Dark-rearing uncovers novel gene expression patterns in an obligate cave-dwelling fish

Abstract

Extreme environments often result in the evolution of dramatic adaptive features. The Mexican tetra, Astyanax mexicanus, includes 30 different populations of cave-dwelling forms that live in perpetual darkness. As a consequence, many populations have evolved eye loss, reduced pigmentation, and amplification of nonvisual sensory systems. Closely-related surface-dwelling morphs demonstrate typical vision, pigmentation, and sensation. Transcriptomic assessments in this system have revealed important developmental changes associated with the cave morph, however, they have not accounted for photic rearing conditions. Prior studies reared individuals under a 12:12 hr light/dark (LD) cycle. Here, we reared cavefish under constant darkness (DD) for 5+ years. From these experimental individuals, we performed mRNA sequencing and compared gene expression of surface fish reared under LD conditions to cavefish reared under DD conditions to identify photic-dependent gene expression differences. Gene Ontology enrichment analyses revealed a number of previously underappreciated cave-associated changes impacting blood physiology and olfaction. We further evaluated the position of differentially expressed genes relative to QTL positions from prior studies and found several candidate genes associated with these ecologically relevant lighting conditions. In sum, this work highlights photic conditions as a key environmental factor impacting gene expression patterns in blind cave-dwelling fish.

Keywords: Mexican tetra; RNA-seq; regressive evolution; troglomorphy.

Figure 1.. Correlations between morphotypes are least similar under natural conditions.

Each point on this scatterplot represents an individual gene. Some genes (red) have a higher expression in the condition on the y-axis. Other genes (blue) have a higher expression in the condition on the x-axis. LD vs. DD (A) is more similar than light/dark conditions (B), while natural conditions (C) are the least related based on the R² and spread of points about the line of regression (purple). As each of these scatterplots encapsulates all 25,271 genes, any visible changes correlate to large scale changes in global gene expression. The increasing distance between the line of regression (purple) and the zero-fold line (green) viewed between panels A to C yields an approximation of the increase in difference between the two conditions.

Figure 2.. More genes are under-expressed in cavefish, regardless of rearing conditions.

Genes with a higher (plus) or lower (minus) expression in cavefish in comparison to surface fish under both natural and light/dark conditions reveal a higher number of genes with a lower expression in cavefish regardless of the lighting condition. This pattern may indicate that the function of many genes is reduced in the cavefish compared to the surface fish. The replication of this pattern in the dark indicates that this pattern of under-expression in cavefish is conserved regardless of lighting condition.

Figure 3.. Rearing under natural photic conditions reveals novel changes to gene expression.

A) The number of differentially expressed genes unique to either light/dark conditions or natural conditions is represented in the 4^th and 5^th columns of the table, respectively. These DEGs are inclusive of genes with a higher expression in either cave or surface fish on their respective lighting conditions, represented diagrammatically to the right of the table (Venn diagram). There are a large number of differentially expressed genes that are revealed under natural conditions alone. This large number of DEGs represent a set of genes that have not been previously explored B) Test sets of genes were selected based on their expression at a 4-fold threshold. DEGs unique to natural conditions were assessed. Under light/dark conditions, 345 genes have a higher expression in C^DD compared to S^LD and 399 have a lower expression in C^DD compared to S^LD. The proportion of GO terms in these test sets was compared to the proportion of GO terms found in the whole transcriptome as a reference. The observed occurrence of each term is taken over the expected occurrence of each term based on is proportion in the reference. Each white bar to the right of a term represents an “Enrichment Score” (observed/expected). This indicates that dark-rearing may potentially play a role in the development and function of the circulatory, olfactory, and metabolic systems in A. mexicanus cavefish.

Figure 4.. Vision and pigmentation genes localized to previously identified QTL providing novel candidate genes.

The distribution of vision-related (A) and pigmentation-related (B) QTL (light orange or light pink boxes) across the 25 chromosomes of the draft Astyanax genome (gray boxes, CH:1–25). Gene identity and function are represented in the tables to the right. Values are given in RPKM (normalized expression). Some genes fall remarkably close to QTL (represented as bold hash marks and text) and demonstrate relevant alterations in development when function is disrupted in other systems. * indicates gene name was derived from Danio rerio based on sequence similarity. ** indicates RPKM was obtained via RNA-seq alignment to Astyanax NCBI draft genome by chromosome, and cannot be directly compared to other values, although intra-gene comparisons of expression based on morphotype and lighting condition are valid. ^† indicates an A. mexicanus gene that aligns to the same protein in D. rerio as another A. mexicanus gene by BLAST. *** is used in place of expression values for the duplicate mc1r gene due to incomplete functional and coding information for this copy number variant.