Transcriptomic analysis of the effect of dark-rearing on Astyanax
mexicanus
A thesis submitted to the
Graduate School
of the University of Cincinnati
in partial fulfillment of the
requirements of the degree of
Master of Science
in the Department of Biological Sciences
of the College of Arts & Sciences
by
Connor R. Sears
B.S. Biology, University of Kentucky, 2015
Committee:
Dr. Joshua Gross, chair
Dr. Eric Tepe
Dr. Dennis Grogan
March 4th, 2019 THESIS ABSTRACT
This thesis examines the role of light in global RNA architecture. To accomplish this, the blind Mexican cavefish, Astyanax mexicanus, has been reared in total darkness for ~5 years. This species, which encompasses both river-dwelling and cave-dwelling morphotypes, is notable for the dramatically different phenotypes apparent in each of these morphotypes. The cavefish, in contrast to surface fish, exhibits a dramatic reduction of eyes and pigmentation in addition to an expansion of non-visual sensory systems, amongst other traits. Additionally, lighting conditions experienced by the cave and surface populations of fish differ dramatically between the extreme darkness of the cave and the diurnal light cycle of surface waters. This project has examined the effect of light on gene expression by investigating differential expression seen when a cavefish is restored to “Natural Conditions”—a comparative paradigm of lighting condition encompassing total darkness for cavefish and a diurnal light/dark cycle for surface fish. By examining the role of light in gene expression in this system, it expands the paradigm of how surface fish were able to successfully colonize the cave environment. Global gene expression is least similar under Natural
Conditions by correlative comparison, and the number of genes differentially expressed between the two morphotypes is greatly expanded. This expansion of differentially expressed genes represents a subset of expression that is likely inducible by dark lighting conditions. The functional repertoire of this gene set implicates the circulatory and olfactory systems as potential down- stream targets of light-affected gene expression. Additional comparisons of gene expression were made at locations of QTL markers which have been implicated in eye or pigmentation reduction in the cavefish compared to the surface fish. Comparisons between cave and surface fish reared on
Light/Dark Conditions are not the same as comparisons conducted on Natural (photic) Conditions when examined both globally and functionally. This comparison provides a greater understanding
ii of how these inherently regressive traits may have been lost in the dark environment, identifying novel candidate genes mediating eye or pigmentation loss. In sum, examining the effect of light on gene expression in this system grants a better understanding of the complex genetic architecture accompanying life in the extreme cave environment. This study illuminates the need to understand the effect that rearing under light and non-natural conditions has on animals whose natural environment is one of total darkness.
iii
iv THESIS ACKNOWLEDGEMENTS
As my graduate studies come to a close, I want to take the time to acknowledge the people that have been instrumental to my success here at the University of Cincinnati. First and foremost,
I would like to thank my advisor, Dr. Joshua Gross. Through his instruction and guidance, and with much patience, he has provided me with the skills to succeed not only in the laboratory setting, but also in life. You have helped in invaluable and incalculable ways, but most importantly you have helped me hone a critical eye to better my own work, and you have taught me how to be a kind and patient mentor.
Thank you to Dr. Dennis Grogan, for the help and ideas you have provided along this journey. To Dr. Eric Tepe, thank you for your support along the last 2+ years, and for the opportunity to teach under your guidance. Thank you to the many other faculty members of UC, including (but not limited to) Drs. Josh Benoit, Dan Buchholz, and Stephanie Rollmann, who have encouraged me not only as a scientist but as a professional, and provided me with many opportunities to succeed.
I would like to thank my family for pushing me to succeed and for always cheering for me in my studies and in my life choices. I would not have made it to the end of this degree without my mother, Debbie Sears, who has taught me to be a strong, independent woman and who has best demonstrated how to surround myself with people who support me unconditionally. Mom, I have watched you build a life that I admire, and you have been instrumental in my success in completing this thesis. I also wish to thank the entire Phillips family—my aunts, uncles, cousins, and grandparents—for being my allies, support system, and role models—few people are fortunate enough to have an extended family this amazing. A huge thank you goes to my father, Terry Sears and my grandmother Nina Sears, for their enthusiastic curiosity about my work. Dad, thank you
v for your unwavering support of and confidence in my academic pursuits. You have always assumed my success and told me to shoot for the moon, without in any way believing that I will land amongst the stars.
Both my oldest friends and the friends that I have made here have been essential to my success along this journey. To my lab-mates-turned-friends, Mandy Powers, Danny Berning, Tyler
Boggs, Heidi Luc, and Amy Manning, thank you so much for your support, insightful comments, and honest desire to help me better my work. I cannot begin to tell you all how much I appreciate everything that you have given me and done for me during my time here. To Julianne Horn, my chosen sister, you have always believed in me and supported me, but seeing your drive first-hand as you ceaselessly pursued your dreams gave me more inspiration to pursue my own than you even know. To Cody Patterson, I truly do not know how I would have done any of this without you.
Thank you for being my best friend and my partner through this wild journey and for indulging every crazy dream I have for the future. Lastly, and perhaps most importantly, thank you to my fur-babies Brody and Remy for their unconditional love—to you both, I promise more walkies now.
Thank you to the many organizations who have generously supported this work through funding including the Wieman-Benedict Award, Department of Biological Sciences, University of
Cincinnati to CRS and a grant from the National Science Foundation (DEB-1457630) to JBG.
vi TABLE OF CONTENTS
THESIS ABSTRACT ...... ii
THESIS ACKNOWLEDGEMENTS ...... v
LIST OF TABLES AND FIGURES ...... 1
INTRODUCTORY STATEMENTS ...... 2
REFERENCES ...... 6
Transcriptomic assessment of dark-reared Astyanax mexicanus cavefish reveals dramatic alterations to global RNA architecture ABSTRACT ...... 11 INTRODUCTION ...... 12 METHODS ...... 17 RESULTS ...... 23 DISCUSSION ...... 28 CONCLUSIONS ...... 43 ACKNOWLEDGEMENTS ...... 44 REFERENCES ...... 45
GENERAL CONCLUSIONS ...... 59
REFERENCES ...... 61
VISUAL MATERIALS ...... 62
TABLES ...... 75
vii LIST OF TABLES AND FIGURES
Figure 1: Comparisons between morphotypes are most divergent under Natural Conditions
Figure 2: More genes are under-expressed in cavefish, regardless of rearing conditions
Figure 3: Rearing under Natural Conditions reveals novel changes to gene expression
Figure 4: Enriched GO terms under Light/Dark Conditions reveal prototypic cave phenotypes
Figure 5: Enriched GO terms under Natural Conditions reveal terms referring to circulatory and olfactory systems
Figure 6: Vision related genes map closely to vision related QTL
Figure 7: Pigmentation related genes map near pigmentation related QTL
Figure 8: Gene expression determined by qPCR validates RNA-seq expression
Table 1: Comparisons between animals reared on their Natural Conditions are the least similar
Table 2: The expression difference of many genes is amplified under Natural Conditions
Table 3: High degree of relatedness in gene expression between RNA-seq and qPCR methodologies validates RNA-seq expression values
1 INTRODUCTORY STATEMENTS
The relationship between animal form and function has been widely studied throughout scientific history; early descriptions and classifications of animal morphology by Aristotle were further expanded upon by Cuvier and by Linnaean classification (Cuvier 1833; Thompson 1910).
Darwin placed these examinations of form and function in the context of the organism’s environment, describing how form-function relationships may provide an adaptive benefit to the organism (1859). Currently, many relationships between organism form and environment are intuitively understood. What is less understood, however, are the underlying genetic mechanisms that accompany adaptation to an extreme environment. Just as variations in environmental conditions (e.g. temperature, pH, light, and oxygen) can both limit and shape the organisms that inhabit an environment, conditions on the “extreme” end of any of these spectra will certainly have an influence on an organism over evolutionary time.
Across most environments, light is available. Lighting conditions in environments such as the subterranean and deep ocean trenches exemplify environments characterized by the absence of light, a phenomenon demonstrated to affect organism phenotype (Hansen 1967; Lessa 1990; Peters and Thomas 1996; Lefébure et al. 2006; Tierney et al. 2017). Caves are marked by a dramatic reduction of light (Jeffery 2012). Cave-dwelling organisms exhibit a number of unusual morphologies such as a regression or complete loss of eyes and pigmentation, an expansion of non-visual sensory systems (e.g. antennae [Protas and Jeffery 2012] or neuromasts [Yoshizawa
2010], and alterations to craniofacial structure, among other traits [Gross 2012b; Klaus et al.
2013]). Hypotheses of why these characteristics have emerged in cave organisms have been discussed, and the convergence of these characters across both phylogeny and geography has been established (Gross 2012a; Protas and Jeffery 2012). Conversely, the complex genetic
2 underpinnings of the phenotypic changes accompanying life in the dark cave are not fully understood.
The blind Mexican cavefish, Astyanax mexicanus, is an attractive model for understanding animal response to an extreme photic environment. Distributed across 30 named cave localities, this fish exhibits the prototypic morphologies of a cave animal in contrast to the surface-dwelling fish of the same species. Surface fish reside in the rivers and streams of East-Central Mexico surrounding the Sierra de El Abra region of limestone karst caves and are regarded as phenotypically ‘normal’, with characteristics similar to other characiform fish (Gross et al. 2015).
Arguments have been made that fish capable of colonizing these caves must be pre-adapted to the harsh conditions to be able to survive, reproduce, and respond over evolutionary time (Wilkens and Hüppop 1986; Poulson 2010; Salin et al. 2010). Indeed, the accidental-entrapment hypothesis posits that of all the animals to wash into a cave environment (during events such as rapid flooding) only those with traits favorable to the environment would be able to survive and reproduce
(Holsinger 2000; Romero and Green 2005). Cavefish residing in isolated cave populations exhibit the similar cave-related alterations to phenotype as other cave dwelling organisms such as eye loss, pigmentation loss, craniofacial abnormalities, metabolic alterations, and behavioral changes
(Şadoğlu 1967; Jeffery 2001; Yamamoto et al. 2003; Protas et al. 2006; Gross et al. 2009; Jeffery
2009; Yoshizawa et al. 2010; Bilandžija et al. 2013; Gross and Wilkens 2013; Kowalko et al.
2013; Ma et al. 2015; Powers et al. 2017; Riddle et al. 2018). Cavefish and surface fish, the two
“morphotypes” of A. mexicanus, can interbreed and produce viable hybrid offspring in the lab and in the wild (Şadoğlu 1957). This allows for direct comparison of both phenotype and genotype between the two morphotypes. Ancestral surface-dwelling fish colonized the caves of the Sierra de El Abra region ~2-3Mya (Gross 2012b). The genetic similarity of the two morphotypes of A.
3 mexicanus is conducive to understanding phenotype-genotype relationships that differ between the two (Avise and Selander 1972). The genetic underpinnings of cave phenotypes have been examined by hybrid cross analysis, QTL studies, and gene knockdown, revealing candidate genes for many above mentioned traits (Şadoğlu 1957; Borowsky and Wilkens 2002; Protas et al. 2007;
Protas et al. 2008; Yoshizawa et al. 2012; O’Quin et al. 2013; Kowalko et al. 2013a; b; Gross et al. 2014). Mutations are responsible for some of the differences apparent between cave and surface fish, but differences in gene expression likely impact the phenotype of the organism as well.
Indeed, an increased boundary of expression of the gene sonic hedgehog (shh) in cavefish compared to surface fish contributes to lens apoptosis and eye degradation, downstream consequences of the increased gene signal (Yamamoto et al. 2004). Therefore, assessments of gene expression are needed to fully understand the molecular underpinnings of these unique morphological characters.
Previous assessments in this system have uncovered expression level differences between cave and surface fish. Comparison to closely related Danio rerio by microarray analysis revealed many lowly-expressed genes related to eye development in cavefish compared to surface fish
(Strickler and Jeffery 2009). Analysis of the cave and surface fish transcriptomes by RNA-seq revealed differential expression of many genes related to eye maintenance and the presence of many mutations in eye-related genes in cavefish (Hinaux et al. 2013; Gross et al. 2013). A transcriptomic examination of development by RNA-seq analysis revealed both convergent and divergent gene expression patterns in two populations of A. mexicanus cavefish (Stahl and Gross
2017). Gene expression is also affected by external variables. Lighting condition has been shown to drive differential gene expression including responses to cyclical light by bacterial species, light-regulation of many genes in Danio rerio reared under variable photic conditions, and reduced
4 expression of visual system genes in another cavefish species (Weger et al. 2011; Waldbauer et al. 2012; Meng et al. 2013). However, no previous analysis in A. mexicanus has comprehensively investigated the effect of light on gene expression.
This thesis explores the effect of photic rearing condition on the blind Mexican cavefish,
A. mexicanus. Alterations to global RNA architecture, examinations of differentially expressed genes in both number and identity, and analyses of GO term enrichment under differing photic rearing conditions demonstrate the complicated alterations to gene expression that accompany life in the dark. Additionally, this thesis work presents candidate genes for eye and pigment regression in A. mexicanus that may be potentially affected by photic rearing and discusses the effect of photic rearing on previously nominated candidate genes for eye loss. This work exemplifies the need for ecologically relevant photic rearing conditions and explores the diverse gene expression changes incurred by dark photic rearing conditions. In sum, this thesis examines the gene expression changes incurred in the absence of light and reveals potential avenues for phenotypic change in the dark.
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10 ABSTRACT
Adaptation to the extreme environment is often accompanied by the acquisition of extreme characteristics in the organism that occupies it. The complete absence of light marks the cave ecosystem as an extreme environment. Cave dwelling organisms display unusual traits including eye loss, pigmentation loss, and the expansion of non-visual sensory systems. This study evaluates the effect of photic condition on gene expression in the cave-dwelling fish Astyanax mexicanus.
Prior assessments of gene expression in this system have not accounted for photic rearing conditions and were conducted under a 12:12hr light/dark cycle. I restored these organisms to their natural conditions by rearing cave and surface fish on constant darkness (DD) for 5+ years and under a 12:12hr light/dark cycle (LD). Total RNA was extracted from head tissue, poly-A primed, and subjected to Illumina HiSeq 2500 RNA-sequencing to a depth of ~10 million reads. Raw reads were aligned to the draft A. mexicanus (Ensembl, v93) genome to evaluate expression of 25,271 predicted genes. When cavefish are dark-reared, there is a larger difference in gene expression between cave and surface fish (R2=0.8597) compared to the relationship between cave and surface fish reared on Light/Dark Conditions (R2=0.8657). GO enrichment analysis reveals broad scale effects of photic condition on circulatory and olfactory system processing. This study nominates, by comparison of QTL marker to gene location and expression level, candidate genes including rab32b, sox18, pde6ha, gucy2f and gucy2d as potential mediators of trait loss in this system. This work reveals the dramatic effect of light on global RNA architecture. Furthermore, this study reveals that transcriptomic assessments conducted under Light/Dark Conditions are not identical those conducted under Natural Conditions, indicating the need for ecologically relevant practices in photic rearing of cave animals.
11 INTRODUCTION
An organism’s environment plays a role in an organism’s phenotype. This becomes especially clear when considering extreme environments, which can yield extreme characters in the organisms that inhabit these locations. Organisms such as the polar cod (Boreogadus saida) which reside in the extreme cold of the of the Arctic, have antifreeze-type proteins in their blood— a response to this extreme environment (Osuga and Feeney 1978). Reptiles living in the extreme heat of the desert have been shown to regulate their body temperature in response to their environment (Mosauer 1936). Variations in temperature are not the only representations of an extreme environment however, as conditions that are extreme in pH, nutrient availability, or light, among a host of other traits will certainly drive the phenotypes of organisms that inhabit these environments. The cave environment also represents an extreme environment which provides a unique set of pressures that influence the evolution of many traits (Popper 1970; Jones et al. 1992;
Romero & Green 2005). Ecological features of the cave (such as constant temperature, limited nutrients (on average), and low levels of predation) may indeed influence a prototypic cave phenotype. Most dramatically, the cave environment can be characterized by its complete absence of light (Elliot 2015; Jeffery 2015) Additionally, the absence of light itself can drive the trait change in many characters, including trait loss or reduction (Gonzalez and Aston-Jones 2008;
Emerling and Springer 2014).
Cave animals exhibit a number of prototypic traits. Regardless of phylogeny or geography, cave animals exhibit traits such as pigmentation loss, eye loss, and expansion of non-visual sensory systems (Jeffery 2001; 2009a; b; Gross 2012). Across multiple taxa, cave animals exhibit a loss or reduction of eyes and pigmentation and an expansion of non-visual sensory systems among a host
12 of other traits. These dramatic phenotypes have been studied previously; however, the complex genetics governing these phenotypes are not fully understood.
Some of these dramatic conditions of the cave have a measurable effect on cave organisms as well. Cave crayfish exhibit a negative phototactic response to a bright light, preferring to hide in the shadows, and exposure to light alters social behaviors and visual displays (Li and Cooper
2002). Responsiveness to light is not limited to cave-dwelling invertebrates. In a cavefish species
(Sinocyclocheilus anophthalmus), transcriptomic analysis of dark reared fish revealed a reduced expression of many phototransduction genes, proposing a mechanism for eye regression in the
Chinese cavefish (Meng et al. 2013). The blind Mexican cavefish, Astyanax mexicanus maintain light-sensing ability through the pineal gland (Yoshizawa and Jeffery 2007). The pineal gland is an endocrine gland that is responsible for the release of hormones such as melatonin and noradrenaline—the release of these hormones has been associated with downstream effects to melanin pigmentation in the skin of fish and amphibians, and circadian rhythm, respectively
(Axelrod 1974). Additionally, light has an effect on the morphology of the pineal gland in A. mexicanus, as continued exposure to a bright light or continued darkness can induce structural changes to the photoreceptor cells of the pineal gland (Omura 1975). Further studies on the effect of light on the behavior of A. mexicanus reveal that cavefish reared in constant darkness exhibit lower daily fluctuations of activity in a dark environment than those of light-reared cavefish when placed in a dark environment (Carlson and Gross 2018). Spatial usage of the tank is also altered by lighting condition as cavefish spend more time at the bottom of the tank during periods of light compared to dark-reared cavefish which do not alter their tank usage in response to change in time of day (Carlson and Gross 2018).
13 A. mexicanus surface fish also respond to light. Retina size decreases under dark-rearing in surface fish (Wilkens 1988). Surface fish also do not exhibit the normal shoaling behaviors typical of these fish when in the dark (Gregson and Burt de Perera 2007). Additional studies on the morphotype of surface fish reared under constant darkness reveals alterations to the quantity and density of melanophores, as dark-reared fish (adults and juveniles) appear darker (Gross et al.
2016; D. Berning, unpublished). The eyes of dark-reared surface juveniles are also smaller and appear darker due to the visible reduction of guanine deposits in the eye (D. Berning, unpublished).
The effect of light on cave and surface fish is especially important, as the species A. mexicanus encompasses both cave- and surface-dwellers. Despite differences in phenotype, these fish are regarded as members of the same species due to the genetic similarity between the two morphotypes, as well as the maintenance of the ability to interbreed and produce viable offspring
(Şadoğlu 1957; Avise and Selander 1972). Across 30 named cave localities in the El Abra region of Mexico, cavefish have independently evolved a number of regressive and constructive traits
(Protas et al. 2006; Jeffery 2009; Doboué et al. 2011). A. mexicanus is an excellent model to study evolution under the selective pressures of the extreme cave environment. Cavefish are easily reared in the lab and have been reared on a 12:12hr light/dark cycle for many years (see: Herwig 1976).
However, the effect that this lighting regimen has on gene expression remains to be investigated.
Previous studies have explored the ‘preadaptation’ of this system to the cave environment, as ancestral surface populations of Astyanax fish were able to successfully colonize the cave, in contrast to many other species (Wilkens and Hüppop 1986; Poulson 2010). However, the cave likely shaped a number of the unusual phenotypes seen in the cavefish, over evolutionary time.
The cavefish exhibits the loss of eyes accompanied with skin coverage over the remnants of the eye socket (Jeffery 2001; Yamamoto et al. 2004; Wilkens 2007). Each of these cavefish
14 populations also demonstrates a severe reduction or complete loss of melanic pigmentation
(Şadoğlu 1957; 1967; Jeffery 2008). An expansion of non-visual sensory systems (specifically, in the lateral line) have been proposed to aid this organism in navigation and food finding ability in the dark (Hassan 1989; Yoshizawa et al. 2014). Additionally, cavefish harbor distinct craniofacial abnormalities compared to surface-dwelling relatives including the fragmentation and fusion of bones surrounding the eye orbit and craniofacial asymmetry (Yamamoto et al. 2003; Gross et al.
2014; Powers et al. 2017). Furthermore, while cavefish exhibit morphological differences compared to surface-dwelling fish, behavioral changes can be observed as well. Cavefish are less aggressive than surface fish, do not school or shoal as surface fish do, and exhibit continual wall- following behavior (Espinasa et al. 2001; Sharma et al. 2009; Yoshizawa et al. 2010; 2012;
Kowalko et al. 2013). Some of the genetic differences contributing to cave traits in this system are understood; notably, deletions in oca2 resulting in oculocutaneous albinism, and alterations to mc1r resulting in brown melanophores (Protas et al. 2006; Gross et al. 2009; Gross and Wilkens
2013). Understanding cave evolution in the context of orchestrated gene-expression requires further study.
Previous studies have capitalized on the genetic similarity between cave and surface fish to characterize differential gene expression, specifically in genes with roles in vision and metabolism (Gross et al. 2013). Gene expression differences contributing to eye loss in cavefish have been evaluated and described (Hinaux et al. 2013), as well as the characterization of gene expression throughout early embryonic development in two populations of A. mexicanus cavefish
(Stahl and Gross 2017). It has been established in this system that differences in gene expression
(such as differences in shh, twhh, and pax6) can contribute to the phenotypic differences existent between cave and surface fish such as forebrain size, and eye size (Strickler et al. 2001; Yamamoto
15 et al. 2004; Pottin et al. 2011). Despite this, the effects of light on global gene expression have not been characterized.
A transcriptomic analysis of gene expression was conducted under lighting conditions that closer mimics the organisms’ ‘natural’ photic environment. Cavefish were reared under near blackout conditions (DD), and surface fish under a 12:12hr light/dark photoperiod (LD).
Comparing the gene expression profile of animals reared on their “Natural Conditions” to the profile of cave and surface fish reared under exclusively Light/Dark Conditions yields the effect that non-natural lighting condition has on gene expression. This analysis is the first comprehensive assessment of the effect of light on global gene expression in this species. As previous studies have described the changes sustained to phenotype in both cave and surface fish in DD conditions, evaluating this organism under its natural photic environment is crucial to understanding the genetics of adaptation to the extreme cave environment. By conducting a transcriptomic assessment on the organism’s natural conditions, I will examine if differential expression on this comparative paradigm is simply an expansion of previously established differences and determine if novel gene expression patterns are emerging in the dark. I aim to 1) assess if light impacts minor or global gene expression patterns before 2) characterizing the effect of light on the polarity of differential gene expression. Then I will 3) determine the genes and functions impacted by darkness in this system and 4) compare differential expression to regions of genome associated with cave biology.
16 METHODS
Rearing Conditions for Cave and Surface Fish
The practiced rearing conditions of A. mexicanus are a 12:12hr light/dark cycle (LD).
Under this regime, the animals receive light (494.62 ± 28.43 Lux) from 8:00a to 8:00p EST followed by darkness (0.034 ± 0.007 Lux) in the remaining hours of a 24-hr cycle (Light Meter
LT300, Extech Instruments, Nashua, NH). Fish were contained in 5-gallon tanks on a continuous flow system (Aquaneering, San Diego, CA) under standard pH (7.4±0.2) and conductivity (~800
µS/cm), regardless of lighting condition. All fish used were fed a pre-prepared mixture of ground flake food and husbandry system water once, daily (TetraMin Tropical Flakes, Tetra, Melle,
Germany). A subset of both cave and surface individuals were reared under dark-dark (DD), conditions for ~five years. DD conditions began when embryos were less than 12 hours post fertilization (hpf). 5-gallon husbandry tanks covered completely in black construction paper were utilized for DD rearing. All non-light variables were held constant between LD and DD tanks. A
180-fold difference in light availability was observed between LD tanks (48.59 ± 6.32 Lux) and
DD tanks (0.27 ± 0.06 Lux). Comparisons are drawn between cavefish and surface fish reared in
LD tanks (Cave LD, Surface LD) and cave and surface fish reared on DD tanks (Cave DD, Surface
DD). “Natural Conditions” refers to comparisons between Cave DD and Surface LD, while
“Light/Dark Conditions” refers to comparisons drawn between Cave LD and Surface LD. This approach allowed us to parse out gene expression differences unique to lighting condition. This work was conducted in accordance with a protocol approved by the Institutional Animal Care and
Use Committee of the University of Cincinnati (Protocol: 10-01-21-01).
RNA Isolations
17 RNA-seq analysis was conducted on (n=4) individuals from each Surface LD, Surface DD,
Cave LD and Cave DD tanks, encompassing 16 total samples. RNA isolations were performed between 11am and 2pm to limit variation in gene expression, as the expression in many genes (like those related to circadian rhythm) has been shown to oscillate based upon the time of day in other fish species (Idda et al. 2012). Fish were anesthetized on ice until no response to mechanical stimulus was observed (~30s-1min on ice). The head was separated from the body immediately posterior to the opercle bone and flash frozen with liquid nitrogen. Using a mortar and pestle, the head was ground into a fine powder. To prevent light-driven gene expression prior to euthanasia, this process was conducted under standard lighting conditions for LD individuals and under red- light illumination for DD individuals. Whole RNA isolation was performed on powdered tissue and immediately placed on ice (RNEasy Universal Mini Kit, Qiagen, Germantown, MD). The isolated total RNA was analyzed prior to RNA-sequencing for quantity and quality of RNA.
Samples were stored at -80°C prior to sequencing or cDNA synthesis. mRNA Sequencing and Processing
The 16 RNA isolate samples were sequenced in duplicate at Cincinnati Children’s Hospital and Medical Center DNA Sequencing and Genotyping Core. Prior to sequencing, samples were subjected to QC analysis and all samples surpassed the quality threshold determined by RQN>7 and were included in further analysis. The RNA was prepped for sequencing at the facility using the Illumina TruSeq Poly-A stranded tail kit, and sequenced in paired-end, 75bp reads to a depth of 10 million reads per sample on an Illumina HiSeq 2500 Rapid sequencer. Raw reads (.fastq) were analyzed for quality (FastQC, v0.11.5), and adapter sequences and poor sequence quality were trimmed off (Trimmomatic, V0.32), screening for TruSeq3-PE adapter sequences, and trimming low quality leading and trailing bases below quality 3, sliding window 4-15, and reads
18 below 36 bases long (Bolger et al. 2014). Additional analysis by FastQC following trimming ensured removal of adapter and poor quality sequences. qPCR Comparison
16 genes were selected for evaluation by qPCR. qPCR was conducted using a BioRad
CFX96 Touch™ Real-Time PCR Detection System (Hercules, California). Reserved RNA isolates from each LD & DD Surface and LD & DD Cave were utilized for cDNA synthesis. The same
RNA isolates were used for both RNA-seq and qPCR analyses. cDNA synthesis was performed using 1µg of RNA, thawed on packed ice for ~15 minutes until fully liquid before 0.25µg of
Oligo(dT)12-18 Primer (Invitrogen) was added to each reaction, and total volume was brought to
13µL with sterile, molecular grade water. These reactions were incubated at 65°C for 10 minutes
(C1000 Thermocycler, BioRad) prior to the addition of 4µL Transcriptor RT Reaction Buffer, 5x
(Millipore-Sigma), 0.5µL Protector RNase Inhibitor (Millipore-Sigma), 2µL dNTP mix (10 mM;
Roche), and 0.5µL Transcriptor Reverse Transcriptase (Millipore-Sigma). 20µL reactions were incubated at 50°C for 60 minutes, followed by inactivation of the enzyme at 85°C for 5 minutes.
Resultant cDNA was stored at -20°C until use.
For qPCR, 20µL reactions were performed using SsoFast EvaGreen Supermix (BioRad,
Hercules, CA.) for each of 16 chosen genes. Cycling conditions were enforced based on suggested specifications for EvaGreen Supermix (activation: 95°C for 30s, denaturation: 95°C for 5s, annealing: 60°C for 5s, with the addition of a melt curve: 65-95°C in 0.5°C increments of 5s/step).
Normalized expression was determined by comparison to the housekeeping gene bactin1 (actb1
(ENSAMXG00000004264.1; McCurley and Callard 2008); CFX Maestro v4.0.2325.0418,
BioRad, Hercules, CA). Prior to qPCR analysis, it was ensured that all primers mapped only to gene of interest using Primer-BLAST (NCBI, Primer3, Ye et al. 2012) and confirmed by the
19 appearance of a single melt temperature per amplicon. Correlation between RNA-seq (RPKM) and qPCR expression (ΔΔCt) was determined by Pearson’s correlation (Microsoft Excel, 2016).
Differential Expression Analyses
RNA-sequencing reads were aligned to the draft A. mexicanus genome (Ensembl Genome
Browser [v93]) for 25,271 unique genes using ArrayStar software (v15.0, DNAStar, Madison,
WI). Sequences from duplicate individuals were grouped, and normalized expression was determined for each experimental group (LD cavefish, LD surface fish, DD cavefish and DD surface fish) by RPKM normalization methodology by ArrayStar (Mortazavi et al. 2008).
Measures of correlative value were assessed by R2 value (determined by ArrayStar) to determine global similarity in expression between two conditions of interest (e.g. LD Cave to LD Surface).
Numbers of differentially expressed genes (DEGs) were determined at both fold-change and confidence interval thresholds, all numbers of DEGs were reported at a 99% confidence interval.
This threshold was also employed to determine the pattern of differentially expressed genes in both Light/Dark Conditions and Natural Conditions for fish of both morphotypes. The number of genes with relatively high and low expression was determined at this threshold by a count of the total of unique gene identities. Genes sensitive to lighting condition were selected based upon differences in fold-change between Natural Conditions and Light/Dark Conditions; genes with an
RPKM <1 for all four experimental conditions were excluded in this analysis. Gene identity was not a factor for inclusion in the table, and genes were chosen based on RPKM and fold change patterns that exemplified a sensitivity to light. Representative genes of both high and low average
RPKM values were chosen.
Gene Ontology Enrichment Analysis
20 Gene ontology (GO) enrichment analysis was performed using Blast2GO (v5.0)
(www.blast2go.com; Ashburner et al. 2000; Harris et al. 2004). The whole transcriptome (25,271 genes) was used as a reference for expected quantity and proportion of GO terms. The local database was compiled from the Danio rerio proteome obtained from the UniProt-Proteomes
Database (UniProt ID: UP000000437, modified June 27, 2018). This database includes proteins on all 25 D. rerio chromosomes, mitochondrion, and unplaced sequences. The database was formatted via Blast2GO. BLAST was performed through ‘BLAST’ function (NCBI BLAST
(blast+) service against UniProt Database in .psq formatting. DEGs were selected for GO enrichment analysis if they met a fold-change threshold of 4-fold or higher between two experimental conditions (Natural Conditions or Light/Dark Conditions) and were separated into groups by direction of fold-change in cavefish relative to surface fish (e.g. higher expression in
Cave LD vs. lower expression in Cave LD). These genes were compiled into test sets and enrichment of terms in these test sets was determined by comparison to the reference set ontology terms in the whole transcriptome. Enriched categories of GO Terms were then filtered by their involvement in cave biology.
QTL Comparison
To determine the location of QTL markers relative to potential candidate genes identified by expression, the draft genome available on NCBI (Astyanax_mexicanus-2.0, release 102) was compared to the Ensembl database draft genome (v93). The location of markers associated with known QTL was compiled by BLAST of the nucleotide sequence of the marker to the NCBI
Astyanax genome (NCBI, Astyanax_mexicanus-2.0, release 102; McGaugh et al. 2014). Trimmed sequencing reads were aligned to each chromosome where a QTL marker resides of the Astyanax genome individually (NCBI). As prior analyses of the dark-reared transcriptome were conducted
21 using the Ensemble Astyanax genome database, Blast2GO was utilized to compare both Ensembl and NCBI databases to the Danio rerio UniProt Database (UniProt ID: UP000000437). This was used to determine the relative chromosomal position of draft Astyanax Ensembl database genes based on the known chromosomal position of the gene in the NCBI draft Astyanax genome. Prior analyses in this system have identified the genes melanosome protein PMEL (pmela) and 5,6- dihydroxyindole-2-carboxylic acid oxidase-like (tyrp1b) as potential mediators of melanophore number in Astyanax cavefish (Stahl et al. 2018). As such, this analysis notes the known location of these genes on the current draft of the Astyanax genome (NCBI, Astyanax_mexicanus=2.0, release 102), and location of the associated QTL markers ASTYANAX_28 and TP33309. To identify genes potentially related to eye loss or vision loss, all genes residing on a chromosome with a QTL and an expression difference of 2-fold or higher between cave and surface, under any lighting regime, were considered. Vision-related genes were selected from this group based on GO
Term. Genes with GO terms inclusive of ‘eye’, ‘lens’, ‘visual’, and ‘retina’ were included (‘vision’ was excluded due to the presence of the word ‘division’ in many GO terms). Pigmentation-related genes with attached GO terms which include the words ‘chromophore’, ‘melanocyte’,
‘melanosome’, ‘melanin’, or ‘pigmentation’ were considered. The positional information of QTL compared to selected genes is visualized using Circos (Circos, RCircos, R, Krzywinski, et al. 2009,
Fig. 6 & 7)
22 RESULTS
Variability in lighting conditions reveals light-sensitivity in the Astyanax transcriptome
Light sensitivity is first assessed by examining differential expression as plotted on a logarithmic scale (Figure 1). Each gene is plotted on this x-y axis (representing 25,271 total genes).
Red genes in the top half of the graph represent genes with a higher expression in the condition represented on the y-axis while blue genes represent genes with a higher expression in the condition represented on the x-axis (Figure 1). Three metrics are used to assess similarity in global gene expression: 1) spread of points, 2) R2 value, and 3) the difference between the line of regression (Figure 1, purple lines) and the 0-fold change line (Figure 1, green lines). Genes residing on this 0-fold change line are not differentially expressed between the two conditions of interest.
Comparisons between closely related conditions such as all LD-reared fish to all DD-reared fish
(both cave and surface for each; Figure 1, A) reveal a high R2 value (0.9751; Figure 1, A; Table
1), a small spread of points about the line of regression, and an overlap between the line of regression and the 0-fold change line. Comparisons of similarity in expression between all cave and all surface fish yield an R2=0.8873 (Table 1), which decreases when accounting for differences between the two morphotypes exclusively on Light/Dark Conditions. Comparisons of global differential gene expression under Light/Dark Conditions reveal a lower relative R2 value (0.8657;
Figure 1, B; Table 1). Additionally, the spread of points about the line of regression is higher than comparisons between all LD- and DD-reared fish and the distance between the line of regression and 0-fold change line is noticeable (Figure 1, B). The R2 value (0.8597; Figure 1, C; Table 1) of
Natural Conditions is lowest of any comparisons drawn (Table 1). In comparison to Light/Dark
Conditions, the spread of points about the line of regression is higher, and the distance between
23 the 0-fold change line and the line of regression is greater (Figure 1, B and C). Comparisons of differential gene expression on a global scale are least similar under Natural Conditions.
To evaluate the polarity of differential gene expression, the number of DEGs that have a higher expression on either cave or surface fish is examined. At each fold-change threshold (2-, 4-
, 8-, or 16-fold) there are more genes with a higher expression in surface fish than cavefish, conversely more genes have a lower expression in cavefish than in surface fish (Figure 2). The number of genes with lower expression in surface fish on Light/Dark Conditions: 322; lower expression in cavefish on Light/Dark Conditions: 785; lower expression in surface fish on Natural
Conditions: 224; lower expression in cavefish on Natural Conditions: 621 (Figure 2).
“Natural” photic rearing reveals novel differences in gene expression
The number and identity of DEGs is examined to assess if DEGs seen under Natural
Conditions are identical to those seen under Light/Dark Conditions. These DEGs represent genes with a differential expression at each fold-change threshold (2-, 4-, 8- and 16-fold) regardless of which experimental condition has higher expression (Figure 3, bottom). The number of DEGs seen under Natural Conditions is expanded under each fold-change threshold compared to genes that have a differential expression under both Natural and Light/Dark Conditions (Figure 3, bottom).
DEGs at a 2-fold threshold display a 160.8% increase in the number of DEGs found unique to
Natural Conditions compared to total shared genes. This is confounded by the large number (2495) of genes unique to Light/Dark Conditions. Genes with a 4-fold or higher difference between cave and surface fish display a 61.1% increase in the number of differentially expressed genes under
Natural Conditions compared to Light/Dark Conditions (Figure 3). This pattern is best represented at a 4-fold threshold. The number of DEGs is expanded under Natural Conditions (Figure 3, top, blue/red hash) compared to DEGs that are shared between the conditions (Figure 3, top, green) or
24 to DEGs unique to Light/Dark Conditions (Figure 3, top, yellow). At a 4-fold threshold and 99%
Confidence Interval (C.I), under Light/Dark Conditions, 845 DEGs were identified with 158 genes unique to this comparison (Figure 3). Comparisons on Natural Conditions revealed 1107 DEGs, with 420 DEGs unique to Natural Conditions also at a 4-fold difference and 99% C.I. threshold.
Darkness impacts genes with functions in blood, metabolism and olfaction
Genes most impacted by lighting condition are examined to begin to determine the genes and functions impacted by darkness in this system. Twenty genes dramatically impacted by lighting condition were selected (Table 2). The fold-change difference of these genes under
Natural Conditions (Table 2, last column) increases in magnitude compared to the fold-change apparent under Light/Dark Conditions (Table 2, second column from right). The identity of these genes indicates an effect of light on pigmentation (gja5b), metabolism (apoa1), and blood (hbaa1, ba1, hbae1.3, hbba2, hpx, serpina1, fgb, fgg).
GO enrichment analysis of DEGs with a higher expression in cavefish on Light/Dark
Conditions reveals the enrichment of terms referring to olfaction (Figure 4, blue) and “other” cave biology traits (Figure 4, green) as indicated by the literature. This represents a set of genes that have a diverse function, but that may aid in adaptation to the cave through various biological processes related to sensory, development, eye development, or other prototypic cave traits. Within
DEGs with a higher expression in surface fish under Light/Dark Conditions, terms referring to pigmentation (Figure 4, red), metabolism (Figure 4, purple), vision (Figure 4, orange), and “other” cave traits (Figure 4, green) are enriched.
In contrast, on DEGs unique to Natural Conditions within genes that have a higher expression in DD Cavefish compared to LD Surface fish, terms referring to olfaction (Figure 5, blue), blood (Figure 5, pink) and “other” cave related traits (Figure 5, green) are enriched. Within
25 Natural Conditions-specific DEGs with a lower expression in DD Cavefish compared to LD
Surface fish, terms referring to metabolism (Figure 5, purple), blood (Figure 5, pink) and “other” cave related traits (Figure 5, green) are enriched. GO enrichment analysis reveals the involvement of light in blood and the olfactory system.
Novel DEGs map near previously discovered loci associated with regressive loss
The location of 15 unique markers associated with vision was determined (Figure 6, orange boxes). Across the transcriptome, 268 vision-related genes with differential expression between cave and surface fish were determined based on GO term, and the position of each gene plotted against the position of vision-related QTL (Figure 6, black lines). Due to position relative to a QTL and function in other organisms, 18 genes are selected and highlighted (Figure 6, table). Some of these genes have a function that indicates a particularly compelling role in eye development or associated processes (Figure 6, table, bold). Genes such as gucy2d, gucy2f, and pde6ha are implicated as potential candidate genes for eye loss in this system due to their function and position relative to eye-related QTL (Figure 6).
The location of 13 markers associated with regressive pigmentation was determined by
BLAST (Figure 7, red boxes). By filtering of associated GO terms, 61 pigmentation genes with differential expression between cave and surface fish were found within the transcriptome (Figure
7, black lines). Based on similarity in location to a pigmentation-related QTL, 19 genes were selected (Figure 7, table). A number of these genes were further examined based on their role in pigmentation development (Figure 7, table, bold). Genes including rab32b, and sox18 have been implicated as potential mediators of pigmentation loss based on their chromosomal position and expression in LD and DD cavefish compared to LD surface fish (Figure 7). qPCR analysis validates dark-sensitive differential gene expression in A. mexicanus
26 Gene expression of 16 genes is assessed through qPCR. These genes represent a diverse set of patterns of gene expression (i.e. expression higher in cave, higher in surface, higher in the dark, etc.). These genes were selected based upon overall gene expression as evaluated through
RNA-seq. Expression values across both lighting condition and morphotype were evaluated
(Figure 8). The average Pearson’s correlation for gene expression between qPCR and RNA-seq methodologies was 86.8% (Table 3). This indicates that expression evaluated on both qPCR and
RNA-seq methodologies is highly correlated (Figure 8). A number of the genes evaluated in both
RNA-seq and qPCR demonstrate an extraordinarily high correlate relationship (>95% Pearson’s correlation). Genes such as f8 and rho demonstrate that, despite large differences in average gene expression between all four conditions, both methodologies are able to discriminate these differences. qPCR analysis confirms the expression data obtained through RNA-seq methodologies.
27 DISCUSSION
This transcriptomic assessment on the Natural Conditions of A. mexicanus indicates that darkness does affect global RNA architecture. The number of DEGs is expanded under these
Natural Conditions, and darkness affects blood, metabolism, and olfaction based on analysis of gene function. Additionally, some genes affected by light may contribute to trait loss (such as eye and pigmentation loss) in this system. Taken together, these results indicate global, and functional alterations to gene expression between Light/Dark and Natural Conditions.
Dark-rearing drives broad scale changes to gene expression in Astyanax cavefish
Previous assessments of the transcriptome in the A. mexicanus system have characterized the variability in gene expression that exists between cave and surface fish (Gross et al. 2013;
Hinaux et al. 2013). These gene expression differences exist throughout development and may contribute to recognizable phenotypic differences between cave and surface fish (Stahl & Gross
2017). Investigation of the effect of light on a system marked by extreme darkness is important to the understanding of cave-related traits in cavefish, as this fish has evolved under millions of years of darkness (Porter et al. 2007).
To assess the global RNA architecture of A. mexicanus cavefish reared in DD conditions,
I have examined the expression of all 25,271 genes in concert (Figure 1; Table 1). The highest diversity in gene expression is seen when animals are reared under Natural Conditions by comparison of R2 value (Table 1). Natural Conditions have the lowest R2 value of any comparisons drawn (R2 =0.8567; Table 1). As these comparisons are conducted on all represented genes in the genome, any noticeable perturbations to R2 value represent a change in expression in many genes.
Inter-morphotypic global gene expression is least similar under natural conditions. This indicates that lighting condition plays much more of a role than previously anticipated.
28 Regardless of rearing condition, DEGs predominantly exhibit a decrease in expression in cavefish, with few genes showing increased expression relative to surface fish (Figure 2). Further examination of dark-inducible differences to global gene expression revealed a stereotypical pattern in DEGs, most notable in DEGs with a 16-fold change or greater between cave and surface fish reared in Light/Dark or Natural Conditions. Under this 16-fold threshold, more than twice as many of these DEGs have a lower expression in cavefish than surface fish, regardless of lighting condition. As the majority of the most differentially expressed genes across the transcriptome have a lower expression in cavefish, these genes with a dramatically lower expression in cavefish than in surface fish may be driving some of the prototypic cave phenotypes apparent in A. mexicanus.
Previous work supports this finding (Strickler & Jeffery 2009), indicating that this pattern of polarity of differential expression exists independent of lighting condition. Interestingly, this pattern of differential gene expression is not observed in transcriptomic analyses of development in cavefish compared to surface fish (Stahl & Gross 2017). In fact, the inverse appears to be true, with cavefish demonstrating a higher expression in more genes than surface fish; although, this pattern seems to flip as developmental age increases—10hpf cavefish embryos harbor a lower number of genes with reduced expression than surface fish, while 72hpf cavefish embryos harbor a higher number of DEGs much more similar to the pattern of expression exhibited in this dark- sensitive transcriptome in adults (Stahl & Gross 2017). Something is likely occurring during this developmental gap between 72hpf embryos and adults to drive the expression of many genes lower in cavefish compared to surface fish. This large set of under-expressed genes in cavefish may indicate that a lower expression of genes contributes to trait loss, or that the accumulation of mutations related to regressive traits results in the under-expression of many genes. However, it
29 cannot be ruled out that relaxed selective pressures due to some features of the cave environment
(such as low predation) may be more permissive of the under-expression of many genes.
Natural Conditions reveal substantial effects of darkness on many biological systems
Out of a subset of genes selected for their sensitivity to light, a near-majority (10/20) of the selected genes that increase in expression in the dark are hemoglobin subunits, or blood related proteins (Table 2). The expression of these genes is vulnerable to changes in lighting condition, and each of these genes were chosen based on the increase in the magnitude of differences in expression, not based on identity. In genes that have a reduced expression in DD Cave, two are related to fibrinogen proteins (fgg and fgb), which function in blood clot formation (Table 2).
Additionally, there is a dramatic increase in the expression of hemoglobin subunits (ba1, hbaa1, hbba2, hbae1.3) exclusively in dark-reared cavefish (Table 2). This indicates a response to lighting condition unique to cavefish. Following GO term enrichment analysis, two terms, “blood coagulation” and “fibrinogen”, were enriched in down-regulated genes of DD Cave (Figure 5).
Analysis of the genes underlying these enriched terms reveals dark-reared cavefish differentially expressed a number of hemoglobin subunits (e.g. hbaa1, hbbe2; higher expression in DD cave), and coagulation factors (e.g. fgg, f9b; lower expression in DD cave) compared to LD-reared counterparts (Figure 4; Figure 5). Under Natural Conditions, there is altered expression of olfaction-related genes, as indicated by the enrichment of terms such as ‘olfactory receptor activity’ and ‘response to odorant’ in genes that have a higher expression in dark-reared cavefish
(Figure 5).
Previous work in A. mexicanus has focused on exploration of traits such as pigmentation and eye loss, and non-visual sensory systems; however, the blood composition of cavefish has been particularly understudied. Rearing animals under Natural Conditions may drive differences
30 in blood composition. Lighting condition has an effect on the expression levels of blood related genes. The functional role of this phenomenon requires further investigation. These functional differences indicate that differences in the expression of blood genes (likely among other traits) are revealed under Natural Conditions. Caves are often marked by their low oxygen levels perhaps as a result of their extreme darkness, and lack of photosynthetic species. In fact, some of the main abiotic factors characterizing life in caves is a limited oxygen supply and lack of seasonal or daily fluctuations in oxygen level (Coineau 2000). Investigations into the effect of cave-dwelling on hemoglobin content, or blood composition have been limited. Previous assessments in another cave species, Triplophysa rosa, have indicated that hemoglobin content is less affected by light than it is body mass (Shi et al. 2018). However, this study did not rear cave animals in the dark, they were acclimated to the conditions for three days. In another species of the Astyanax genus, the river dwelling fish Astyanax scabripinnis, altitude was not found to have a significant effect on hemoglobin oxygen affinity, as measured by the Bohr effect (Landini et al. 2002). Furthermore, prior assessments of blood in Astyanax cavefish have been limited to glucose concentration (Riddle et al. 2017). These data suggest that there is perhaps a phenotypic change of the blood associated with hemoglobin when cavefish are reared in the dark, though further analysis is required.
The coordinated up-regulation of oxygen carrier-related genes in DD cavefish may indicate a dark-dependent response to the decreased amount of dissolved oxygen found in caves (Ornelas-
Garcia et al. 2018). Should cavefish be changing their hemoglobin content in response to photic condition, it would shed light on how this organism successfully responded to the cave environment. This informs on the concept of preadaptation in cave biology. An upregulation of hbaa and other alpha-like hemoglobin genes may indeed be one of these features should the expression level correspond to a functional increase in oxygen carrying capacity. These findings
31 on the increase of expression of blood-related genes on DD conditions in cavefish may indicate rearing cave animals under light-dark conditions represses the expression of key blood-related genes and rearing the animal on Natural Conditions restores the environmentally normative expression of these genes. This finding reveals an unappreciated light-dependency of the circulatory system in cavefish. This may explain an important, and previously unknown, adaptive mechanism of A. mexicanus to the extreme, dark, cave environment.
The appearance of olfaction-related terms in enrichment analyses of dark-reared cavefish indicates a vulnerability of olfaction-related gene expression to lighting condition, including particular odorant receptor genes expressed in olfactory neurons (Vogt et al. 1997; Alioto and Ngai
2005). While these terms are also enriched in LD Cave, the enrichment of olfaction-related terms in DD Cave indicates that additional olfaction-related genes may have a higher expression in DD
Cave relative to their LD reared counterparts. The identity of these olfaction-related genes with a higher expression in cavefish is almost exclusively odorant-receptor genes (e.g. or101-1, or118-
2; higher expression in LD Cave compared to LD Surface). This is recapitulated in DD cavefish compared to LD surface fish (e.g. or126-1, or125-5; higher expression in DD cavefish compared to LD surface fish). the number of differentially expressed olfactory and odorant receptor genes in dark-reared cavefish as evidenced by their contribution to the enrichment of olfaction related GO terms may indicate alterations to the number of olfactory neurons in DD reared cavefish. While the enhancement of the gustatory and olfactory system processing in cavefish compared to surface fish has been established—analysis of the cavefish brain indicates an expanded telencephalon, wherein the olfactory bulbs are located (Rétaux et al. 2008). The impact of light on these systems has not been thoroughly addressed in A. mexicanus cavefish. Perhaps the lack of light acts as a cue to increase the processing ability of non-visual sensory systems—a phenomenon believed to be
32 the reason for the increase of other non-visual senses in this system, such as neuromasts
(Yoshizawa et al. 2014). Importantly, the function of these olfaction- and odorant-receptor genes has not been verified, and further analysis of these genes and olfactory neurons is needed to yield a clear picture of the role of light in olfaction in cavefish.
Additionally, light appears to affect pigmentation-related genes such as gap junction protein 5b (gja5b) which exhibits a 3.35-fold increase in expression in dark-reared cavefish in comparison to surface fish (this gene is also referred to as connexin 41.8 (cx41.8)). Interestingly, this gene retains a very low level of expression in cave and surface fish reared on Light/Dark
Conditions (0.73 and 0.55 RPKM, respectively; Table 3); however, the expression of this gene is higher in cavefish reared under DD conditions compared to LD surface fish. Mutations in gja5 in humans are associated with idiopathic atrial fibrillation due to the role of gja5 in activation of the atria (Gollob et al. 2006), which may indicate a role of this gene in the circulatory system as well as in body pigmentation. In zebrafish, gja5b plays an important role in pigmentation; defects in this gene result in the leopard zebrafish mutant, devoid of its characteristic stripes which have instead been organized into leopard-like spots (Irion et al. 2014). Though cavefish do not display the same melanin pigmentation as surface fish, cavefish have melanophores that do not produce melanin, though these melanophores are reduced in number compared to surface fish (Jeffery
2009). The altered expression in gja5b in dark-reared cavefish may indicate an effect of light on melanophore distribution or quantity in cavefish. Taken together, the sensitivity of gja5b to light and the involvement of this gene in the organization of pigmentation is of particular interest due to the hypermelanism demonstrated in hybrid progeny of cave and surface fish, and in DD-reared surface juveniles which exhibit a significant increase in the melanophore number and melanophore darkness (Gross et al. 2016; Berning, unpublished data).
33 Comparison of DEGs to QTL marker location nominates potential candidate genes for vision and pigmentation loss
Previous studies in Astyanax have capitalized on the inter-fertility of the two morphotypes to create hybrids, allowing the nomination of candidate genes contributing to trait loss by QTL analysis (Şadoğlu 1957; Borowsky & Wilkins 2002). Some of these previous studies have proposed genes that play a role in pigmentation loss (tyrp1b, pmela, mc1r, among others) or eye loss (shisa2, crxa, cryaa, etc.) (Gross et al. 2009; McGaugh et al. 2014; Stahl et al. 2018).
Analyzing RNA-seq expression data in pigmentation- and vision-related genes is valuable to understanding what genes may be potentially contributing to feature loss in cavefish.
211 total markers associated with known QTL were considered in this study. 59 QTL markers have been found to be associated with regressive eye phenotypes in eight prior QTL analyses (Borowsky and Wilkens 2002; Protas et al. 2007; Protas et al. 2008; Yoshizawa et al.
2012; O’Quin et al. 2013; Kowalko et al. 2013a; b; Gross et al. 2014). This represents a wealth of data on which regions of the chromosome may potentially influence eye size, pupil size, lens size, or eye orbit diameter, depending on the marker. Subsequent studies have identified the same marker for multiple similar or identical traits; as such, there exists 48 unique QTL markers referring to phenotypes associated with eye or vision reduction or loss. Three of these unique QTL were found as a result of a backcross between Pachón cavefish and surface fish and subsequent random amplified polymorphic DNA (RAPD) fingerprinting to generate anonymous markers, and have no associated positional information reported (Borowsky & Wilkens 2002). These QTL markers refer to phenotypic traits such as “Eye Size”, “Lens Size”, and “Retinal thickness” among other traits (Borowsky & Wilkins 2002; Protas et al. 2007; Protas et al. 2008; Yoshizawa et al.
34 2012; O’Quin et al. 2013; Kowalko et al. 2013a; b; Gross et al. 2014). The positional information for 15 unique vision-related QTL was determined (Figure 6).
307 vision-related genes were determined by GO term. These 307 genes were then directly compared to the location of eye-related QTL. One such gene, rx3 lies ~5Mb from marker 55A on
Chromosome 14 (Figure 6). This gene has been found to be critical for eye formation in both zebrafish and medaka (Loosli et al. 2003). This may be a potential candidate gene for eye loss in
Astyanax cavefish, and has been discussed previously (McGaugh et al. 2014). Two genes, guanylate cyclase 2F and 2D (gucy2f, gucy2d), are located ~6Mb downstream of marker 55B on chromosome 1 and ~7Mb downstream of marker 234B on Chromosome 3, respectively, and present as promising candidates due to their role in regressive vision in humans. Another gene,
Phosphodiesterase-6 (pde6ha), resides on Chromosome 13 on the draft Astyanax genome ~1.6Mb from marker 30C. This gene has been identified based on its role in phototransduction.
Genes gucy2d and gucy2f are nominated due to their function in vision and retina development. Defects in human gucy2d are linked to Leber congenital amaurosis, a disorder that causes severe visual impairment due to defects in the retina (Milam et al. 2003). Previous studies have also established that knockdown of gucy2f in zebrafish creates a similar visual dysfunction model, accompanied with histological changes to the eye (Stiebel-Kalish et al. 2012). Prior investigations into the function of gucy2d and gucy2f in subterranean mammals revealed the inactivation of either or both genes in multiple species of mole (Heterocephalus glaber and
Chrysochloris asiatica); ultimately indicating that the inactivation of gucy2d may have greater consequences to vision and cone degradation than gucy2f in these underground mammals
(Emerling and Springer 2014). These genes have not been thoroughly investigated in Astyanax
35 fish to date but may present candidates for retinal and cone degeneration, as rendering these genes non-functional in other systems results in optical degradation.
The gene pde6h encodes a subunit that resides in cones and is activated by light stimulation
(Ionita & Pitler 2006; Collery & Kennedy 2009). Pde6h has been implicated in cone phototransduction, specifically as a causative gene in human achromatopsia, which results in not only color blindness but also reduced visual acuity (Kohl et al. 2012). However, functional analyses of pde6h in mouse suggest a species-specific effect of the gene, as mouse knockouts did not exhibit noticeable regressions to phototransduction (Brennenstuhl et al. 2015). In cavefish, as in zebrafish and other teleost species, pde6h has two paralogs pde6ha and pde6hb, and both A. mexicanus and D. rerio have lost the first intron of pde6ha, unlike other teleost species indicating a closer relationship between these two organisms than other teleost fish (Lagman et al. 2016).
While there is limited information about the full role that pde6ha plays in phototransduction in zebrafish, this gene appears to be a part of the light reception cascade and is upregulated in response to light in D. rerio (Weger et al. 2011). Further functional analyses of this gene have not yet been examined, but due to its role in reduced visual acuity in human disease this may indeed be a promising candidate gene for vision loss in cavefish.
Previous studies have compared gene location and QTL location to nominate candidate genes. Specifically, genes such as rx3 and crx were nominated as potential mediators of eye loss in this system (McGaugh et al. 2014). These genes have been found to be strong contenders in not just this system, but also in Sinocyclocheilus cavefish as well. Interestingly, DD rearing in these
Chinese cavefish causes a down-regulation of these transcription factors and downstream regulators of retina and eye size (Meng et al. 2013). Additionally, this novel transcriptomic assessment investigated the position of genes cryaa and tbx2b for their implication in eye loss
36 (McGaugh et al. 2014). While these genes have been nominated as potential mediators of eye and vision loss in cavefish in the past, these genes do not fall near or within the set boundary of a known QTL, given the restrictions in place on differential expression (2-fold or higher to be included). However, rx3 and crx each fall near the QTL boundaries of markers 55A and 216F respectively (Figure 6). crx has been implicated previously as promoting retinogenesis (Shen &
Raymond 2004). This gene may indeed play an important role in eye loss in this system.
In pigmentation, 29 QTL markers have been linked to this regressive trait in previous studies. Multiple studies have identified the same QTL marker, as such 22 unique loci are associated with pigmentation. Of these, two refer to albinism, and one refers to marker OCA2 which refers to gene oca2 (Figure 7) from four studies (Borowsky & Wilkens 2002; Protas et al.
2006; Yoshizawa et al. 2012; Gross et al. 2014); loss of function in this gene results in albinism, and this gene is found to be the cause of albinism in multiple cave populations (Protas et al. 2006;
Gross and Wilkens 2013). Of the remaining 20 QTL associated with regressive pigmentation in cavefish, one refers to the trait of brown melanophores, marker 230D on chromosome 18 (Figure
7; Gross et al. 2009). This trait is found to be the result of alterations in the coding sequence of the gene Mc1r, resulting in reduced melanin content within brown melanophores and the brown phenotype (Gross et al. 2009). Interestingly, marker 230D is also associated with melanophore number above the left eye and in the middle of the dorsal fin (Protas et al. 2007; 2008). One albinism QTL marker was identified subsequent to a backcross of Pachón cavefish with surface fish and have no positional information associated (Borowsky and Wilkens 2002). The remaining
19 unique QTL refer to a standardized score of melanophores on the back and dorsal fin, and melanic pigmentation above the left eye, in the middle of the dorsal fin, slightly above the middle of the anal fin and in the lightly pigmented region between the heavily melanized lateral stripe and
37 the more heavily pigmented area below the stripe (Borowsky and Wilkens 2002; Protas et al. 2007;
2008). Of these 19 QTL, two were identified via a Pachón x surface backcross and RAPD sequencing, much the same as one albinism QTL mentioned above; as such, the positional information of these QTL was not obtained (Borowsky and Wilkens 2002). The positional information of 6 other QTL markers was unable to be obtained. The 11 remaining unique pigmentation related QTL are depicted on Figure 7. This study also includes the location and associated expression of genes pmela and tyrp1b, as these genes have been previously implicated for their role in melanic pigmentation in Astyanax cavefish (Stahl et al. 2018). BLAST of marker sequence against the Astyanax genome conforms to the previously established location of tyrp1b and pmela in comparison to QTL markers ASTYANAX_28 and TP33309, respectively. Although, in an updated draft of the Astyanax genome, the markers and genes have changed chromosomal position from the previously reported location (Stahl et al. 2018). Interestingly, the expression of these genes decreases slightly in cavefish under dark rearing, although this decrease is nominal in each (tyrp1b: LD Cave: 0.124, DD Cave: 0.074, pmela: LD Cave: 0.157, DD Cave: 0.143, Figure
7). This may indicate a role of light on melanosome organization, but this has not been confirmed by any phenotypic or functional analyses.
The positional information in the draft genome for 61 pigmentation-related genes was determined. Located ~5Mb upstream of marker 206A is gene Rab32b (Figure 7). LD reared cavefish exhibit a 9.34-fold increase in rab32b expression compared to LD reared surface fish.
Rab proteins, a family of small GTPases, are involved in membrane trafficking between organelles in eukaryotic cells (Diekmann et al. 2011; Coppola et al. 2016). Rab32a and b (redundantly with closely related Rab38, possibly resultant from vertebrate whole genome duplication [Wasmeier et al. 2006; Coppola et al. 2016]) function as a component of melanocytes and melanosomes and
38 transport tyrosinase (Tyr) and tyrosinase related protein (Tyrp1) to maturing melanosomes
(Braasch et al. 2009; Wasmeier et al. 2006). Rab38 defects in mice were found to produce the
“chocolate” phenotype, characterized by hypopigmentation of the eyes and coat (Loftus et al.
2002; Brooks et al. 2007). In zebrafish embryos, Rab32b paralog Rab32a is expressed in the retinal pigment epithelium and in cells positionally determined to be migrating melanoblasts at 24hpf
(Coppola et al. 2016). Defects in tyrp1b have been found to contribute to melanophore darkness and position in Astyanax cavefish (Stahl et al. 2018); potential defects in Rab32b (as the protein that would transport Tyrp1b into the melanosome) further complicates Tyrp1b’s role in complex pigmentation loss. Interestingly, LD reared cavefish exhibit a 9.34-fold increase in rab32b expression compared to LD reared surface fish. This increase in expression may mitigate any defects in tyrp1 expression by increasing Tryp1 protein in the cell due to an increase in Rab32 protein quantity, should the expression increase coincide with an increase in protein production.
However, this increase in expression all but disappears in DD reared cavefish, where there is a mere 1.24-fold increase in rab32b expression in DD reared cavefish compared to LD reared surface fish (Figure 7). Again, rearing cavefish on their natural conditions may restore the expression of this gene to its ‘normative’ (based on surface fish expression) condition. Rab32b may indeed be a potential candidate gene for pigmentation regression in Astyanax cavefish.
Another marker 214C, found on chromosome 15, has been linked to anal fin melanophore number. The gene SRY-related HMG-box 18 (sox18) resides ~5Mb downstream from this marker
(Figure 7). Sox-family transcription factors regulate a diverse set of features and events throughout early development including neural crest development (Kiefer 2007). Sox18 is involved in vascular and lymphatic system development and sox18 has been found to be the causative gene in human
Hypotrichosis-Lymphedema-Telangiectasia, which is characterized by abnormalities in the hair
39 and the cardiovascular and lymphatic systems (Irrthum et al. 2003; François et al. 2008; Cermenati et al. 2008). Most importantly, sox18 defective mice have been shown to demonstrate defective coats, including an alteration to pheomelanin content in hair shafts, which results in a darker appearance overall (Pennisi et al. 2000). Beyond the involvement of sox18 in these diverse traits, little is known of the transcription factor’s role in complex pigmentation development. The expression of sox18 increases in dark-reared compared to light-reared cavefish. The expression of sox18 more closely resembles surface fish in LD cavefish, although there is only a 2.05-fold increase in gene expression in cavefish compared to surface fish (Figure 7). Due to the alterations to pheomelanin content in mice hair in sox18 mutants, a role of this gene in pigmentation development cannot be ruled out without further study.
As noted, mc1r (brown melanophores; Gross et al. 2013) resides on chromosome 18 in the draft Astyanax genome. Interestingly, a copy number variant of mc1r is located just 0.06Mb up- stream from mc1r (based on NCBI genome assembly ‘Astyanax_mexicanus-2.0’). This mc1r duplicate has been explored previously (Gross et al. 2017) In both genes, expression is higher in surface fish than cavefish, although expression of mc1r appears to decrease slightly in the dark, while mc1r-like increases slightly. Previous studies have indicated that the duplicate mc1r gene may indeed slightly rescue the brown phenotype observed in cavefish (Gross et al. 2017). Why this gene is differentially expressed in response to lighting condition needs further exploration before the function of this change can be hypothesized. This indicates a complexity to pigmentation related gene expression that will require further functional analysis to parse out.
Evolution under the pressure of constant darkness
The investigation of the influence of darkness on the transcriptome of A. mexicanus raises interesting questions of what behaviors and traits need to be investigated under DD conditions. An
40 eye-focused transcriptome in Sinocyclocheilus indicates that DD conditions cause a down- regulation of transcription factors, resulting in retinal and eye size reduction; however, this in contrast to A. mexicanus (Meng et al. 2013). The findings support this contrast; there is an increase in expression of both rx3 and crx in dark-reared cavefish compared to LD cavefish, however this increase is marginal. The increase in expression in these genes is below 0.5 RPKM for either gene.
The largest fold change in transcription factors such as crx and rx3 is seen under Light/Dark
Conditions. In crx specifically, this is likely due to the lower expression in LD and DD cavefish
(0.293 and 0.479 RPKM, respectively) compared to higher expression in LD surface fish (4.688
RPKM).
Previous studies have established the morphological effect of dark-rearing on the phenotype of A. mexicanus cave and surface fish, but expression level differences have not been previously explored. This study examines and describes the effect of dark-rearing on global gene expression and on the expression of genes related to pigmentation, vision, olfaction, and blood, among other traits. As such, a diverse group of traits may be affected by dark-induced gene expression, this study makes a compelling argument for the additional study of phenotype under environmentally relevant rearing conditions. Gene expression is affected by light on a global scale.
Furthermore, more diverse gene expression is apparent under Natural Conditions, revealing the effect of light on blood. Expression level differences of hemoglobin subunit genes have not been appreciated under Light/Dark conditions—this is especially relevant given the lighting regimen under which the cavefish evolved. By capitalizing on the differential expression of many pigmentation- and vision-related genes, I have nominated candidate genes that may contribute to the regression of these traits in cavefish. The differential expression of such genes as rab32b and crx in cavefish indicates a potential role of light in vision and pigmentation development that may
41 differ between LD- and DD-reared cavefish. These novel differences seen only on Natural
Conditions may represent methods of phenotypic change in response to an environment devoid of light. Through additional study of these responses to lighting condition, we may begin to understand how A. mexicanus was able to change in response to the extreme cave environment. In sum, this study establishes the importance of photic condition on gene expression in the blind
Mexican cavefish. These results indicate that photic rearing condition may play a much larger role in gene expression or gene regulation than previously appreciated. Importantly, this study establishes the importance of accounting for natural lighting condition when examining gene expression and phenotype in this system.
Evaluation of the transcriptome of dark-reared cavefish indicates that rearing under Natural
Conditions does not drive identical gene expression to rearing these animals under Light/Dark
Conditions. Gene expression differences may indicate a subsequent phenotypic change in these organisms in response to the dark environment. Light does influence differential gene expression in A. mexicanus. Most importantly, this study illuminates novel information on the genetic underpinnings of cave adaptation. This study focused on just one cave-dwelling organism, A. mexicanus. While other efforts have been made to understand the effect of light on both the phenotype and transcriptome of other cave dwelling organisms (Li and Cooper 2002; Meng et al.
2013), this study raises the importance of understanding the effect that rearing under light and non- natural conditions have on animals whose natural environment is one of total darkness.
42 CONCLUSIONS
I conducted an assessment of global gene expression under Natural Conditions in Astyanax mexicanus. By rearing cave animals on DD conditions, I have restored these animals to their natural lighting condition; the effect light on gene expression had not been explored in this system previously. I have identified novel patterns of gene expression associated with lighting conditions by exploring the RNA architecture of this system under both standard laboratory and ecologically relevant conditions. I have uncovered substantial effects of light on global RNA architecture— rearing animals on ‘natural’ conditions causes substantial changes to the number and identity of differentially expressed genes. In fact, the greatest difference in global gene expression between cave and surface morphs is seen when cavefish are restored to their normative photic conditions.
Rearing cavefish on DD conditions revealed an expansion of the number of DEGs compared to the number of DEGs seen on Light/Dark Conditions. I uncovered the types of genes that are differentially expressed under ‘natural’ conditions and discovered new information about how blood, and perhaps oxygen carrying capacity, are affected. The functional repertoire of the DEGs seen under this expansion indicates broad scale changes to the circulatory and olfactory systems.
I discuss the candidate genes that possibly contribute to regressive evolution in this system.
Comparing the location of known QTL markers with the location of vision- or pigmentation- related genes identifies several candidate genes related to eye or pigmentation loss, respectively.
Traditional rearing methodologies have not sufficiently accounted for the complex interactions that light holds on the expression of genes related to many traits involved in cave biology. Overall, rearing cave animals under DD conditions yields a more accurate representation of gene expression in the dark and illustrates the need for environmentally relevant rearing conditions in RNA-seq analyses.
43 ACKNOWLEDGEMENTS
The authors wish to thank Amanda Powers, Heidi Luc, Amy Manning, and the many members of the Gross Lab, past and present, for their help with this project. A special thanks goes to Tyler Boggs for his assistance with QTL marker BLAST and Circos coding. Daniel Berning was instrumental in fish husbandry practices and phenotypic analysis of dark-reared surface juveniles. We would like to thank the Cincinnati Children’s Hospital and Medical Center DNA
Sequencing Core for their assistance in RNA sequencing. Additionally, we are grateful to Brian
Carlson for his help in data management. Thank you to the many organizations who have generously supported this work through funding including the Wieman-Benedict Award,
Department of Biological Sciences, University of Cincinnati to CRS and a grant from the National
Science Foundation (DEB-1457630) to JBG.
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58 GENERAL CONCLUSIONS
This work presents an assessment of global differential gene expression under ‘natural’ rearing conditions in Astyanax mexicanus. This assessment reveals which genes may have increased and reduced expression in the cave environment than in the ‘standard’ lighting conditions that these cavefish have been reared in prior to this study. Rearing under Light/Dark
Conditions does not account for the substantial effect that light holds on the expression of genes related to cave biology. Though previous studies have indicated the complex role that light plays on both phenotype and genotype in Astyanax and other cave organisms under dramatic lighting conditions, light affects global RNA architecture in a number of surprising ways. The least similarity between cave and surface morphotypes is apparent when cavefish are restored to their normative photic conditions by examinations of total relatedness by R2. Additionally, rearing cavefish on DD conditions revealed an expansion of the number of differentially expressed genes; this presents a novel set of genes wherein differences in expression may be dark-induced.
Examinations of these dark-inducible differences in gene expression by GO enrichment analysis indicate that light may play more of a role in the circulatory and olfactory systems than previously appreciated. Additionally, comparing the location of known QTL markers with the location of vision- or pigmentation-related genes identifies several candidate genes related to eye or pigmentation loss, respectively. Overall, this study examined the role of darkness in gene expression in a system marked in nature by exposure to total darkness.
This examination was made possible by the increased availability of genetic resources such as the draft genome of Astyanax (McGaugh et al. 2014) and NGS sequencing. As these resources improve, future studies may capitalize on the comparisons between cave and surface fish to complete more acute transcriptomic assessments on specific phenotypes. Single-cell RNA-seq
59 may prove to be a valuable tool to investigate pigmentation and eye regression in this system and including light into these analyses will prove vital to understanding how these phenotypes evolved in total darkness. Additionally, as this analysis revealed the complex effect that light holds on many genes related to the circulatory system, further investigation of this system in Astyanax fish is required to gather a full understanding of how darkness may play a role in respiration, metabolism, or blood clotting.
This is not the first examination of RNA architecture of life in the dark, as previous studies have conducted evaluations of dark-induced gene expression in cave animals. In contrast to the findings of Meng et al. 2013, the transcriptomic assessment presented here sought to understand how cave animals reared on light-dark conditions differ from those reared in total darkness. We now understand that gene expression is least similar between cave and surface fish under ‘natural’ lighting conditions, which indicates that this system may benefit from analyzing dark-reared fish for any alterations to phenotype or genotype. We might expect metabolism to differ in dark-reared individuals (as previously seen, Hüppop 1986), but other phenotypes may differ as well.
Additionally, establishing a line of both dark-reared cave and surface fish in the laboratory setting may be beneficial to the study of evolution in the dark as well—as established in dark-reared
Drosophila, alterations to coding sequences may occur as early as generation 14 (Isutzu et al.
2016). This system is a very attractive model species for establishing such a long-running study, as comparisons can be drawn directly to both the light-reared cave and surface fish and may prove important to modeling colonization of the cave environment. In sum, this thesis presents evidence that evolution in an extreme environment may influence characters that are under-appreciated when that animal is restored to a non-natural light cycle.
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61 VISUAL MATERIALS
Figure 1. Comparisons between morphotypes are most divergent 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 Rearing (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.
62 Figure 2. More genes are under-expressed in cavefish, regardless of rearing conditions. Genes with a higher (red plus) or lower (blue 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 is especially apparent at a 16- fold difference threshold. Under Natural Conditions 352 genes have a lower expression in DD
Cavefish compared to LD Surface fish, while under Light/Dark Conditions 340 genes have a lower expression in LD Cavefish than LD Surface fish. 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 is conserved within the cave morphotype regardless of lighting condition.
Figure 3. Rearing under Natural Conditions reveals novel changes to gene expression. The number of differentially expressed genes under Light/Dark Conditions or Natural Conditions is represented in the 2nd and 3rd columns of the table, respectively. These DEGs are inclusive of
63 genes with a higher expression in either cave or surface fish on their respective lighting conditions, represented diagrammatically below the table (Venn diagram). There are a large number of differentially expressed genes that are revealed under Natural Conditions alone (see row: 4x). This 61.1% expansion of DEGs represents a set of genes that are influenced by darkness, and previously unexplored. This expanded number of DEGs is best viewed at 4-fold change or higher (see: Venn diagram, bottom). The blue/red hashed crescent represents the number of DEGs that are unique to Natural Conditions, while the green overlap is genes that are differentially expressed under the comparative paradigms of both Natural Conditions and
Light/Dark Conditions, while the yellow crescent represents genes that are differentially expressed under Light/Dark Conditions alone.
64
65 Figure 4. Enriched GO terms under Light/Dark Conditions reveal prototypic cave phenotypes.
Test sets of genes were selected based on their expression at a 4-fold threshold (no C.I.). Genes were divided based on the polarity of expression as either having a higher expression in cavefish or surface fish. Under Light/Dark Conditions, 673 genes have a higher expression in cavefish and 1197 have a higher expression in surface fish. 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). Under light/dark conditions, enriched GO terms reveal patterns of cave evolution (as demonstrated by the enrichment of these terms in surface fish) such as a loss of melanic pigmentation, and a reduction of eye development and phototransduction in cavefish.
This pattern follows previously established analyses of enriched GO terms in cavefish (Gross et al. 2013; Hinaux et al. 2013). In genes that have a higher expression in surface fish than cave fish, genes referring to pigmentation (red) or vision (orange), along with metabolism (purple) and terms referring to processes in “other” cave biology traits (green) are enriched compared to the whole transcriptome. In cavefish, enriched GO terms refer to olfaction and general cave biology. Additionally, the enrichment of these terms in either cave or surface fish confirms that analyses of enriched GO terms support the underlying biology of the organism.
66
67 Figure 5. Enriched GO terms under Natural Conditions reveal terms referring to circulatory and olfactory systems. Test sets of genes were selected based on their expression at a 4-fold threshold (no C.I.). Genes were divided based on the polarity of expression as either having a higher expression in cavefish or surface fish. Under Light/Dark Conditions, 345 genes have a higher expression in DD Cavefish compared to LD Surface fish and 399 have a lower expression in DD Cavefish compared to LD Surface fish. 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). GO terms with (*) include an axis break. In genes that have a higher expression in cavefish in the dark (compared to LD surface fish), we see enrichment of blood- related (pink) GO terms referring to oxygen binding, transport, and carrier activity, as well as enrichment of olfaction (blue) related terms along with terms referring to “other” cave related traits (green). In genes with a lower expression in DD cavefish, we see enrichment of terms referring to blood (pink) and metabolism (purple) and “other” cave related traits (green).
Fibrinogen complex* displays a large enrichment score, based on the inclusion of all “fibrinogen complex” genes in the test set, * denotes this distinction. This indicates that dark-rearing may potentially play a role in the development and function of the circulatory system in A. mexicanus cavefish. Additionally, the enrichment of these terms indicates that there may be an expansion of the olfaction system in dark-reared cavefish either through an increase in functioning olfactory receptor genes or in an increase in olfactory receptor neurons.
68
69 Figure 6. Vision related genes map closely to vision related QTL. The distribution of vision- related QTL (light orange boxes) across the 25 chromosomes of the draft Astyanax genome (gray boxes, CH:1-25). * indicates gene name was derived from Danio rerio based on sequence similarity. Gene identity and function are represented in the table to the right. Expression of each of these genes differs between cave and surface fish. Values are given in RPKM (normalized expression). Some vision-related genes (black hash marks) fall remarkably close to vision related
QTL (represented in bold) and demonstrate relevant alterations to eye development when function is disrupted in other systems. Genes such as gucy2f, gucy2d and pde6ha are presented as potential candidate genes mediating eye regression in A. mexicanus cavefish. Analysis of gene location integrated with normalized expression derived from RNA-seq yields novel insight into how gene function may change in the dark environment. This allows for projection of how eyeless phenotypes resulted from colonization of the cave by ancestral surface-dwelling forms by way of differential gene expression.
70
71 Figure 7. Pigmentation related genes map near pigmentation related QTL. The distribution of pigmentation-related QTL (light pink boxes) and genes (black hash marks) across 25 chromosomes (gray boxes, CH:1-25) in the draft Astyanax genome. Gene identity and function are represented in the table to the right. Expression of each of these genes differs between cave and surface fish. Values are given in RPKM (normalized expression). * 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 still valid. Inclusion of the markers ASTYANAX_28 and TP33309 and genes pmela and tyrp1b validates previous syntenic studies of genes mediating pigmentation loss in A. mexicanus cavefish. † indicates an A. mexicanus gene that BLASTs to the same protein in D. rerio as another A. mexicanus gene, this may signify a functional duplication of the gene.
*** is used in place of expression values for the duplicate mc1r gene due to incomplete functional and coding information for this copy number variant. Comparison of gene location,
QTL location, and expression information leads to the proposition of candidate genes rab32b and sox18 as potential causative genes of complex pigmentation loss in Astyanax cavefish (bolded).
These genes were selected as potential candidates for trait loss due to their related functions and the influence of light on the expression of these genes.
72
Figure 8. Gene expression determined by qPCR validates RNA-seq expression. Qualitative (gel, inset, A; B) expression and quantitative RNA-seq (in RPKM) expression is highly similar in both genes with a such as rhodopsin (A; C) and in genes with more similar expression between conditions the housekeeping/reference gene bactin1 (B). Comparisons of RNA-seq (RPKM, x- axes) and qPCR-derived (DDc(t), y-axes) quantitative expression indicate a high degree of similarity in expression for many genes as indicated by Pearson’s correlation. This is exemplified
73 for the genes rho (C), nfil3-5 (D), f8 (E) and cepbp (F), although this high degree of correlation is recapitulated across 16 tested genes, with a mean correlative value of 86.8%.
74 TABLES
Table 1. Comparisons between animals reared on their Natural Conditions are the least similar.
We examine the R2 values of each comparison to get a sense of global similarity in gene expression. Amongst comparisons of both lighting condition and morphotype, Natural
Conditions have the lowest similarity in global gene expression, indicating higher levels of differential gene expression between LD surface fish and DD cave than that of Light/Dark
Conditions (LD cave to LD surface), inter-morphotypic comparisons, and comparisons of morphotype within one lighting condition.
2 Comparison Variation (R )
LD x DD 0.9751 Cave x Surface 0.8873 CLD x CDD 0.9599 SLD x SDD 0.9548 CLD x SDD 0.8720 CDD x SDD 0.8759 LD Conditions 0.8657 Natural Conditions 0.8597
Table 2. The expression difference of many genes is amplified under Natural Conditions. The ten
“most-sensitive” genes are listed. Each of these genes is more differentially expressed under
Natural Conditions (NC) than it is under Light/Dark (LD) Conditions. These genes were chosen based on fold change and RPKM (normalized expression) values, regardless of gene identity.
Representative genes of high and low average RPKM were selected. Interestingly, a near- majority (10/20) of these genes are implicated in circulatory system processes (hbaa1, hbae1.3, ba1, hbba2, hpx, fgb, fgg), indicating a potential role of light in blood composition and clotting,
75 and oxygen carrying. Analysis of precisely how the circulatory system is affected by lighting
condition will require further functional study.
LD Natural LD Conditions Conditions Gene Gene Description LD Cave DD Cave Surface Fold Fold Change Change
hbaa1 Hemoglobin subunit alpha 1 63.3 1337.4 98.3 1.55 up 21.11 up Adhesion G protein-coupled adgre6 0.2 2.3 1.7 9.91 up 13.55 up receptor E6 (Fragment) Hemoglobin, alpha embryonic hbae1.3 0.2 2.5 1.1 5.41 up 11.81 up 1.3 (Fragment) ba1 Hemoglobin subunit beta-1 399.3 3996.7 530.4 1.33 up 10.01 up faua 40S ribosomal protein S30 12.4 116.5 17.2 1.39 up 9.39 up hbaa1 Hemoglobin subunit alpha 1 337.6 2828.7 527.1 1.56 up 8.38 up ba1 Hemoglobin subunit beta-1 32.2 151.1 43.1 1.34 up 4.69 up tnni2a.4 Fast muscle troponin I 26.9 117.6 70.7 2.63 up 4.37 up gja5b Gap junction protein 5b 0.6 1.8 0.7 1.33 up 3.35 up hbba2 Hemoglobin, beta adult 2 585.5 1527.1 1111.1 1.9 up 2.61 up ckbb Brain-subtype creatine kinase 134.0 41.8 68.0 1.97 down 3.21 down hpx Hemopexin 530.1 151.2 329.8 1.61 down 3.51 down apoa1 Apolipoprotein A-I 3891.5 1057.4 2905.4 1.34 down 3.68 down Solute carrier family 25 member slc25a48 2.1 0.5 1.8 1.17 down 4.03 down 48 Serpin peptidase inhibitor, clade serpina1 A (alpha-1 antiproteinase, 730.8 168.6 282.2 2.59 down 4.33 down antitrypsin), member 1 fgb Fibrinogen beta chain 172.6 38.9 105.3 1.64 down 4.43 down fgg Fgg protein 177.3 35.1 91.3 1.94 down 5.05 down abl2 Tyrosine-protein kinase 1.8 0.2 1.4 1.33 down 7.56 down CCAAT enhancer-binding cebpb 224.1 22.2 123.1 1.82 down 10.07 down protein beta CCAAT enhancer-binding cebpd 323.7 30.1 200.5 1.61 down 10.74 down protein delta
Table 3. High degree of relatedness in gene expression between RNA-seq and qPCR
methodologies validates expression values from RNA-seq. Correlation of expression between
76 RNA-seq and qPCR was obtained by Pearson’s correlation in Microsoft Excel (2016). RNA-seq expression (RPKM normalization) is directly compared to qPCR expression (DDc(t), normalization to bactin1). The average correlation is 87.3% across 16 genes encapsulating 9
‘patterns’ of differential gene expression. Across each of these patterns, comparison of expression by RNA-seq and qPCR is highly correlated, validating the expression derived by
RNA-seq.
Pattern Gene Ensembl ID Correlation nfil3-6 ENSAMXG00000025841.1 0.81 cebpb ENSAMXG00000018723.1 0.93 Down-regulated in the dark rgs2 ENSAMXG00000017666.1 0.99 ddit4 ENSAMXG00000011697.1 0.45 Up-regulated in the dark smtlb ENSAMXG00000006382.1 0.85
Exclusively up-regulated in the dark in hbaa1 ENSAMXG00000016426.1 0.97 Cave f8 ENSAMXG00000003967.1 0.99 Exclusively up-regulated in the dark in col10a1b ENSAMXG00000003922.1 0.98 Surface Exclusively down-regulated in the dark atp5pd ENSAMXG00000020844.1 0.75 in Cave Exclusively down-regulated in the light serpinh1b ENSAMXG00000000056.1 0.51 in Surface Inversely regulated in Cave and Surface nfil3-5 ENSAMXG00000025528.1 1 rho ENSAMXG00000026346.1 0.99 Up-regulated in Surface opn1|w1 ENSAMXG00000006368.1 1 Gene 'A' ENSAMXG00000025407.1 0.87 Up-regulated in Cave Gene 'B' ENSAMXG00000009667.1 0.94 olfml3a ENSAMXG00000025778.1 0.86
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