fig 1. zrps3a, zrpl5a or zrpl11 knockdown heightens vulnerability to alcohol-induced craniofacial deficits in zebrafish embryos.

(A-E) Untreated embryos (A) had normal development of cranial cartilage elements, and these were largely unaffected by treatment with a nonsense morpholino (B) or morpholinos directed against zrps3a (C), zrpl5a (D), or zrpl11 (E). (F-J) Alcohol treatment modestly reduced the size and shape of cranial cartilage elements in otherwise normal embryos. Treatment with a nonsense morpholino (G) did not further worsen cranial development. However, the combination of alcohol with morpholino directed against zrps3a (H), zrpl5a (I), or zrpl11 (J) resulted in ablation of many cranial cartilage elements and reduced ocular size. Embryos are either 4 dpf (A-D, F-I) or 3 dpf (E, J). All views are ventral with equivalent magnification. Abbreviations used: c, cardiac edema; cb, ceratobranchial; ch, ceratohyal; ep, ethmoid plate; m, Meckel’s cartilage.

Fig 2. zrps3a, zrpl5a, or zrpl11 Knockdown Heightens Vulnerability to Alcohol-Induced Apoptosis in 12 hpf Zebrafish Embryos.

(A-E) Untreated embryos (A) had few TUNEL+ cells in the cranial region (arrow). Treatment with a nonsense morpholino (B) caused a modest increase in TUNEL⁺ cells within the cranial region (arrow), as did morpholinos directed against zrps3a (C), zrpl5a (D), or zrpl11 (E). (F-J) Alcohol treatment (F) caused appreciable apoptosis within both cranial and somatic regions. The addition of nonsense morpholino treatment (G) did not further increase TUNEL⁺ cell numbers in alcohol-treated embryos. However, the combination of alcohol with morpholino directed against zrps3a (H), zrpl5a (I), or zrpl11 (J) resulted in higher levels of apoptosis within the cranial region as compared with embryos that received the same morpholino and no alcohol (C-E), or alcohol and no morpholino (F). All views are lateral at equivalent magnification, with rostral at the top and the embryo ‘looking’ left. Arrow indicates the cranial region. (K) Enumeration of TUNEL⁺ cranial cells in alcohol- and morpholino-treated embryos. Values are mean ± S.D. with 10–12 embryos per treatment. ^* Alcohol group differs from no-alcohol control within a treatment at P<0.05. ^† Morpholino-treated differs from its irrelevant-morpholino control at P<0.05. Abbreviation used: Alc, alcohol; C, control; MO, morpholino; y, yolk.