Brain of the blind: transcriptomics of the golden-line cavefish brain

Brain of the blind: transcriptomics of the golden-line cavefish brain The genus Sinocyclocheilus (golden-line barbel) includes 25 species of cave-dwelling blind fish (cavefish) and more than 30 surface-dwelling species with normal vision. Cave environments are dark and generally nutrient-poor with few predators. Cavefish of several genera evolved conver- gent morphological adaptations in visual, pigmentation, brain, olfactory, and digestive systems. We compared brain morphology and gene expression patterns in a cavefish Sinocyclocheilus anophthalmus with those of a closely related surface-dwelling species S. angustiporus. Results showed that cavefish have a longer olfactory tract and a much smaller optic tectum than surface fish. Transcriptomics by RNA-seq revealed that many genes upregulated in cavefish are related to lysosomes and the degradation and metabolism of proteins, amino acids, and lipids. Genes down- regulated in cavefish tended to involve “activation of gene expression in cholesterol biosynthesis” and cholesterol degradation in the brain. Genes encoding Srebfs (sterol regulatory element- binding transcription factors) and Srebf targets, including enzymes in cholesterol synthesis, were downregulated in cavefish brains compared with surface fish brains. The gene encoding Cyp46a1, which eliminates cholesterol from the brain, was also downregulated in cavefish brains, while the total level of cholesterol in the brain remained unchanged. Cavefish brains misexpressed several genes encoding proteins in the hypothalamus–pituitary axis, including Trh, Sst, Crh, Pomc, and Mc4r. These results suggest that the rate of lipid biosynthesis and breakdown may both be depressed in golden-line cavefish brains but that the lysosome recycling rate may be increased in cavefish; properties that might be related to differences in nutrient availability in caves. Key words: cavefish, cholesterol, cyp46a, optic tectum, Sinocyclocheilus, transcriptomics. Caves present organisms with an extreme environment, but various (Protas et al. 2008; Windsor et al. 2008; Jeffery 2009; Romero et al. lineages of fish have independently colonized these perpetually dark 2009; Gross 2012; Krishnan and Rohner 2017). Among cavefish realms. Teleost fish from at least 19 families have successfully colon- species, investigations of the Mexican tetra Astyanax mexicanus ized caves from 40 N to the Tropic of Capricorn (Zhao et al. 2011). have led to many important advancements concerning the evolution Cave-dwelling fish have evolved a suite of troglomorphic traits, of troglodyte traits, for example, the eyes and optic tectum of including reduction or loss of eyes, pigmentation, and scales, Astyanax cavefish are greatly reduced (Rohner et al. 2013; Elipot coupled with the enhancement of chemoreceptors in olfactory et al. 2014; McGaugh et al. 2014; Simon et al. 2017; Stahl and organs and mechanoreceptors including the lateral-line system Gross 2017). V C The Author(s) (2018). Published by Oxford University Press. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2 Current Zoology, 2018, Vol. 0, No. 0 ’The cyprinid Sinocyclocheilus (golden-line barbel) is the most Materials and Methods species-rich cavefish genus with more than 55 known species, about 25 Animals of which live in energy-limited cave environments (Romero et al. 2009; Golden-line cavefish S. anophthalmus were collected in Jiuxiang cave, Zhao et al. 2011; Meng et al. 2013 b; Yang et al. 2016). Phylogenetic Yiliang County (N 25.05478 , E 103.37975 , Yunnan, China) and analyses based on mitochondrial cytochrome b and ND4 gene sequences maintained in the laboratory in a dark environment. The surface spe- have shown that the known Sinocyclocheilus species cluster into 5 major cies, S. angustiporus were collected from Huangnihe River in Agang monophyletic clades (Xiao et al. 2005), several of which have cave- Town (N 25.00905 , E 103.59256 , Yunnan, China). Both collection dwelling forms, suggesting that different lineages of Sinocyclocheilus sites were in the Nanpanjiang River drainage, the largest tributary of have adapted to cave environments independently several times. Xijiang River in the Pearl River basin. Although the two sites are only Comparative transcriptomic and genomic analyses of Sinocyclocheilus about 30 km apart in a straight line, they are separated by about 100 species have revealed many genetic changes that were associated with river-kilometers (Supplementary Figure S1). The obligatory cave adaptive features such as eye degeneration, albinism, rudimentary scales, species and the surface species are closely related phylogenetically circadian rhythm, and enhanced taste buds (Meng et al. 2013a; Yang (Xiao et al. 2005; Zhao and Zhang 2009) and the sequences of their et al. 2016). Thus, Sinocyclocheilus provides an ideal model genus to orthologs are highly similar, 98.126 0.9% identical at the nucleotide evaluate the mechanisms of adaptation in cave animals. level (Meng et al. 2013a), suggesting that differences between them in Like cavefish, some people are born blind and the brains of blind terms of gene expression levels are more likely to be due to evolved dif- people develop a compensatory reorganization, especially in areas that ferences adapting to habitat rather than to random neutral differences appear to help improve spatial resolution of sounds (Roder et al. 1999). that occur over time. While the two congeners S. grahami and S. tingi Similar types of brain reorganization may occur in blind cavefish. are more closely related to S. anophthalmus than is S. angustiporus, Several studies have investigated the eye, brain, and behavior in cavefish unfortunately, these more closely related species are not available in (Soares et al. 2004; Menuet et al. 2007; Yoshizawa et al. 2010; Strecker sufficient numbers to perform the required experiments. Surface et al. 2012; Yoshizawa et al. 2015). These studies show that cavefish animals were maintained on a 12: 12 Light: Dark cycle. Cave and have a larger hypothalamus region, reduced the size of the optic tectum, surface fish were fed twice per day with the same carp food (Sanyou well-developed olfactory bulbs, and more sensory hair cells in the Chuangmei company, Beijing) in mini pellets, which delivered a nutri- neuromasts of the lateral line system. An additional feature of many tionally complete formula. Uneaten food was removed 15 min after cave habitats is low and sporadic nutrient availability, and several feeding. All experimental procedures involving animals were con- studies have investigated cavefish energy metabolism. For example, ducted and approved by the Animal Care and Use Committee of Meng et al. (2013a) found that several genes in the mitochondrial gen- Institute of Zoology, Chinese Academy of Sciences. ome that are relevant to energy metabolism are downregulated in the cavefish eye. These results raised the hypothesis that these adaptations Brain volume analysis contribute to the regulation of energy metabolism in golden-line cave- To investigate the effects of constant darkness on brain morphology, fish in their perpetually dark, clear, slow moving, and presumably we measured various regions of cavefish and surface fish brains. nutrient-poor streams, where bat guano or periodic flooding are the Surface fish and cavefish were euthanized with 0.05% tricaine metha- only sources of outside nutrients. Although the brain expends a nesulfonate and decapitated (3 individuals per species). The dorsal substantial fraction of an animal’s whole-body energy budget surface of the head was dissected away to expose the brain directly to (Ivanisevic and Siuzdak 2015), it is unknown whether energy metabol- prefix in 4% paraformaldehyde (PFA) for 6 h, and then brains were ism in the cavefish brain has adapted to the food-limited cave environ- dissected from the head, fixed again in 4% PFA overnight at 4 C, ment. This situation led us to wonder how the greatly reduced eyes in and finally embedded in paraffin. Transverse sections (10 lmthick) congenitally blind golden-line cavefish (Meng et al. 2013a, 2013 b), of whole brains of both species were mounted and stained with hema- which would be pathogenic in a surface-dwelling fish, would affect toxylin and eosin. The area of various regions of the brain was meas- brain structure and gene expression patterns over evolutionary time. ured on every 8th section (80 lm) using ImageJ (version 10.2). The In this study, we first showed that the volume of the optic tectum volume was estimated by calculating the area of each section and the was significantly smaller in the cavefish Sinocyclocheilus anophthalmus distance between the sections (Rosen and Harry 1990). To measure than in the surface fish S. angustiporus and the length of the olfactory the volume of different brain regions, we calculated the volume of 6 tract was significantly greater in cavefish than in surface fish. Next, to brain areas (olfactory bulb, telencephalon, diencephalon, optic tec- quantify differences in gene expression, we compared the transcrip- tum, cerebellum, and medulla oblongata). The telencephalon has two tomes of surface and cavefish by RNA-seq analyses. We identified subdivisions, the area dorsalis and the area ventralis telencephali; differentially expressed genes, and found that upregulated genes in the cerebellum measurements encompassed the crista cerebellaris, corpus cavefish brain were involved in several pathways related to the lyso- cerebelli, and valvula cerebelli. The diencephalon and medulla oblon- some and the degradation and metabolism of proteins, amino acids, gata were distinguished by the nuclei of the preglomerular complex. and lipids. Downregulated genes in cavefish brains included the sterol The end of the medulla oblongata was the medial funicular nucleus. regulatory element-binding transcription factor genes (srebf1, srebf2) The volume of different brain regions was normalized to fish standard and their transcriptionally regulated targets involved in cholesterol length (from the tip of the snout to the end of the caudal peduncle). biosynthesis, and in addition cyp46a1, which encodes the enzyme Statistical analysis was performed using a two-tailed Student’s t-test responsible for cholesterol elimination from the brain. Direct measure- in Microsoft Excel. All data met the assumption of normality. ments of cholesterol levels showed no differences between cavefish and surface fish brains, suggesting that decreased biosynthesis and decreased degradation of cholesterol balanced each other. We specu- Differential gene expression analysis late that transcriptome evolution in the cavefish brain may have led to To obtain insights into the molecular genetic mechanisms involved in energy savings in cholesterol metabolism, which might help this species the evolution of the cavefish brain, we profiled gene transcription in to adapt to a presumably resource-poor cave environment. dissected brains. We generated total RNA from the brain of two adult Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Meng et al.  Transcriptomics of the golden-line cavefish brain 3 individuals of each species (surface and cave dwelling species). Poly Identification of enriched pathways (A) RNA was isolated from total RNA samples using MicroPoly(A) Enriched pathways were identified from differentially expressed PuristTM (Ambion) according to the manufacturer’s protocol. Brain genes in the brains of cavefish and surface fish using KOBAS 3.0 cDNA libraries were obtained from two individuals for each species (updated 26 January 2015) (Xie et al. 2011), a program that assigns following established protocols (Meng et al. 2013a). Samples (200- to putative pathways and disease relationships to a gene set and pro- 400-bp inserts) were sequenced using 80-nt paired-end reads from an vides statistically significant enriched pathways from 5 pathway Illumina sequencer GA-II (Illumina Inc., San Diego, CA, USA). databases. Sequences of differentially expressed genes were com- Barcodes were identified and nucleotides with low-quality base pared to the Homo sapiens database using the “annotate” feature in calls were removed, and then reads were mapped to the previously KOBAS 3.0 to allow inferences from the data on human pathways. assembled golden-line transcriptome (Meng et al. 2013a) using We then used “identify” to find significantly enriched pathways; Bowtie (Langmead 2010). In Bowtie, “the maximum mismatches “inputs” were the output of “annotate” for upregulated and down- per read” was set to 3 while other parameters were left as default. regulated gene sets, and the “background” was the entire set of Accession numbers are GAHO01000000 for S. angustiporus and 11,471 unique genes expressed in the golden-line brain identified by GAHL01000000 for S. anophthalmus at DDBJ/EMBL/GenBank. RNA-seq. Data were analyzed using a hypergeometric test and Mapped reads of both surface and cave species were converted to Benjamini–Hochberg FDR (false discovery rate) correction, and RPKM (reads per kilobase of exon per million mapped sequence only pathways or diseases with a corrected P < 0.05 were considered reads) values and normalized (Mortazavi et al. 2008). To enhance to be enriched. statistical robustness, genes with fewer than 5 RPKM in either spe- cies were excluded from the pathway enrichment and gene ontology Cholesterol content (GO) analyses, but these genes are recorded in a subsheet of Brain, liver, and muscle tissues of cavefish and surface fish were Supplementary Table S1. P-values were obtained for each gene by homogenized in 50 mM NaCl. The lipid fraction was then extracted computing a conditional probability of observing N1 reads for a through multiple washes with a 2:1 chloroform: methanol solution. gene given that we obtained N2 reads from the controls and experi- Samples were dried down with 10% triton-X 100/acetone mentals (Audic and Claverie 1997). Genes were identified as (Suzuki et al. 2013). Cholesterol content was assayed by enzymatic differentially expressed when fold change (FC) was> 2 and assay according to the manufacturer’s protocol (Wako Chemicals, P< 0.05. We used WebGestalt (Wang et al. 2013) to identify cat: 439-17501). functional categories among the differentially expressed genes. Real-time polymerase chain reaction Results cDNA samples were created from mRNAs isolated from the brains of surface and cave-dwelling individuals. Real-time polymerase chain reac- The optic tectum is smaller in cavefish than in surface fish tion (PCR) was conducted using SYBR Green (TaKaRa) chemistry. The cave-dwelling species S. anophthalmus has small internal eyes Real-time PCR primers were designed based on the golden-line tran- in contrast with those of its closely related surface-dwelling species scriptome sequence assembled previously (Meng et al. 2013a). Primer S. angustiporus (Figure 1A,B, Supplementary Figure S2A). Laser sequences were as follows (forward and reverse): b-actin:5 -GAA light (wavelengths are 650 nm for red and 532 nm for green) was 0 0 GATCAAGATCATTGCTCCC-3 and 5 -ATGTCATCTTGTTCGAG shined into the eyes of cavefish and surface fish. Surface fish re- 0 0 0 0 AGGT-3 ; cyp46a1.3:5 -GGAAACGCTGCGTCTGTA-3 and 5 -GG sponded by moving to avoid the light. In contrast, cavefish treated 0 0 TTCGTGGACCAAGTGC-3 ; cyp51:5 -CATCCTGCAAACGCTGAT in the same way made no response (cavefish n ¼ 12, surface fish 0 0 0 0 AG-3 and 5 -AGTGCAGAGGAGGCAGATGT-3 ; dhcr24:5 -ATGG n ¼ 9). We conclude that the eyes of cavefish do not detect light or 0 0 GAACAGGCATTGAGTC-3 and 5 -TAGCGAAGCTTCACCCAT that cavefish fail to react to light. 0 0 0 0 TT-3 ; ebp:5 -AACGCGGGAAATAATCACAT-3 and 5 -TGAAC Measurements showed that cavefish brains were slimmer than 0 0 GGTCATTAGCCACAT-3 ; faxdc2:5 -GTTGTTTAACGCCCTCC those of surface fish (Figures 1C,D and 2A). The results of brain 0 0 0 0 TGA-3 and 5 -CGCCTCTTCATCACCATGTA-3 ; fdft1:5 -TGGG morphological analysis showed that the volume of the optic tectum 0 0 TCTGTTCCTCCAGAAG-3 and 5 -TCTGGGACGTGATGGAGAG- in golden-line cavefish was significantly smaller than in surface fish, 0 0 0 0 3 ; fdps:5 -CTCCTGGAGGCAAGAGAAAC-3 and 5 -ATGTCAT about a third as large (Figures 1E,F and 2B), a result also found in 0 0 CAGCCACCAAGAA-3 ; hmgcra:5 -CCCAAGAGAATTGAGCCA Astyanax cavefish. In addition to a difference in the volume of the 0 0 0 0 GA-3 and 5 -CAGCACTGATCAGGAGACCA-3 ; hmgcs1:5 -GGGA optic tectum, the olfactory tract was over twice as long in cavefish 0 0 TGATCAAGGAGATCCA-3 and 5 -CAACACTGGACTGGGAT than in surface fish (2.466 0.12-fold longer in cavefish than surface 0 0 0 0 TAGC-3 ; nsdhl:5 -GGAACCGACATCAAGAATGG-3 and 5 -GC fish) (Figures 1C,D and 2C). This change in brain morphology re- 0 0 GGGCTGTATCTATCAGGA-3 ; pmvk:5 -ATAGATGTGTCTGCGT flects differences in the morphology of the whole head in cavefish 0 0 0 0 TCGG-3 and 5 -TTCTCCAGAACGTCATCAGC-3 ; srebf1:5 -GATT and surface fish (Figure 1A,B, Supplementary Figure S2B), because 0 0 GGATGTTGCCCACTCT-3 and 5 -GACGACCAAGAGCTTTGAG the head of cavefish (30.9%6 0.9% of standard length) was longer 0 0 0 0 G-3 ; srebf2:5 -CTCTCCTGGAACCTGATTCG-3 and 5 -AGACA than the head of surface fish (27.2%6 1.0%) (Head length: the dis- 0 0 CACACACACCCCAGA-3 ; tm7sf2:5 -TCGGGTATCTGTTTGAG tance between the snout tip and posterior edge of operculum). 0 0 0 CTG-3 and 5 -CTCGACCCATGAAGAAGTCA-3 .We used bsed in Brain volume analysis has shown that the volume of the optic tec- as a reference gene. All PCR reactions were run in a 96-well block with tum was significantly smaller (Figure 2B:33.46 1.4%, P< 0.001) in 20 lL reactions in each well. All assays included reference samples and cavefish than in surface fish. The volume of the other 5 brain regions negative controls in which cDNAs were replaced by water. Target genes was not significantly different while comparing cavefish to surface were normalized to the reference gene and expression levels were quan- fish (Figure 2B, P> 0.05). These results are consistent with the tified using the relative Ct method. Each reaction (samples and primers) hypothesis that reduced inputs of visual signals led to a reduction in involved at least 3 replicates. the volume of the optic tectum in the cavefish. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4 Current Zoology, 2018, Vol. 0, No. 0 category, cavefish gene expression was downregulated relative to surface fish in the subcategories of biological regulation, multicellu- lar organismal processes, developmental processes, cell communica- tion, and growth (Figure 3); in the cellular component category, cavefish were substantially downregulated in the nucleus, extracellu- lar matrix, and chromosome subcategories (Figure 3); and in the molecular function category, cavefish were downregulated in the nu- cleic acid binding, molecular transducer activity, and chromatin binding subcategories (Figure 3). These differences likely reflect genes that are related to the function and development of the cave- fish brain. Identification of enriched pathways among differentially expressed genes Results showed that 17 pathways had significant P-values (<0.01) in the downregulated gene group and 20 pathways had significant P-values (<0.01) in the upregulated group. Pathways relevant to cholesterol biosynthesis were significantly enriched in the downregu- lated group, including “activation of gene expression by Srebf” (sterol regulatory element-binding protein, Reactome pathway data- base) and “cholesterol biosynthesis” (Reactome) (Supplementary Table S2). In the upregulated group, 8 of 20 enriched pathways were involved in degradation and metabolism of proteins, amino acids, and lipids. These pathways included “sumoylation” (BioCarta), “lysosome” (KEGG), “phenylalanine and tyrosine ca- tabolism” (Reactome), “eicosanoid metabolism” (BioCarta), and “other glycan degradation” (KEGG) (Supplementary Table S2). These findings suggested that, compared with the surface fish brain, the Figure 1. Cavefish phenotypes. Surface fish S. angustiporus (A) and cavefish cavefish brain reduces the synthesis and metabolism of organic com- S. anophthalmus (B). Dissected brains of surface (C) and cavefish (D). Green pounds and/or enhances the degradation and recycling of materials. lines indicate the locations of sections in E and F. Hematoxylin and eosin stained sections of adult surface fish brain (E) and cavefish brain (F). CC: Enhanced material recycling by the lysosome in the crista cerebellaris; CCe: corpus cerebelli; Di: diencephalon; IL: inferior lobe; MO: medulla oblongata; OB: olfactory bulb; OT: optic tectum; OTr: olfactory cavefish brain tract; Tel: telencephalon; TL: torus longitudinalis; Tla: torus lateralis; Val: val- Several genes upregulated in cavefish brains encode members of the vula cerebelli. Scale bar in (A and B): 1 cm; (C and D): 2 mm; (E and F) 1 mm. adaptor-related protein complex, a part of the clathrin coat assem- bly [ap1s1{3.93 FC up}, ap1s2 {2.22 up}, ap1s3b {3.26 up}, ap3m1 {3.26 up}, and ap4s1 {2.52 up}] (Supplementary Table S1). RNA-seq Differential gene expression comparing brains of results also showed that lysosome-related genes were significantly cavefish and surface fish upregulated in cavefish brains compared with the brains of surface The Illumina sequencing reads were deposited in the Short Read fish, including genes encoding several lysosomal enzymes, such as Archive as accession numbers SRR788094 for S. angustiporus; aga (2.01 up), asah1a (3.92 up), ctsk (2.89 up), galcb (3.54 up), gla SRR788095 for S. anophthalmus. For the cavefish brain, a total of (2.56 up), glb1l (2.16 up), ppt1 (2.81 up) and, sgsh (2.36 up) 12,895,766 reads, corresponding to 43.63% of all high-quality (Supplementary Table S1). These results suggested that the cavefish cavefish reads, mapped to 55,362 golden-line transcriptome contigs brain may be more active in the conduction of materials and (98.73%) and matched to 13,957 zebrafish UniGenes. For the sur- destruction of cell components than the surface fish brain. face fish brain, 7,642,283 reads (49.66%) mapped to the golden- line transcriptome, which aligned to 53,115 golden-line reference transcriptome contigs (94.72%) and matched to 13,768 zebrafish Reduced expression of genes regulated by srebfs UniGenes. Genes that were downregulated in the cavefish brain were signifi- We obtained 11,471 unique genes with expression values  5 cantly enriched in the “activation of gene expression by Srebf” pathway RPKM in at least one of the golden-line brain transcriptomes. (13/41, P ¼ 0.000091). In the cavefish brain, 13 genes in the “activa- Among these unique genes, 2,147 were identified as differentially tion of gene expression by Srebf” category were downregulated, and 7 expressed (2-FC in transcript level between cavefish and surface of these genes encode cholesterol-synthesizing enzymes, cyp51 (3.51 FC fish with a P< 0.05). Of the differentially expressed genes, 1,080 down), dhcr7 (2.16 down), fdft1 (4.26 down), hmgcs1 (7.89 down), were downregulated and 1,067 were upregulated in the cavefish idi1 (2.03 down), lss (2.18 down), and sqle (5.41 down). Srebp brain. Supplementary Table S1 lists differentially expressed genes. also regulates the nuclear gene encoding mitochondrial glycerol-3- Of the 1,067 upregulated genes, 949 mapped to unique human phosphate acyltransferase (gpam), which was downregulated Entrez Gene IDs. Of the 1,080 downregulated genes, 758 mapped to (2.04 down). Additional factors that co-activate Srebf target genes were unique human Entrez Gene IDs. Enrichment analyses were con- also downregulated, including CREB binding protein a (crebbpa,2.23 ducted and GO Slim classifications were assigned based on the down), crebbpb (2.07 down), nuclear receptor coactivator 1 (ncoa1, mapped unique Entrez Gene IDs (Figure 3). In the biological process 2.51 down), and retinoid X receptor-a-a (rxraa, 2.00 down). Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Meng et al.  Transcriptomics of the golden-line cavefish brain 5 and our RNA-seq data showed that cyp46a1 was significantly downregulated (2.91 down) in the cavefish brain relative to the sur- face fish brain (Figure 4 and Supplementary Table S1). To test whether the downregulation of genes for both the synthe- sis and breakdown of cholesterol affect cholesterol homeostasis in cavefish, we extracted lipids from brain, liver, and muscle of both species and assayed their cholesterol content. Results revealed no significant difference between cholesterol levels in the brains, livers, or muscles of cavefish versus surface fish (Figure 6A,B). This result would be expected if the rate of synthesis and the rate of breakdown of cholesterol were both lower in cavefish than in surface fish, as suggested by the RNA-seq data. Differential expression of genes in the mitochondrial genome and hypothalamic hormones In the cavefish brain, none of the 13 genes in the mitochondrial genome were downregulated, but 4 genes in the mitochondrial genome were upregulated (mt-atp6,2.08 up; mt-atp8,4.71 up; mt-co3,2.23 up; mt- nd3,2.13 up) (Supplementary Figure S3 and Supplementary Table S1). Orthologs of 3 of 7 genes encoding secreted hypothalamic hor- mones were annotated in our RNA-seq dataset, and all 3 were strongly upregulated in golden-line cavefish compared with golden-line surface fish (Supplementary Table S1). Thyrotropin-releasing hormone (trh, 2.38 up) and its receptor in the pituitary (trhrb, 3.13 up) were signifi- cantly upregulated, as was somatostatin (sst1, 2.42upand sst3,4.05 up). Corticotropin-releasing hormone (crhb, 4.06 up) was also signifi- cantly upregulated, but its downstream target in the pituitary, proopio- Figure 2. Morphometrics of cavefish brains. (A) Cavefish have a slender brain melanocortin [pomca {alias acth}], was substantially downregulated compared to surface species. (B) Comparison of the volume of different brain (7.16 down). The gene encoding the melanocortin-4-receptor regions normalized to fish standard length (excludes the length of the caudal (mc4r) was also downregulated (2.68 down) in golden-line cavefish fin of fish). (C) Comparison of the length of olfactory tract to fish standard brains relative to surface fish brains. length. Values expressed as mean6 SD, *P< 0.01. CC: crista cerebellaris; CCe: corpus cerebelli; Di: diencephalon; MO: medulla oblongata; OB: olfac- tory bulb; OTr: olfactory tract; Tel: telencephalon; TeO: optic tectum. Discussion Additionally, in cavefish brains, both srebf1 and srebf2 themselves were To investigate the effects of a cave habitat on the brain of a cave- downregulated(2.90 down and1.68down, respectively) withrespect adapted species, we first compared brain morphology between to surface fish brains (Supplementary Table S1). Real-time quantitative golden-line cavefish and golden-line surface fish. Results showed PCR confirmed the direction and approximate magnitude of gene that the cavefish brain is narrower than the surface fish brain. expression change for all of the 14 genes tested (Figure 4). Furthermore, the volume of the optic tectum in the cavefish brain was about a third of the size of the optic tectum in surface fish. Downregulation of genes involved in cholesterol Other brain regions, however, were roughly of the same size in the 2 biosynthesis and catabolism in the cavefish brain species. Our finding is consistent with previous studies that showed RNA-seq analyses revealed that many genes involved in cholesterol that adult cave-dwelling Astyanax is longer and slimmer than that biosynthesis were downregulated in the cavefish brain relative to the of the surface population and the size of the optic tectum is smaller surface fish brain. Figure 5 displays our RNA-seq results superim- in Astyanax cavefish due to reduced numbers of retino-tectal fibers posed onto the biosynthetic pathway of cholesterol synthesis. The compared with surface controls (Riedel 1997; Soares et al. 2004). gene encoding Hmgcr, which catalyzes the rate-limiting step of chol- Reduced retino-tectal fiber input and/or enhanced programmed cell esterol biosynthesis, was downregulated 2.3-fold along with 9 add- death or reduced proliferation of optic tectum cells might also gener- itional enzymes in cholesterol biosynthesis that were significantly ate the smaller optic tectum of golden-line barbel cavefish. The con- reduced in cavefish brain (Figure 5). The gene encoding Pmvk was vergent small-tectum phenotype is consistent with the idea that the only cholesterol synthesis gene that was upregulated (2.51 up, common changes in brain morphology evolve independently mul- P< 0.001) in cavefish brains relative to surface fish brains (Figure 5 tiple times in cave-dwelling fish, but a gap in our knowledge is and Supplementary Table S1). Many cholesterol biosynthesis genes whether these common morphologies reflect shared developmental that were downregulated in the cavefish brain are downstream tar- genetic mechanisms. Further research is needed to document the gets of Srebfs, including hmgcs1 (7.89 down), hmgcra (2.31 down), morphology and molecular genetics of brain development during sqle (5.41 down), fdft1 (4.26 down), cyp51 (3.51 down), lss early life stages of obligatory cave-dwelling Sinocyclocheilus. (2.18 down), dhcr7 (2.16 down), and idi1 (2.03 down), suggesting Our RNA-seq results showed that a cave-dwelling fish species dif- that the downregulation of cholesterol biosynthesis genes is likely to fers from surface fish in the expression levels of genes involved be facilitated by the reduced expression of srebfs we found in cave- in brain lipid metabolism, secretion of hypothalamic peptide hor- fish brains. The enzyme Cyp46a1 eliminates cholesterol in the brain mones, and mitochondrial activity. We found that genes encoding 3 Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6 Current Zoology, 2018, Vol. 0, No. 0 Figure 3. Gene ontology (GO) ID representations for downregulated genes (blue) and upregulated group (red). Three comparisons are shown: biological process ontology, cellular component ontology, and molecular function ontology. Proportion represents the ratio of a gene set in differentially expressed genes com- pared with a reference gene set in the category. peptide hormones (Trh, Sst, Crh) secreted by the hypothalamus were have been found in Astyanax cavefish in the gene encoding Mc4r upregulated over 3-fold in cavefish compared with surface fish. Trh (Aspiras et al. 2015), which integrates leptin and insulin levels in the stimulates the release of thyrotropin (thyroid-stimulating hormone) hypothalamus to regulate feeding and metabolism (Tao 2010)and from the pituitary, which causes the thyroid to produce thyroid hor- contributes likely to the insatiable appetite of some Astyanax cavefish mones, which accelerate metabolism in most cells of the body. populations (Aspiras et al. 2015); correspondingly, our data showed Somatostatin (Sst) has an effect opposite to that of Trh: Sst decreases that mc4r expression was downregulated in golden-line cavefish rela- or inhibits the release of thyrotropin from the pituitary (Harris et al. tive to surface fish brains. The upregulation of the pituitary protein 1978; De Groef et al. 2003; Bodo et al. 2010). The third hypothal- Trh-receptor that our data identified might be expected from the amic peptide hormone in our dataset, Crh, is usually secreted in re- upregulation of thr, but the upregulation of the hypothalamic gene sponse to stress and it depresses appetite, so its upregulation in the encoding Crh followed by the downregulation of the gene encoding cavefish brain is surprising given the usual expectation that cavefish its downstream hormone Pomc, shows that the regulation of the often have increased appetites (Hu ¨ ppop 2005). Indeed, mutations hypothalamus–pituitary axis in cavefish is likely to be complex and Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Meng et al.  Transcriptomics of the golden-line cavefish brain 7 might not be fully described by examining gene expression at the level of mRNA rather than protein. Experiments reported here revealed low expression of srebf genes and downregulation of downstream target genes of srebfs in the golden-line cavefish brain. These reduced gene expression levels may lead to decreased cholesterol biosynthesis in the cavefish brain. Cholesterol is a key component of cell membranes that is import- ant for the maintenance and function of neurons and is most concentrated in the brain (Pfrieger 2003; Dietschy and Turley 2004). Correspondingly, Srebf transcription factors can activate the expres- sion of at least 30 genes involved in cholesterol and lipid synthesis (Weber et al. 2004; Porter and Herman 2011; Faust and Kovacs 2014; Martin et al. 2014; Mitsche et al. 2015). The cholesterol con- tent of the brain, however, was similar between cavefish and surface fish brains (Figure 6B), and the likely reason for similar cholesterol contents despite different levels of expression of cholesterol biosynthe- Figure 4. Validation of RNA-seq results by real-time PCR using RNA isolated sis genes is the decreased expression our data show for the gene from brains of each species. Expression levels of target genes were quanti- encoding Cyp46a1, which is the enzyme responsible for eliminating fied and normalized to beta-actin1. Relative expression values are mean6 SD of at least 3 independent experiments. most of the cholesterol removed from the central nervous system (Lund et al. 2003; Russell et al. 2009). Cavefish inhabiting karstic caves, which lack production by autotrophs and experience only spor- adic food availability, often exhibit behaviors that maximize energy intake and minimize energy expenditure (Hu ¨ ppop 2005; Salin et al. 2010). Reduced expression of cholesterol biosynthetic genes might help to reduce energy expenditure in golden-line barbel cavefish. An additional measure of energy metabolism is the expression level of mitochondrial genes. Our golden-line RNA-seq experiments showed that 4 genes in the mitochondrial genome were upregulated in the cave- fish brain. This result for the golden-line cavefish brain contrasts with previous results for the golden-line cavefish eye, in which 7 mitochon- drial genes were downregulated with respect to the eye of surface fish (Meng et al. 2013a). Decreased mitochondrial activity in the cavefish eye is likely related to reduced eye size, its internal location, and dimin- ished function; in contrast, increased activity of mitochondrial genes in the brain may reflect increased effort directed toward detecting the envir- onment by nonvisual sense organs, such as lateral line organs and other detectors in the skin, which are increased in some cavefish (Yoshizawa et al. 2010). While changes in the activity of mitochondrial genes may represent adaptations for survival in cave conditions, this hypothesis re- quires testing by direct measurements of energy expenditures. Compared with surface aquatic habitats, cave habitats are often nutrient-poor and have seasonal periods of nutrient input (Aspiras et al. 2015). Adaptations to fluctuating environments in Astyanax cavefish appear to include a highly efficient metabolism (Moran et al. 2014), and in golden-line cavefish, some of this energy effi- ciency might involve lower rates of both the synthesis and the break- down of cholesterol, and/or enhanced degradation and recycling of cellular debris by lysosomes, which contain several enzymes whose genes were upregulated in our data. Studies examining energy ex- penditures over the entire animal have not yet been conducted in golden-line cavefish and surface fish, so we do not yet know whether the total energy budget of golden-line cavefish is reduced compared to surface congeners, however, the changes we observed in the cave- fish brain transcriptome could contribute to more efficient use of Figure 5. Cholesterol biosynthetic pathway and relative expression changes limited and sporadic resources in cave environments. for genes encoding related enzymes. Hmgcr catalyzes the rate-limiting step of cholesterol biosynthesis. Because the two post-lanosterol pathways (Bloch vs. Kandutsch–Russell) share enzymatic stages, the figure shows only the Kandutsch–Russel pathway, which is the one the brain uses most (Mitsche Ethics Statement et al. 2015). Numbers following genes indicate the values of FCs of gene ex- All experimental procedures involving animals were conducted and pression. Red numbers and arrows represent upregulation of cavefish rela- approved by the Animal Care and Use Committee of Institute of tive to surface fish. Green numbers and arrows represent downregulation of cavefish relative to surface fish (P< 0.01). P: phosphate, PP: pyrophosphate. Zoology, Chinese Academy of Sciences. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 8 Current Zoology, 2018, Vol. 0, No. 0 Glossary acat1 acetyl-coenzyme A acetyltransferase 1 (thiolase) aga aspartylglucosaminidase ap1s1 adaptor-related protein complex 1, sigma 1 subunit ap1s2 adaptor-related protein complex 1, sigma 2 subunit ap1s3b adaptor-related protein complex 1, sigma 3 subunit, b ap3m1 adaptor-related protein complex 3, mu 1 subunit ap4s1 adaptor-related protein complex 4, sigma 1 subunit asah1a N-acylsphingosine amidohydrolase (acid ceramidase) 1a crh corticotropin releasing hormone cyp46a1 cytochrome P450, family 46, subfamily A, polypeptide 1 cyp51 cytochrome P450, family 51 (lanosterol demethylase) ctsk cathepsin K dhcr24 24-dehydrocholesterol reductase dhcr7 7-dehydrocholesterol reductase ebp emopamil binding protein (sterol isomerase) faxdc2 fatty acid hydroxylase domain containing 2 Figure 6. Total cholesterol content in the liver, muscle, and brain of surface fdft1 farnesyldiphosphate farnesyltransferase 1 fish and cavefish. The cholesterol content of liver and muscle (A) and brain fdps farnesyl-diphosphate synthase (B) were quantified by enzymatic assay according to the manufacturer’s galcb galactosylceramidase b protocol and normalized to tissue weight. Relative values are mean6 SD of at gla galactosidase, alpha least 3 independent experiments. Cavefish did not differ significantly from glb1l galactosidase, beta 1-like surface fish in any of the 3 tissues. hmgcr 3-hydroxy-3-methylglutaryl-CoA reductase hmgcs 3-hydroxy-3-methylglutaryl-CoA synthase Availability of Data and Material hsd17b7 hydroxysteroid (17-beta) dehydrogenase 7 idi1 isopentenyldiphosphate isomerase 1 The authors confirm that all data underlying the findings are fully lbr lamin-B receptor available without restriction. All relevant data are within the lss lanosterol synthase Methods, in the Additional files section, and in the SRA under acces- mc4r melanocortin-4-receptor sion numbers: SRR788094 for S. angustiporus and SRR788095 for msmo1 methylsterol monooxygenase 1 S. anophthalmus. mvd mevalonate-diphosphate decarboxylase mvk mevalonate kinase nsdhl NAD(P) dependent steroid dehydrogenase-like Conflict of Interest pmvk phosphomevalonate kinase pomc proopiomelanocortin The authors declare that they have no competing interests. ppt1 palmitoyl-protein thioesterase 1 sc5d sterol-c5-desaturase (lathosterol oxidase) sgsh N-sulfoglucosamine sulfohydrolase Authors’ Contributions sqle squalene monooxygenase F.W.M. and J.H.P. conceived this study and designed the experi- srebf sterol regulatory element-binding factor ments. F.W.M., Y.H.Z. and C.G.Z. collected the fish samples. sst somatostatin trh thyrotropin releasing hormone F.W.M. carried out the H&E staining, real-time PCR and cholesterol tm7sf2 transmembrane 7 superfamily member 2 test. F.W.M. and T.T. prepared the cDNA libraries for RNA-seq. F.W.M. and J.H.P. performed computer analysis of RNA-seq data. F.W.M. generated all images and F.W.M. and J.H.P. wrote the manu- script. All authors read, revised, and approved the final manuscript. References Aspiras AC, Rohner N, Martineau B, Borowsky RL, Tabin CJ, 2015. Melanocortin 4 receptor mutations contribute to the adaptation of cave- Acknowledgments fish to nutrient-poor conditions. Proc Natl Acad Sci USA 112:9668–9673. We thank R. BreMiller (University of Oregon) and J. Ganz (Michigan State Audic S, Claverie JM, 1997. The significance of digital gene expression pro- University) for help with neuroanatomy. files. Genome Res 7:986–995. Bodo E, Kany B, Gaspar E, Knuver J, Kromminga A et al., 2010. 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Brain of the blind: transcriptomics of the golden-line cavefish brain

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Abstract

The genus Sinocyclocheilus (golden-line barbel) includes 25 species of cave-dwelling blind fish (cavefish) and more than 30 surface-dwelling species with normal vision. Cave environments are dark and generally nutrient-poor with few predators. Cavefish of several genera evolved conver- gent morphological adaptations in visual, pigmentation, brain, olfactory, and digestive systems. We compared brain morphology and gene expression patterns in a cavefish Sinocyclocheilus anophthalmus with those of a closely related surface-dwelling species S. angustiporus. Results showed that cavefish have a longer olfactory tract and a much smaller optic tectum than surface fish. Transcriptomics by RNA-seq revealed that many genes upregulated in cavefish are related to lysosomes and the degradation and metabolism of proteins, amino acids, and lipids. Genes down- regulated in cavefish tended to involve “activation of gene expression in cholesterol biosynthesis” and cholesterol degradation in the brain. Genes encoding Srebfs (sterol regulatory element- binding transcription factors) and Srebf targets, including enzymes in cholesterol synthesis, were downregulated in cavefish brains compared with surface fish brains. The gene encoding Cyp46a1, which eliminates cholesterol from the brain, was also downregulated in cavefish brains, while the total level of cholesterol in the brain remained unchanged. Cavefish brains misexpressed several genes encoding proteins in the hypothalamus–pituitary axis, including Trh, Sst, Crh, Pomc, and Mc4r. These results suggest that the rate of lipid biosynthesis and breakdown may both be depressed in golden-line cavefish brains but that the lysosome recycling rate may be increased in cavefish; properties that might be related to differences in nutrient availability in caves. Key words: cavefish, cholesterol, cyp46a, optic tectum, Sinocyclocheilus, transcriptomics. Caves present organisms with an extreme environment, but various (Protas et al. 2008; Windsor et al. 2008; Jeffery 2009; Romero et al. lineages of fish have independently colonized these perpetually dark 2009; Gross 2012; Krishnan and Rohner 2017). Among cavefish realms. Teleost fish from at least 19 families have successfully colon- species, investigations of the Mexican tetra Astyanax mexicanus ized caves from 40 N to the Tropic of Capricorn (Zhao et al. 2011). have led to many important advancements concerning the evolution Cave-dwelling fish have evolved a suite of troglomorphic traits, of troglodyte traits, for example, the eyes and optic tectum of including reduction or loss of eyes, pigmentation, and scales, Astyanax cavefish are greatly reduced (Rohner et al. 2013; Elipot coupled with the enhancement of chemoreceptors in olfactory et al. 2014; McGaugh et al. 2014; Simon et al. 2017; Stahl and organs and mechanoreceptors including the lateral-line system Gross 2017). V C The Author(s) (2018). Published by Oxford University Press. 1 This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 2 Current Zoology, 2018, Vol. 0, No. 0 ’The cyprinid Sinocyclocheilus (golden-line barbel) is the most Materials and Methods species-rich cavefish genus with more than 55 known species, about 25 Animals of which live in energy-limited cave environments (Romero et al. 2009; Golden-line cavefish S. anophthalmus were collected in Jiuxiang cave, Zhao et al. 2011; Meng et al. 2013 b; Yang et al. 2016). Phylogenetic Yiliang County (N 25.05478 , E 103.37975 , Yunnan, China) and analyses based on mitochondrial cytochrome b and ND4 gene sequences maintained in the laboratory in a dark environment. The surface spe- have shown that the known Sinocyclocheilus species cluster into 5 major cies, S. angustiporus were collected from Huangnihe River in Agang monophyletic clades (Xiao et al. 2005), several of which have cave- Town (N 25.00905 , E 103.59256 , Yunnan, China). Both collection dwelling forms, suggesting that different lineages of Sinocyclocheilus sites were in the Nanpanjiang River drainage, the largest tributary of have adapted to cave environments independently several times. Xijiang River in the Pearl River basin. Although the two sites are only Comparative transcriptomic and genomic analyses of Sinocyclocheilus about 30 km apart in a straight line, they are separated by about 100 species have revealed many genetic changes that were associated with river-kilometers (Supplementary Figure S1). The obligatory cave adaptive features such as eye degeneration, albinism, rudimentary scales, species and the surface species are closely related phylogenetically circadian rhythm, and enhanced taste buds (Meng et al. 2013a; Yang (Xiao et al. 2005; Zhao and Zhang 2009) and the sequences of their et al. 2016). Thus, Sinocyclocheilus provides an ideal model genus to orthologs are highly similar, 98.126 0.9% identical at the nucleotide evaluate the mechanisms of adaptation in cave animals. level (Meng et al. 2013a), suggesting that differences between them in Like cavefish, some people are born blind and the brains of blind terms of gene expression levels are more likely to be due to evolved dif- people develop a compensatory reorganization, especially in areas that ferences adapting to habitat rather than to random neutral differences appear to help improve spatial resolution of sounds (Roder et al. 1999). that occur over time. While the two congeners S. grahami and S. tingi Similar types of brain reorganization may occur in blind cavefish. are more closely related to S. anophthalmus than is S. angustiporus, Several studies have investigated the eye, brain, and behavior in cavefish unfortunately, these more closely related species are not available in (Soares et al. 2004; Menuet et al. 2007; Yoshizawa et al. 2010; Strecker sufficient numbers to perform the required experiments. Surface et al. 2012; Yoshizawa et al. 2015). These studies show that cavefish animals were maintained on a 12: 12 Light: Dark cycle. Cave and have a larger hypothalamus region, reduced the size of the optic tectum, surface fish were fed twice per day with the same carp food (Sanyou well-developed olfactory bulbs, and more sensory hair cells in the Chuangmei company, Beijing) in mini pellets, which delivered a nutri- neuromasts of the lateral line system. An additional feature of many tionally complete formula. Uneaten food was removed 15 min after cave habitats is low and sporadic nutrient availability, and several feeding. All experimental procedures involving animals were con- studies have investigated cavefish energy metabolism. For example, ducted and approved by the Animal Care and Use Committee of Meng et al. (2013a) found that several genes in the mitochondrial gen- Institute of Zoology, Chinese Academy of Sciences. ome that are relevant to energy metabolism are downregulated in the cavefish eye. These results raised the hypothesis that these adaptations Brain volume analysis contribute to the regulation of energy metabolism in golden-line cave- To investigate the effects of constant darkness on brain morphology, fish in their perpetually dark, clear, slow moving, and presumably we measured various regions of cavefish and surface fish brains. nutrient-poor streams, where bat guano or periodic flooding are the Surface fish and cavefish were euthanized with 0.05% tricaine metha- only sources of outside nutrients. Although the brain expends a nesulfonate and decapitated (3 individuals per species). The dorsal substantial fraction of an animal’s whole-body energy budget surface of the head was dissected away to expose the brain directly to (Ivanisevic and Siuzdak 2015), it is unknown whether energy metabol- prefix in 4% paraformaldehyde (PFA) for 6 h, and then brains were ism in the cavefish brain has adapted to the food-limited cave environ- dissected from the head, fixed again in 4% PFA overnight at 4 C, ment. This situation led us to wonder how the greatly reduced eyes in and finally embedded in paraffin. Transverse sections (10 lmthick) congenitally blind golden-line cavefish (Meng et al. 2013a, 2013 b), of whole brains of both species were mounted and stained with hema- which would be pathogenic in a surface-dwelling fish, would affect toxylin and eosin. The area of various regions of the brain was meas- brain structure and gene expression patterns over evolutionary time. ured on every 8th section (80 lm) using ImageJ (version 10.2). The In this study, we first showed that the volume of the optic tectum volume was estimated by calculating the area of each section and the was significantly smaller in the cavefish Sinocyclocheilus anophthalmus distance between the sections (Rosen and Harry 1990). To measure than in the surface fish S. angustiporus and the length of the olfactory the volume of different brain regions, we calculated the volume of 6 tract was significantly greater in cavefish than in surface fish. Next, to brain areas (olfactory bulb, telencephalon, diencephalon, optic tec- quantify differences in gene expression, we compared the transcrip- tum, cerebellum, and medulla oblongata). The telencephalon has two tomes of surface and cavefish by RNA-seq analyses. We identified subdivisions, the area dorsalis and the area ventralis telencephali; differentially expressed genes, and found that upregulated genes in the cerebellum measurements encompassed the crista cerebellaris, corpus cavefish brain were involved in several pathways related to the lyso- cerebelli, and valvula cerebelli. The diencephalon and medulla oblon- some and the degradation and metabolism of proteins, amino acids, gata were distinguished by the nuclei of the preglomerular complex. and lipids. Downregulated genes in cavefish brains included the sterol The end of the medulla oblongata was the medial funicular nucleus. regulatory element-binding transcription factor genes (srebf1, srebf2) The volume of different brain regions was normalized to fish standard and their transcriptionally regulated targets involved in cholesterol length (from the tip of the snout to the end of the caudal peduncle). biosynthesis, and in addition cyp46a1, which encodes the enzyme Statistical analysis was performed using a two-tailed Student’s t-test responsible for cholesterol elimination from the brain. Direct measure- in Microsoft Excel. All data met the assumption of normality. ments of cholesterol levels showed no differences between cavefish and surface fish brains, suggesting that decreased biosynthesis and decreased degradation of cholesterol balanced each other. We specu- Differential gene expression analysis late that transcriptome evolution in the cavefish brain may have led to To obtain insights into the molecular genetic mechanisms involved in energy savings in cholesterol metabolism, which might help this species the evolution of the cavefish brain, we profiled gene transcription in to adapt to a presumably resource-poor cave environment. dissected brains. We generated total RNA from the brain of two adult Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Meng et al.  Transcriptomics of the golden-line cavefish brain 3 individuals of each species (surface and cave dwelling species). Poly Identification of enriched pathways (A) RNA was isolated from total RNA samples using MicroPoly(A) Enriched pathways were identified from differentially expressed PuristTM (Ambion) according to the manufacturer’s protocol. Brain genes in the brains of cavefish and surface fish using KOBAS 3.0 cDNA libraries were obtained from two individuals for each species (updated 26 January 2015) (Xie et al. 2011), a program that assigns following established protocols (Meng et al. 2013a). Samples (200- to putative pathways and disease relationships to a gene set and pro- 400-bp inserts) were sequenced using 80-nt paired-end reads from an vides statistically significant enriched pathways from 5 pathway Illumina sequencer GA-II (Illumina Inc., San Diego, CA, USA). databases. Sequences of differentially expressed genes were com- Barcodes were identified and nucleotides with low-quality base pared to the Homo sapiens database using the “annotate” feature in calls were removed, and then reads were mapped to the previously KOBAS 3.0 to allow inferences from the data on human pathways. assembled golden-line transcriptome (Meng et al. 2013a) using We then used “identify” to find significantly enriched pathways; Bowtie (Langmead 2010). In Bowtie, “the maximum mismatches “inputs” were the output of “annotate” for upregulated and down- per read” was set to 3 while other parameters were left as default. regulated gene sets, and the “background” was the entire set of Accession numbers are GAHO01000000 for S. angustiporus and 11,471 unique genes expressed in the golden-line brain identified by GAHL01000000 for S. anophthalmus at DDBJ/EMBL/GenBank. RNA-seq. Data were analyzed using a hypergeometric test and Mapped reads of both surface and cave species were converted to Benjamini–Hochberg FDR (false discovery rate) correction, and RPKM (reads per kilobase of exon per million mapped sequence only pathways or diseases with a corrected P < 0.05 were considered reads) values and normalized (Mortazavi et al. 2008). To enhance to be enriched. statistical robustness, genes with fewer than 5 RPKM in either spe- cies were excluded from the pathway enrichment and gene ontology Cholesterol content (GO) analyses, but these genes are recorded in a subsheet of Brain, liver, and muscle tissues of cavefish and surface fish were Supplementary Table S1. P-values were obtained for each gene by homogenized in 50 mM NaCl. The lipid fraction was then extracted computing a conditional probability of observing N1 reads for a through multiple washes with a 2:1 chloroform: methanol solution. gene given that we obtained N2 reads from the controls and experi- Samples were dried down with 10% triton-X 100/acetone mentals (Audic and Claverie 1997). Genes were identified as (Suzuki et al. 2013). Cholesterol content was assayed by enzymatic differentially expressed when fold change (FC) was> 2 and assay according to the manufacturer’s protocol (Wako Chemicals, P< 0.05. We used WebGestalt (Wang et al. 2013) to identify cat: 439-17501). functional categories among the differentially expressed genes. Real-time polymerase chain reaction Results cDNA samples were created from mRNAs isolated from the brains of surface and cave-dwelling individuals. Real-time polymerase chain reac- The optic tectum is smaller in cavefish than in surface fish tion (PCR) was conducted using SYBR Green (TaKaRa) chemistry. The cave-dwelling species S. anophthalmus has small internal eyes Real-time PCR primers were designed based on the golden-line tran- in contrast with those of its closely related surface-dwelling species scriptome sequence assembled previously (Meng et al. 2013a). Primer S. angustiporus (Figure 1A,B, Supplementary Figure S2A). Laser sequences were as follows (forward and reverse): b-actin:5 -GAA light (wavelengths are 650 nm for red and 532 nm for green) was 0 0 GATCAAGATCATTGCTCCC-3 and 5 -ATGTCATCTTGTTCGAG shined into the eyes of cavefish and surface fish. Surface fish re- 0 0 0 0 AGGT-3 ; cyp46a1.3:5 -GGAAACGCTGCGTCTGTA-3 and 5 -GG sponded by moving to avoid the light. In contrast, cavefish treated 0 0 TTCGTGGACCAAGTGC-3 ; cyp51:5 -CATCCTGCAAACGCTGAT in the same way made no response (cavefish n ¼ 12, surface fish 0 0 0 0 AG-3 and 5 -AGTGCAGAGGAGGCAGATGT-3 ; dhcr24:5 -ATGG n ¼ 9). We conclude that the eyes of cavefish do not detect light or 0 0 GAACAGGCATTGAGTC-3 and 5 -TAGCGAAGCTTCACCCAT that cavefish fail to react to light. 0 0 0 0 TT-3 ; ebp:5 -AACGCGGGAAATAATCACAT-3 and 5 -TGAAC Measurements showed that cavefish brains were slimmer than 0 0 GGTCATTAGCCACAT-3 ; faxdc2:5 -GTTGTTTAACGCCCTCC those of surface fish (Figures 1C,D and 2A). The results of brain 0 0 0 0 TGA-3 and 5 -CGCCTCTTCATCACCATGTA-3 ; fdft1:5 -TGGG morphological analysis showed that the volume of the optic tectum 0 0 TCTGTTCCTCCAGAAG-3 and 5 -TCTGGGACGTGATGGAGAG- in golden-line cavefish was significantly smaller than in surface fish, 0 0 0 0 3 ; fdps:5 -CTCCTGGAGGCAAGAGAAAC-3 and 5 -ATGTCAT about a third as large (Figures 1E,F and 2B), a result also found in 0 0 CAGCCACCAAGAA-3 ; hmgcra:5 -CCCAAGAGAATTGAGCCA Astyanax cavefish. In addition to a difference in the volume of the 0 0 0 0 GA-3 and 5 -CAGCACTGATCAGGAGACCA-3 ; hmgcs1:5 -GGGA optic tectum, the olfactory tract was over twice as long in cavefish 0 0 TGATCAAGGAGATCCA-3 and 5 -CAACACTGGACTGGGAT than in surface fish (2.466 0.12-fold longer in cavefish than surface 0 0 0 0 TAGC-3 ; nsdhl:5 -GGAACCGACATCAAGAATGG-3 and 5 -GC fish) (Figures 1C,D and 2C). This change in brain morphology re- 0 0 GGGCTGTATCTATCAGGA-3 ; pmvk:5 -ATAGATGTGTCTGCGT flects differences in the morphology of the whole head in cavefish 0 0 0 0 TCGG-3 and 5 -TTCTCCAGAACGTCATCAGC-3 ; srebf1:5 -GATT and surface fish (Figure 1A,B, Supplementary Figure S2B), because 0 0 GGATGTTGCCCACTCT-3 and 5 -GACGACCAAGAGCTTTGAG the head of cavefish (30.9%6 0.9% of standard length) was longer 0 0 0 0 G-3 ; srebf2:5 -CTCTCCTGGAACCTGATTCG-3 and 5 -AGACA than the head of surface fish (27.2%6 1.0%) (Head length: the dis- 0 0 CACACACACCCCAGA-3 ; tm7sf2:5 -TCGGGTATCTGTTTGAG tance between the snout tip and posterior edge of operculum). 0 0 0 CTG-3 and 5 -CTCGACCCATGAAGAAGTCA-3 .We used bsed in Brain volume analysis has shown that the volume of the optic tec- as a reference gene. All PCR reactions were run in a 96-well block with tum was significantly smaller (Figure 2B:33.46 1.4%, P< 0.001) in 20 lL reactions in each well. All assays included reference samples and cavefish than in surface fish. The volume of the other 5 brain regions negative controls in which cDNAs were replaced by water. Target genes was not significantly different while comparing cavefish to surface were normalized to the reference gene and expression levels were quan- fish (Figure 2B, P> 0.05). These results are consistent with the tified using the relative Ct method. Each reaction (samples and primers) hypothesis that reduced inputs of visual signals led to a reduction in involved at least 3 replicates. the volume of the optic tectum in the cavefish. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 4 Current Zoology, 2018, Vol. 0, No. 0 category, cavefish gene expression was downregulated relative to surface fish in the subcategories of biological regulation, multicellu- lar organismal processes, developmental processes, cell communica- tion, and growth (Figure 3); in the cellular component category, cavefish were substantially downregulated in the nucleus, extracellu- lar matrix, and chromosome subcategories (Figure 3); and in the molecular function category, cavefish were downregulated in the nu- cleic acid binding, molecular transducer activity, and chromatin binding subcategories (Figure 3). These differences likely reflect genes that are related to the function and development of the cave- fish brain. Identification of enriched pathways among differentially expressed genes Results showed that 17 pathways had significant P-values (<0.01) in the downregulated gene group and 20 pathways had significant P-values (<0.01) in the upregulated group. Pathways relevant to cholesterol biosynthesis were significantly enriched in the downregu- lated group, including “activation of gene expression by Srebf” (sterol regulatory element-binding protein, Reactome pathway data- base) and “cholesterol biosynthesis” (Reactome) (Supplementary Table S2). In the upregulated group, 8 of 20 enriched pathways were involved in degradation and metabolism of proteins, amino acids, and lipids. These pathways included “sumoylation” (BioCarta), “lysosome” (KEGG), “phenylalanine and tyrosine ca- tabolism” (Reactome), “eicosanoid metabolism” (BioCarta), and “other glycan degradation” (KEGG) (Supplementary Table S2). These findings suggested that, compared with the surface fish brain, the Figure 1. Cavefish phenotypes. Surface fish S. angustiporus (A) and cavefish cavefish brain reduces the synthesis and metabolism of organic com- S. anophthalmus (B). Dissected brains of surface (C) and cavefish (D). Green pounds and/or enhances the degradation and recycling of materials. lines indicate the locations of sections in E and F. Hematoxylin and eosin stained sections of adult surface fish brain (E) and cavefish brain (F). CC: Enhanced material recycling by the lysosome in the crista cerebellaris; CCe: corpus cerebelli; Di: diencephalon; IL: inferior lobe; MO: medulla oblongata; OB: olfactory bulb; OT: optic tectum; OTr: olfactory cavefish brain tract; Tel: telencephalon; TL: torus longitudinalis; Tla: torus lateralis; Val: val- Several genes upregulated in cavefish brains encode members of the vula cerebelli. Scale bar in (A and B): 1 cm; (C and D): 2 mm; (E and F) 1 mm. adaptor-related protein complex, a part of the clathrin coat assem- bly [ap1s1{3.93 FC up}, ap1s2 {2.22 up}, ap1s3b {3.26 up}, ap3m1 {3.26 up}, and ap4s1 {2.52 up}] (Supplementary Table S1). RNA-seq Differential gene expression comparing brains of results also showed that lysosome-related genes were significantly cavefish and surface fish upregulated in cavefish brains compared with the brains of surface The Illumina sequencing reads were deposited in the Short Read fish, including genes encoding several lysosomal enzymes, such as Archive as accession numbers SRR788094 for S. angustiporus; aga (2.01 up), asah1a (3.92 up), ctsk (2.89 up), galcb (3.54 up), gla SRR788095 for S. anophthalmus. For the cavefish brain, a total of (2.56 up), glb1l (2.16 up), ppt1 (2.81 up) and, sgsh (2.36 up) 12,895,766 reads, corresponding to 43.63% of all high-quality (Supplementary Table S1). These results suggested that the cavefish cavefish reads, mapped to 55,362 golden-line transcriptome contigs brain may be more active in the conduction of materials and (98.73%) and matched to 13,957 zebrafish UniGenes. For the sur- destruction of cell components than the surface fish brain. face fish brain, 7,642,283 reads (49.66%) mapped to the golden- line transcriptome, which aligned to 53,115 golden-line reference transcriptome contigs (94.72%) and matched to 13,768 zebrafish Reduced expression of genes regulated by srebfs UniGenes. Genes that were downregulated in the cavefish brain were signifi- We obtained 11,471 unique genes with expression values  5 cantly enriched in the “activation of gene expression by Srebf” pathway RPKM in at least one of the golden-line brain transcriptomes. (13/41, P ¼ 0.000091). In the cavefish brain, 13 genes in the “activa- Among these unique genes, 2,147 were identified as differentially tion of gene expression by Srebf” category were downregulated, and 7 expressed (2-FC in transcript level between cavefish and surface of these genes encode cholesterol-synthesizing enzymes, cyp51 (3.51 FC fish with a P< 0.05). Of the differentially expressed genes, 1,080 down), dhcr7 (2.16 down), fdft1 (4.26 down), hmgcs1 (7.89 down), were downregulated and 1,067 were upregulated in the cavefish idi1 (2.03 down), lss (2.18 down), and sqle (5.41 down). Srebp brain. Supplementary Table S1 lists differentially expressed genes. also regulates the nuclear gene encoding mitochondrial glycerol-3- Of the 1,067 upregulated genes, 949 mapped to unique human phosphate acyltransferase (gpam), which was downregulated Entrez Gene IDs. Of the 1,080 downregulated genes, 758 mapped to (2.04 down). Additional factors that co-activate Srebf target genes were unique human Entrez Gene IDs. Enrichment analyses were con- also downregulated, including CREB binding protein a (crebbpa,2.23 ducted and GO Slim classifications were assigned based on the down), crebbpb (2.07 down), nuclear receptor coactivator 1 (ncoa1, mapped unique Entrez Gene IDs (Figure 3). In the biological process 2.51 down), and retinoid X receptor-a-a (rxraa, 2.00 down). Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Meng et al.  Transcriptomics of the golden-line cavefish brain 5 and our RNA-seq data showed that cyp46a1 was significantly downregulated (2.91 down) in the cavefish brain relative to the sur- face fish brain (Figure 4 and Supplementary Table S1). To test whether the downregulation of genes for both the synthe- sis and breakdown of cholesterol affect cholesterol homeostasis in cavefish, we extracted lipids from brain, liver, and muscle of both species and assayed their cholesterol content. Results revealed no significant difference between cholesterol levels in the brains, livers, or muscles of cavefish versus surface fish (Figure 6A,B). This result would be expected if the rate of synthesis and the rate of breakdown of cholesterol were both lower in cavefish than in surface fish, as suggested by the RNA-seq data. Differential expression of genes in the mitochondrial genome and hypothalamic hormones In the cavefish brain, none of the 13 genes in the mitochondrial genome were downregulated, but 4 genes in the mitochondrial genome were upregulated (mt-atp6,2.08 up; mt-atp8,4.71 up; mt-co3,2.23 up; mt- nd3,2.13 up) (Supplementary Figure S3 and Supplementary Table S1). Orthologs of 3 of 7 genes encoding secreted hypothalamic hor- mones were annotated in our RNA-seq dataset, and all 3 were strongly upregulated in golden-line cavefish compared with golden-line surface fish (Supplementary Table S1). Thyrotropin-releasing hormone (trh, 2.38 up) and its receptor in the pituitary (trhrb, 3.13 up) were signifi- cantly upregulated, as was somatostatin (sst1, 2.42upand sst3,4.05 up). Corticotropin-releasing hormone (crhb, 4.06 up) was also signifi- cantly upregulated, but its downstream target in the pituitary, proopio- Figure 2. Morphometrics of cavefish brains. (A) Cavefish have a slender brain melanocortin [pomca {alias acth}], was substantially downregulated compared to surface species. (B) Comparison of the volume of different brain (7.16 down). The gene encoding the melanocortin-4-receptor regions normalized to fish standard length (excludes the length of the caudal (mc4r) was also downregulated (2.68 down) in golden-line cavefish fin of fish). (C) Comparison of the length of olfactory tract to fish standard brains relative to surface fish brains. length. Values expressed as mean6 SD, *P< 0.01. CC: crista cerebellaris; CCe: corpus cerebelli; Di: diencephalon; MO: medulla oblongata; OB: olfac- tory bulb; OTr: olfactory tract; Tel: telencephalon; TeO: optic tectum. Discussion Additionally, in cavefish brains, both srebf1 and srebf2 themselves were To investigate the effects of a cave habitat on the brain of a cave- downregulated(2.90 down and1.68down, respectively) withrespect adapted species, we first compared brain morphology between to surface fish brains (Supplementary Table S1). Real-time quantitative golden-line cavefish and golden-line surface fish. Results showed PCR confirmed the direction and approximate magnitude of gene that the cavefish brain is narrower than the surface fish brain. expression change for all of the 14 genes tested (Figure 4). Furthermore, the volume of the optic tectum in the cavefish brain was about a third of the size of the optic tectum in surface fish. Downregulation of genes involved in cholesterol Other brain regions, however, were roughly of the same size in the 2 biosynthesis and catabolism in the cavefish brain species. Our finding is consistent with previous studies that showed RNA-seq analyses revealed that many genes involved in cholesterol that adult cave-dwelling Astyanax is longer and slimmer than that biosynthesis were downregulated in the cavefish brain relative to the of the surface population and the size of the optic tectum is smaller surface fish brain. Figure 5 displays our RNA-seq results superim- in Astyanax cavefish due to reduced numbers of retino-tectal fibers posed onto the biosynthetic pathway of cholesterol synthesis. The compared with surface controls (Riedel 1997; Soares et al. 2004). gene encoding Hmgcr, which catalyzes the rate-limiting step of chol- Reduced retino-tectal fiber input and/or enhanced programmed cell esterol biosynthesis, was downregulated 2.3-fold along with 9 add- death or reduced proliferation of optic tectum cells might also gener- itional enzymes in cholesterol biosynthesis that were significantly ate the smaller optic tectum of golden-line barbel cavefish. The con- reduced in cavefish brain (Figure 5). The gene encoding Pmvk was vergent small-tectum phenotype is consistent with the idea that the only cholesterol synthesis gene that was upregulated (2.51 up, common changes in brain morphology evolve independently mul- P< 0.001) in cavefish brains relative to surface fish brains (Figure 5 tiple times in cave-dwelling fish, but a gap in our knowledge is and Supplementary Table S1). Many cholesterol biosynthesis genes whether these common morphologies reflect shared developmental that were downregulated in the cavefish brain are downstream tar- genetic mechanisms. Further research is needed to document the gets of Srebfs, including hmgcs1 (7.89 down), hmgcra (2.31 down), morphology and molecular genetics of brain development during sqle (5.41 down), fdft1 (4.26 down), cyp51 (3.51 down), lss early life stages of obligatory cave-dwelling Sinocyclocheilus. (2.18 down), dhcr7 (2.16 down), and idi1 (2.03 down), suggesting Our RNA-seq results showed that a cave-dwelling fish species dif- that the downregulation of cholesterol biosynthesis genes is likely to fers from surface fish in the expression levels of genes involved be facilitated by the reduced expression of srebfs we found in cave- in brain lipid metabolism, secretion of hypothalamic peptide hor- fish brains. The enzyme Cyp46a1 eliminates cholesterol in the brain mones, and mitochondrial activity. We found that genes encoding 3 Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 6 Current Zoology, 2018, Vol. 0, No. 0 Figure 3. Gene ontology (GO) ID representations for downregulated genes (blue) and upregulated group (red). Three comparisons are shown: biological process ontology, cellular component ontology, and molecular function ontology. Proportion represents the ratio of a gene set in differentially expressed genes com- pared with a reference gene set in the category. peptide hormones (Trh, Sst, Crh) secreted by the hypothalamus were have been found in Astyanax cavefish in the gene encoding Mc4r upregulated over 3-fold in cavefish compared with surface fish. Trh (Aspiras et al. 2015), which integrates leptin and insulin levels in the stimulates the release of thyrotropin (thyroid-stimulating hormone) hypothalamus to regulate feeding and metabolism (Tao 2010)and from the pituitary, which causes the thyroid to produce thyroid hor- contributes likely to the insatiable appetite of some Astyanax cavefish mones, which accelerate metabolism in most cells of the body. populations (Aspiras et al. 2015); correspondingly, our data showed Somatostatin (Sst) has an effect opposite to that of Trh: Sst decreases that mc4r expression was downregulated in golden-line cavefish rela- or inhibits the release of thyrotropin from the pituitary (Harris et al. tive to surface fish brains. The upregulation of the pituitary protein 1978; De Groef et al. 2003; Bodo et al. 2010). The third hypothal- Trh-receptor that our data identified might be expected from the amic peptide hormone in our dataset, Crh, is usually secreted in re- upregulation of thr, but the upregulation of the hypothalamic gene sponse to stress and it depresses appetite, so its upregulation in the encoding Crh followed by the downregulation of the gene encoding cavefish brain is surprising given the usual expectation that cavefish its downstream hormone Pomc, shows that the regulation of the often have increased appetites (Hu ¨ ppop 2005). Indeed, mutations hypothalamus–pituitary axis in cavefish is likely to be complex and Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 Meng et al.  Transcriptomics of the golden-line cavefish brain 7 might not be fully described by examining gene expression at the level of mRNA rather than protein. Experiments reported here revealed low expression of srebf genes and downregulation of downstream target genes of srebfs in the golden-line cavefish brain. These reduced gene expression levels may lead to decreased cholesterol biosynthesis in the cavefish brain. Cholesterol is a key component of cell membranes that is import- ant for the maintenance and function of neurons and is most concentrated in the brain (Pfrieger 2003; Dietschy and Turley 2004). Correspondingly, Srebf transcription factors can activate the expres- sion of at least 30 genes involved in cholesterol and lipid synthesis (Weber et al. 2004; Porter and Herman 2011; Faust and Kovacs 2014; Martin et al. 2014; Mitsche et al. 2015). The cholesterol con- tent of the brain, however, was similar between cavefish and surface fish brains (Figure 6B), and the likely reason for similar cholesterol contents despite different levels of expression of cholesterol biosynthe- Figure 4. Validation of RNA-seq results by real-time PCR using RNA isolated sis genes is the decreased expression our data show for the gene from brains of each species. Expression levels of target genes were quanti- encoding Cyp46a1, which is the enzyme responsible for eliminating fied and normalized to beta-actin1. Relative expression values are mean6 SD of at least 3 independent experiments. most of the cholesterol removed from the central nervous system (Lund et al. 2003; Russell et al. 2009). Cavefish inhabiting karstic caves, which lack production by autotrophs and experience only spor- adic food availability, often exhibit behaviors that maximize energy intake and minimize energy expenditure (Hu ¨ ppop 2005; Salin et al. 2010). Reduced expression of cholesterol biosynthetic genes might help to reduce energy expenditure in golden-line barbel cavefish. An additional measure of energy metabolism is the expression level of mitochondrial genes. Our golden-line RNA-seq experiments showed that 4 genes in the mitochondrial genome were upregulated in the cave- fish brain. This result for the golden-line cavefish brain contrasts with previous results for the golden-line cavefish eye, in which 7 mitochon- drial genes were downregulated with respect to the eye of surface fish (Meng et al. 2013a). Decreased mitochondrial activity in the cavefish eye is likely related to reduced eye size, its internal location, and dimin- ished function; in contrast, increased activity of mitochondrial genes in the brain may reflect increased effort directed toward detecting the envir- onment by nonvisual sense organs, such as lateral line organs and other detectors in the skin, which are increased in some cavefish (Yoshizawa et al. 2010). While changes in the activity of mitochondrial genes may represent adaptations for survival in cave conditions, this hypothesis re- quires testing by direct measurements of energy expenditures. Compared with surface aquatic habitats, cave habitats are often nutrient-poor and have seasonal periods of nutrient input (Aspiras et al. 2015). Adaptations to fluctuating environments in Astyanax cavefish appear to include a highly efficient metabolism (Moran et al. 2014), and in golden-line cavefish, some of this energy effi- ciency might involve lower rates of both the synthesis and the break- down of cholesterol, and/or enhanced degradation and recycling of cellular debris by lysosomes, which contain several enzymes whose genes were upregulated in our data. Studies examining energy ex- penditures over the entire animal have not yet been conducted in golden-line cavefish and surface fish, so we do not yet know whether the total energy budget of golden-line cavefish is reduced compared to surface congeners, however, the changes we observed in the cave- fish brain transcriptome could contribute to more efficient use of Figure 5. Cholesterol biosynthetic pathway and relative expression changes limited and sporadic resources in cave environments. for genes encoding related enzymes. Hmgcr catalyzes the rate-limiting step of cholesterol biosynthesis. Because the two post-lanosterol pathways (Bloch vs. Kandutsch–Russell) share enzymatic stages, the figure shows only the Kandutsch–Russel pathway, which is the one the brain uses most (Mitsche Ethics Statement et al. 2015). Numbers following genes indicate the values of FCs of gene ex- All experimental procedures involving animals were conducted and pression. Red numbers and arrows represent upregulation of cavefish rela- approved by the Animal Care and Use Committee of Institute of tive to surface fish. Green numbers and arrows represent downregulation of cavefish relative to surface fish (P< 0.01). P: phosphate, PP: pyrophosphate. Zoology, Chinese Academy of Sciences. Downloaded from https://academic.oup.com/cz/advance-article-abstract/doi/10.1093/cz/zoy005/4802228 by Ed 'DeepDyve' Gillespie user on 08 June 2018 8 Current Zoology, 2018, Vol. 0, No. 0 Glossary acat1 acetyl-coenzyme A acetyltransferase 1 (thiolase) aga aspartylglucosaminidase ap1s1 adaptor-related protein complex 1, sigma 1 subunit ap1s2 adaptor-related protein complex 1, sigma 2 subunit ap1s3b adaptor-related protein complex 1, sigma 3 subunit, b ap3m1 adaptor-related protein complex 3, mu 1 subunit ap4s1 adaptor-related protein complex 4, sigma 1 subunit asah1a N-acylsphingosine amidohydrolase (acid ceramidase) 1a crh corticotropin releasing hormone cyp46a1 cytochrome P450, family 46, subfamily A, polypeptide 1 cyp51 cytochrome P450, family 51 (lanosterol demethylase) ctsk cathepsin K dhcr24 24-dehydrocholesterol reductase dhcr7 7-dehydrocholesterol reductase ebp emopamil binding protein (sterol isomerase) faxdc2 fatty acid hydroxylase domain containing 2 Figure 6. Total cholesterol content in the liver, muscle, and brain of surface fdft1 farnesyldiphosphate farnesyltransferase 1 fish and cavefish. The cholesterol content of liver and muscle (A) and brain fdps farnesyl-diphosphate synthase (B) were quantified by enzymatic assay according to the manufacturer’s galcb galactosylceramidase b protocol and normalized to tissue weight. Relative values are mean6 SD of at gla galactosidase, alpha least 3 independent experiments. Cavefish did not differ significantly from glb1l galactosidase, beta 1-like surface fish in any of the 3 tissues. hmgcr 3-hydroxy-3-methylglutaryl-CoA reductase hmgcs 3-hydroxy-3-methylglutaryl-CoA synthase Availability of Data and Material hsd17b7 hydroxysteroid (17-beta) dehydrogenase 7 idi1 isopentenyldiphosphate isomerase 1 The authors confirm that all data underlying the findings are fully lbr lamin-B receptor available without restriction. All relevant data are within the lss lanosterol synthase Methods, in the Additional files section, and in the SRA under acces- mc4r melanocortin-4-receptor sion numbers: SRR788094 for S. angustiporus and SRR788095 for msmo1 methylsterol monooxygenase 1 S. anophthalmus. mvd mevalonate-diphosphate decarboxylase mvk mevalonate kinase nsdhl NAD(P) dependent steroid dehydrogenase-like Conflict of Interest pmvk phosphomevalonate kinase pomc proopiomelanocortin The authors declare that they have no competing interests. ppt1 palmitoyl-protein thioesterase 1 sc5d sterol-c5-desaturase (lathosterol oxidase) sgsh N-sulfoglucosamine sulfohydrolase Authors’ Contributions sqle squalene monooxygenase F.W.M. and J.H.P. conceived this study and designed the experi- srebf sterol regulatory element-binding factor ments. F.W.M., Y.H.Z. and C.G.Z. collected the fish samples. sst somatostatin trh thyrotropin releasing hormone F.W.M. carried out the H&E staining, real-time PCR and cholesterol tm7sf2 transmembrane 7 superfamily member 2 test. F.W.M. and T.T. prepared the cDNA libraries for RNA-seq. F.W.M. and J.H.P. performed computer analysis of RNA-seq data. F.W.M. generated all images and F.W.M. and J.H.P. wrote the manu- script. All authors read, revised, and approved the final manuscript. References Aspiras AC, Rohner N, Martineau B, Borowsky RL, Tabin CJ, 2015. Melanocortin 4 receptor mutations contribute to the adaptation of cave- Acknowledgments fish to nutrient-poor conditions. Proc Natl Acad Sci USA 112:9668–9673. We thank R. BreMiller (University of Oregon) and J. Ganz (Michigan State Audic S, Claverie JM, 1997. The significance of digital gene expression pro- University) for help with neuroanatomy. files. Genome Res 7:986–995. Bodo E, Kany B, Gaspar E, Knuver J, Kromminga A et al., 2010. 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Current ZoologyOxford University Press

Published: Jan 12, 2018

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