extfieldsofstudy: Chemistry Medicine

text:
Pentameric quaternary structure of the intracellular domain of serotonin type 3A receptors

In spite of extensive efforts over decades an experimentally-derived structure of full-length eukaryotic pentameric ligand-gated ion channels (pLGICs) is still lacking. These pharmaceutically highly-relevant channels contain structurally well-conserved and characterized extracellular and transmembrane domains. The intracellular domain (ICD), however, has been orphaned in structural studies based on the consensus assumption of being largely disordered. In the present study, we demonstrate for the first time that the serotonin type 3A (5-HT3A) ICD assembles into stable pentamers in solution in the absence of the other two domains, thought to be the drivers for oligomerization. Additionally, the soluble 5-HT3A-ICD construct interacted with the protein RIC-3 (resistance to inhibitors of cholinesterase). The interaction provides evidence that the 5-HT3A-ICD is not only required but also sufficient for interaction with RIC-3. Our results suggest the ICD constitutes an oligomerization domain. This novel role significantly adds to its known contributions in receptor trafficking, targeting, and functional fine-tuning. The innate diversity of the ICDs with sizes ranging from 50 to 280 amino acids indicates new methodologies need to be developed to determine the structures of these domains. The use of soluble ICD proteins that we report in the present study constitutes a useful approach to address this gap.

the transmembrane domain of full-length receptors 5 . The chimeric 5-HT 3A -ICD construct, hereafter referred to as 5-HT 3A -ICD, was expressed in E. coli and purified from the soluble fraction after cell disruption by affinity chromatography using amylose resin and size exclusion chromatography (SEC) (Fig. 1). The presence of the complete ICD was confirmed by mass spectrometry (MS/MS, MALDI-TOF/TOF 4800 mass spectrometer, Applied Biosystems). On SDS-PAGE gels the protein ran at 53 kDa, while higher molecular weight bands approximately every 50 kDa were discernible clearly with Western blot detection.
Using SDS-PAGE and denaturing microfluidic capillary gel electrophoresis (Bioanalyzer, Agilent) the weight of purified monomeric 5-HT 3A -ICD construct was determined to be 53 kDa (theoretical weight 54 kDa) (Figs 2 and 3). The peak fraction analyzed by SEC eluted as a single symmetric peak indicating assembly into a defined and stable oligomeric state. The elution volume (11.52 ± 0.02 mL, n = 5) of 5-HT 3A -ICD was significantly smaller than what would correspond to monomeric 5-HT 3A -ICD, indicating higher oligomeric assemblies. MBP elutes at 15.2 mL yielding a predicted weight of 44 kDa (theoretical weight 41 kDa), indicating that MBP is a monomeric protein under the experimental conditions tested 7,8 . Importantly, MBP has been extensively used to create fusion proteins for structural studies, including peptides of similar or even shorter lengths as studied here (Tom20, 61 amino acids 9 ; MCL1, 147 amino acids 10 ; PilA1, 133 amino acids 11 ; Norrin, 103 amino acids 12 , Menangle virus phosphoprotein, 49 amino acids 13 ; adenylate cyclase 1 Receptor-short N-terminal extracellular domain, 93 amino acids 14 ). If oligomeric assemblies were observed in X-ray structures of MBP fusion proteins, oligomerization was always mediated by the fusion protein of interest and not MBP. The elution volume of 5-HT 3A -ICD chimera during SEC corresponds to a weight of 265.1 ± 2.8 kDa (n = 5), indicative of a pentameric assembly. When the purified protein was treated with the cross-linker glutaraldehyde using a vapor diffusion method 15 and then separated by either SDS-PAGE or denaturing capillary gel electrophoresis, a ladder was observed, representing monomer, dimer, trimer, tetramer and pentamer (Fig. 3). Pretreatment of 5-HT 3A -ICD with SDS obliterated the higher oligomeric assemblies (Fig. 3). Dynamic light scattering (DLS) experiments yielded a particle size corresponding to 245 ± 19 kDa (n = 5). To further ascertain the results obtained by SEC, chemical cross-linking and DLS, we performed SEC coupled with multi-angle light scattering (SEC-MALS), a molecular shape-independent method, to directly establish the absolute molecular weight of purified protein in solution. Analysis of four separate experiments yielded a weight-average molecular mass of 250.8 ± 4.4 kDa (mean ± S.E.M., n = 4) with a dispersity term of 1.010 ± 0.004 (mean ± S.E.M., n = 4) (Fig. 2, Table 1), again confirming the pentameric assembly of 5HT 3A -ICD. The result that the 5-HT 3A -ICD chimera conferred pentameric assembly is surprising.
In pentameric ligand-gated ion channels (pLGICs), the three domains found along an axis perpendicular to the membrane are the extracellular (ECD), transmembrane (TMD), and intracellular domain (ICD). Structurally, at the level of each domain within a receptor, five-fold symmetry is found. Previously, ECDs and TMDs have been expressed individually and their oligomerization studied. The present study is the first to show pentameric assembly of the ICD alone. Originally, intersubunit-interactions between ECDs were thought to drive the assembly and stoichiometry of pLGICs [16][17][18][19] . This suggested that the ECD would be an autonomous folding and assembly domain. Indeed, AcetylCholine Binding Protein (AChBP) 20 , a soluble protein found in the snail Lymnaea stagnalis, that is homologous to the ECD of nAChR, forms pentameric assemblies in solution. Similarly, engineered ECD-only constructs of pLGICs that natively form functional homopentamers, including GlyRα 1 and nAChRα 7 subunits, assemble into pentamers in solution, that preserve high-affinity ligand-binding capability 21,22 . Conversely, and unsurprisingly, the ECD of nAChRα 1 that normally is found in heteropentamers does not assemble to higher homooligomeric states and in the X-ray structure nAChRα 1 ECD was monomeric 23 . In summary, these individual ECDs parallel the oligomerization properties of their full-length receptors.
Conversely, a construct of the prokaryotic Gloeobacter violaceus pLGIC (GLIC) that solely consists out of the ECD is monomeric in solution even at very high concentration and only forms hexa-but not pentameric structures in the crystallized form 24 . Full-length GLIC that consists of ECD and TMD has so far been only described as a pentamer 25,26 . It was postulated that the GLIC TMD is required for correct pentameric assembly 24 . Indeed, for diverse eukaryotic pLGICs, aromatic residues in non-channel-lining transmembrane helices (TM1, TM3, and TM4) network to determine α -helical packing and thus subunit stoichiometry 27 . Several pLGIC TMDs expressed individually assemble as pentamers in lipid micelles. For example, cation-conducting nAChRα 7 TMDs as well as nAChRα 4β 2 TMDs with short M3-M4 linkers, GGGEG and G 5 , respectively, assemble as pentamers in lauryldimethylamine-oxide (LDAO) micelles 28,29 , and anion-conducting GlyR TMDs form pentamers in lysophospholipid lyso-1-palmitoylphosphotidylglycerol (LPPG) 30 . Hydrophobic contacts, especially leucine-isoleucine, and leucine-leucine interactions, drive these assemblies. Interestingly, separate nAChR α 4 and β 2 TMDs alone also form homopentamers, whereas the complete subunits do not form such homopentamers, and instead only obligate heteropentamers, indicating that domains apart from the TMD control the defined stoichiometric assembly in full-length receptors. Only a few previous studies investigated expression of solely the ICD. No pentamers were observed and spectroscopic methods as well as limited digests lead to the conclusion that the ICD was unfolded 31,32 . In general, the ICD has largely been thought of as a region with disorganized structure 3,33 . Intriguingly, we show here, that the complete 5-HT 3A -ICD when fused to the soluble monomeric MBP confers pentameric architecture to the resulting soluble chimera. Only stable and defined pentamers were observed, without evidence for other oligomeric assembly states. The driving force behind this observed pentamerization must lie in sequences directly contributed by the ICD.
RIC-3 is a crucial chaperone protein for some pLGICs that influences their functional maturation 34 . For mouse 5-HT 3A receptors expressed in Xenopus laevis oocytes we have shown that RIC-3 co-expression attenuated serotonin-induced currents almost completely, as compared to 5-HT 3A expressed alone. When the 115-amino acid 5-HT 3A -ICD is replaced with the short heptapeptide M3-M4 linker of GLIC, this RIC-3 effect is abolished 6 . In reverse chimeras consisting of GLIC-ECD and TMD with the 5-HT 3A -ICD added to obtain a GLIC-5-HT 3A -ICD chimeras we showed that RIC-3 co-expression similarly only modulated the 5-HT 3A -ICD-containing chimeras, but not GLIC 35,36 . These results indicate that the 5-HT 3A -ICD is required for RIC-3 modulation. By using both proteins, GLIC-5-HT 3A -ICD and RIC-3, individually purified after expression in E. coli, in pull-down experiments, we showed that GLIC-5-HT 3A -ICD interacts with RIC-3 37 . Under the same conditions wild-type GLIC that does not contain any ICD is not pulled down. These experiments demonstrate that the interaction between the 5-HT 3A -ICD and RIC-3 is direct and not mediated by other proteins. Here, the soluble 5-HT 3A -ICD chimera was used in pull-down experiments using metal affinity resin directed towards a C-terminal His 6 tag in a RIC-3 construct (hRIC-3(His) 6 ). The soluble 5-HT 3A -ICD chimera was successfully pulled down in these experiments providing evidence that the 5-HT 3A -ICD is not only required but also sufficient for the interaction with RIC-3 (Fig. 4). These studies can be extended to the multitude of other intracellular proteins that interact with pLGIC ICDs. The characterization of such interaction sites may provide targets for future drug design.
It is astounding that the ICD that is still thought to be a vastly disordered domain drives pentamerization when expressed alone, and it is intriguing to propose that it is also a driver for pentamerization of pLGICs as a whole. Importantly, quaternary polymorphism as observed for many other oligomeric proteins including the GLIC ECD is absent for the 5-HT 3A -ICD. The soluble 5-HT 3A -ICD chimera construct is now a perfect starting point for determining its high-resolution structure and also for identification of molecular features inside the ICD that mediate RIC-3 as well as other cytosolic protein interactions.
The recent 5-HT 3A X-ray structure including the proteolyzed ICD showed that lateral windows identified as ion pathways in the Torpedo nAChR model connecting the intracellular channel cavity with the cytosol were entirely occluded by post-M3 loops 5,38 . Additionally, three MA-helix arginines previously thought to   electrostatically repel conducted cations and be therefore causative for the sub-pSiemens single-channel conductance were involved in inter-subunit salt bridges 5,38 . The structure does also not provide an intracellular pathway for cations, albeit the crystallized construct had a significantly increased single-channel conductance 5 . Clearly, further detailed studies of the 5-HT 3A -ICD are therefore needed to elucidate the intracellular ion pathway as well as opening mechanism, and to reconcile previous functional studies with structure. Due to the diversity of the ICD in this large ion-channel superfamily, more than 40 subunits in humans, investigations are needed for diverse pLGICs. Highly-specialized mechanisms for the fine-tuning of targeting, trafficking or even functional aspects have evolved to be increasingly complex in metazoans over unicellular organisms. Importantly, it is becoming increasingly apparent that the ICD is involved in all aspects of pLGIC from folding and structural assembly to modulation of agonist efficacy and desensitization.

Methods
Plasmids. The cytoplasmic domain of the mouse 5-HT 3A receptor fused to the C-terminus of maltose binding protein (MBP) was generated using a modified pMAl-c2x (New England Biolabs) vector, pMALX 7 . The 5-HT 3A -ICD (QDL… VGY) was amplified by PCR using appropriate NotI and BamHI forward and reverse primers, respectively. The BamHI reverse primer also included a stop codon. The final construct, MBP-5-HT 3A -ICD-pMALX, was obtained after double-digest of the PCR-amplified ICD as well as pMALX, and subsequent ligation.
where V e is the elution volume, V o is the column void volume (8.16 mL), and V c is the geometric column volume (24 mL). For each standard protein, the calculated K av value was plotted against the log of its known molecular weight (Log M r ). The standard curve analysis algorithm of SigmaPlot 13.0 (Systat Software Inc., San Jose, CA) was then employed to obtain a predicted value (x) of the molecular weight of the fusion protein using a linear equation: where a is the slope and y 0 is the y-intercept. The obtained molecular weight of the fusion protein represents the average of at least five separate experiments.
Chemical Cross-Linking. For investigating the oligomeric state of fusion protein, glutaraldehyde-mediated cross-linking experiments were performed at 30 °C by utilizing a vapor-diffusion technique. Briefly, 40 μL of 25 % (v/v) glutaraldehyde (Sigma-Aldrich) was placed in the bottom of a microfuge tube and acidified with 1 μL of 5 N HCl. 15 μL of buffer B containing 0.5 μg of purified fusion protein was placed on the inside of the cap of the same microfuge tube. Upon carefully closing the cap, the protein solution was allowed to form a hanging drop inside the tube, and separated at ≤1 cm from glutaraldehyde. The entire 'closed microfuge tube system' was then incubated in a mini dry-bath at the temperature indicated. The protein solution was then carefully transferred into a separate tube to quench the reaction by adding sodium dodecyl sulfate (SDS) sample buffer (62.5 mM Tris HCl, pH 6.8, 10% (v/v) glycerol, 2% (w/v) SDS, 100 mM dithiothreitol (DTT), 0.01% (w/v) bromophenol blue). All samples were boiled and then resolved by SDS-PAGE. Cross-linked proteins were transferred to polyvinyl difluoride (PVDF) membrane and immunoblotted as described later. The analysis was repeated at various intervals of time after the start of the reaction to reveal a ladder of the oligomeric states. Alternatively, in a similar experiment, protein was exposed to glutaraldehyde vapors for various time intervals as described above and the cross-linked proteins were resolved by 'On-Chip-Electrophoresis' under reducing Scientific RepoRts | 6:23921 | DOI: 10.1038/srep23921 conditions using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). Protein chip and loading samples were prepared using the manufacturer provided kit, and according to instruction manual. Dynamic Light Scattering. Dynamic light scattering was used for particle size determination. Purified protein was diluted to a protein concentration of 1.0 mg/mL, filtered with a 0.22 um Millipore Millex-GV filter (Millipore, Billerica, MA) right before measurements, and subjected to the light scattering measurements at 90° using a Brookhaven Instruments (Brookhaven, CT) BI-200SM device with an avalanche photodetector. The molecular mass of the protein was calculated from the measured hydrodynamic radius and a calibration curve that included seven soluble proteins (ribonuclease A, carbonic anhydrase, bovine serum albumin, conalbumin, aldolase, ovalbumin, and IgG), and three membrane proteins (KcsA, connexin 26 hemichannels, and P-glycoprotein).

Size Exclusion Chromatography in tandem with Multi-angle Light Scattering (SEC-MALS).
SEC-MALS analysis was utilized to determine the weight-average molar mass (M w , g mole −1 ) and dispersity (Ð, M w /M n , where M n is the number-average molar mass) of purified protein independent of its shape. Chromatographic separation of protein samples was obtained under isocratic conditions using a Superdex TM 200 HR 10/300 GL column (GE Healthcare), pre-equilibrated with two column volumes of an SEC buffer (20 mM HEPES, 150 mM NaCl, 5 mM maltose, 0.01% NaN 3 , pH 7.4). The individual run was developed at an ambient temperature while a flow rate of 0.3 mL/min was maintained on an APLC system (LabAlliance, State College, PA) equipped with a solvent degasser. The chromatography system was in a tandem arrangement downstream with a miniDAWN-TREOS light scattering instrument equipped with three detectors positioned at angles 44 º , 90° and 136° (Wyatt Technologies, Santa Barbara, CA) and Optilab T-rEX dRI detector (Wyatt Technologies, Santa Barbara, CA). Both instruments were reading at a wavelength of 658 nm. Scattered angles were normalized using bovine serum albumin (BSA) following the manufacturer's instructions. For a typical experiment, the SEC buffer was injected for a blank run to establish stable baselines for the light scattering and differential refractive index measurements. All purified protein samples were filtered with a 0.22 μm low protein binding Millex-GV filter just before each experimental run. Subsequently, 0.1-1.0 mg of purified and filtered protein samples were loaded onto the column and their elution profiles were monitored at A 280 . The extinction coefficient of purified protein was calculated from the amino acid sequence by using the ExPASy-ProtParam tool. The online measurement of the Rayleigh scattering intensity was processed to determine the weight-average molar mass and dispersity of protein material contained in top one-third of the major A 280 chromatographic peak. Data was acquired with the ASTRA 5.3.4 software (Wyatt Technologies, Santa Barbara, CA) and processed using Debye model as per the manufacturer's guidelines.

SDS-PAGE and Western Blot Analysis. For SDS-electrophoresis, 4-15% precast gradient TGX
Stain-Free TM gels (Bio-Rad Laboratories, Inc.) were used and visualized by stain-free enabled imager (Gel Doc TM EZ Imager, Bio-Rad). After blotting onto PVDF membrane (Bio-Rad), the proteins were visualized by probing with a murine horseradish peroxidase (HRP) conjugated antibody (New England BioLabs) against MBP at 1:50,000 dilution. Bands were detected by chemiluminescence using enhanced chemiluminescent (ECL) substrate (Thermo Scientific) and a digital imaging system (ImageQuant TM LAS 4000, GE Healthcare).


extfieldsofstudy: Biology Medicine

text:
Comparative transcriptomics of anal fin pigmentation patterns in cichlid fishes

Background Understanding the genetic basis of novel traits is a central topic in evolutionary biology. Two novel pigmentation phenotypes, egg-spots and blotches, emerged during the rapid diversification of East African cichlid fishes. Egg-spots are circular pigmentation markings on the anal fins of hundreds of derived haplochromine cichlids species, whereas blotches are patches of conspicuous anal fin pigmentation with ill-defined boundaries that occur in few species that belong to basal cichlid lineages. Both traits play an important role in the breeding behavior of this group of fishes. Knowledge about the origin, homology and underlying genetics of these pigmentation traits is sparse. Results Here, we present a comparative transcriptomic and differential gene expression analysis of egg-spots and blotches. We first conducted an RNA sequencing experiment where we compared egg-spot tissue with the remaining portion of egg-spot-free fin tissue using six individuals of Astatotilapia burtoni. We identified 1229 differentially expressed genes between the two tissue types. We then showed that rates of evolution of these genes are higher than average estimated on whole transcriptome data. Using quantitative real-time PCR, we found that 29 out of a subset of 46 differentially expressed genes showed an analogous expression pattern in another haplochromine species’ egg-spots, Cynotilapia pulpican, strongly suggesting that these genes are involved in the egg-spot phenotype. Among these are the previously identified egg-spot gene fhl2a, two known patterning genes (hoxC12a and bmp3) as well as other pigmentation related genes such as asip. Finally, we analyzed the expression patterns of the same gene subset in two species that feature blotches instead of egg-spots, one haplochromine species (Pseudocrenilabrus philander) and one ectodine species (Callochromis macrops), revealing that the expression patterns in blotches and egg-spots are rather distinct. Conclusions We identified several candidate genes that will serve as an important and useful resource for future research on the emergence and diversification of cichlid fishes’ egg-spots. Only a limited degree of conservation of gene expression patterns was detected between the egg-spots of the derived haplochromines and blotches from ancestral haplochromines, as well as between the two types of blotches, suggesting an independent origin of these traits. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3046-y) contains supplementary material, which is available to authorized users.

Background
Animal pigmentation patterns are highly variable phenotypes both at the intra-and inter-specific level, and represent prominent traits to study the genetics of species diversification and adaptation (reviewed in [1][2][3]). The functionality of color patterns can readily be assessed in most cases, given that these traits often evolve in response to adaptation to the environment via natural selection (e.g. inter-and intra-specific communication, camouflage and mimicry), or co-vary with female choice via sexual selection [4][5][6]. The outcome of these two types of selection regimes can be different, with the former often producing cryptic phenotypes, where coloration mimics the environment, while the latter generates conspicuous phenotypes, where males typically display bright colors driving female choice or malemale competition [4][5][6]. Despite the high evolutionary significance of color patterns, the genetic mechanisms underlying their formation and diversification often remain elusive [1][2][3].
Recent work in fish model systems, especially in zebrafish, has started to uncover the genes and cellular processes involved in pigmentation pattern formation [7][8][9]. Pigmentation patterns are determined by the specification of different types of neural crest derived pigment cellsthe chromatophores [10] that contain different light absorbing pigments: melanophores contain black eumelanin pigments; erythrophores and xantophores contain yellow-red carotenoid and pteridine pigments; cyanophores contain a blue pigment of unknown composition; and finally, iridophores contain purine crystals that produce metallic iridescence [11]. Differences in the arrangement, position, and density of these cells leads to the diversity of color patterns present in nature. These differences depend on a variety of factors including neural crest cell migration, specification, proliferation, and survival [7][8][9]11].
In this study, we address the molecular basis of two novel and conspicuous pigmentation traits found in the anal fin of male cichlid fishesegg-spots and blotches (Fig. 1). Egg-spots represent an evolutionary novelty that emerged only once in the haplochromine lineage, the most species-rich group of East African cichlids [12,13]. These circular markings consist of a central circular area containing xanthophores and iridophores, surrounded by an outer transparent ring [14,15]. They are primarily found in males and show an extreme inter-and intraspecific variability in number, color, and position on the fin [13][14][15][16]. Egg-spots have been the subject of intense studies suggesting a signaling function in the peculiar mating behavior of the mouth-brooding haplochromines. They are likely sexually selected via female choice in some species [17,18] and via male-male competition in others [19][20][21]. Blotches, on the other hand, are patches of conspicuous anal fin pigmentation with ill-defined boundaries and occur only in a handful of cichlid species, including some basal haplochromines [13][14][15] and ectodine cichlids from Lake Tanganyika (Fig. 1). As with egg-spots, they are mostly found in males and their function might also be linked to courtship behavior, although this has been less extensively studied [12]. The origin and evolutionary trajectory of these anal fin patterns remains unclear. Due to the phylogenetic position of the species showing blotches as sister-group to the egg-spot bearing haplochromines [13][14][15], it might be speculated that egg-spots are derived from the blotch-pattern, which would make the two phenotypes homologous.
Convergent evolution is widespread in East African cichlid adaptive radiations, not only between lakes [22,23], but also within a single lake [24]. For example, haplochromine anal fin blotches are phenotypically similar to the ones found in the genus Callochromis (Fig. 1). However, the phylogenetic position of Callochromis, which is nested within the Ectodini [25], suggests that these two types of blotches evolved independently. Overall, we envision two possible scenarios for the origin of egg-spots: in one case they represent a derived state of blotches found in haplochromines, whereas blotches found in ectodines evolved independently (two origins); alternatively egg-spots have evolved independently from the blotches of both basal haplochromines and ectodines (three origins).
Understanding the genetic pathways underlying these pigmentation phenotypes can help us to distinguish between these scenarios. While several studies have addressed pigmentation diversity in East African cichlids, little is known about the genetics underlying their Fig. 1 Representative males from the four species analyzed: two haplochromine species displaying egg-spots in their anal fins (A. burtoni and C. pulpican), a basal haplochromine species (P. philander) and an ectodine species (C. macrops), both showing orange blotches in their anal fin coloration and pigmentation patterning, and only a handful of genes have been studied in detail. Among these genes is hagoromo, which shows a greater diversity of alternatively spliced variants and accelerated protein evolution in the haplochromines compared to other cichlids [26,27]; paired box 7 (pax7), on the other hand, was shown to be linked to a haplochromine female biased pigmentation phenotype [28]. Three genes have so far been associated with the egg-spot phenotype: the xanthophore marker colony stimulating factor 1 receptor A (csf1ra), and the two four and a half lim domain 2 proteins (fhl2a and fhl2b). csf1ra is expressed in haplochromine egg-spots and in the characteristic "Perlfleckmuster" (pearly spotted) pattern present in cichlid fins. This gene underwent adaptive sequence evolution in the ancestral lineage of the haplochromines coinciding with the emergence of egg-spots [14]. However, csf1ra is downstream in the pathway of egg-spot morphogenesis. More recently, we have shown that fhl2a and fhl2b are more causally related to egg-spot development and that an alteration in the cis-regulatory region of fhl2b could have contributed to the emergence of this trait in haplochromines in the first place [15].
In this study, we first addressed the question of the genetic basis of the egg-spots. We then went onto use comparative transcriptomics across species carrying eggspots and blotches to shed light on the origin of this novel trait. Specifically, we identified a total of 1229 genes that were differentially expressed (DE) between egg-spot and non-egg-spot fin tissues in the haplochromine cichlid Astatotilapia burtoni. These genes are evolving at a higher rate than average making this a valuable dataset to study the emergence and rapid diversification of this trait. For a subset of 46 DE genes we measured expression levels in three other species: the egg-spot bearing haplochromine Cynotilapia pulpican, carrying egg-spots on a different region of the anal fin than A. burtoni, and two blotch-bearing species, the basal haplochromine Pseudocrenilabrus philander and the ectodine Callochromis macrops. The rationale is that if egg-spots and blotches in haplochromines are controlled by the same genetic components they might show similar expression profiles.
A total of 29 out of 46 genes were found to be DE in C. pulpican. By comparing the expression in two haplochromine species with different egg-spot arrangements, we confirmed that the expression of the genes is correlated with the presence of egg-spots (irrespective of their position on the anal fin), whilst excluding potential positional genes and therefore confirming their involvement in egg-spots formation. Both types of blotches showed very distinct expression profiles from the egg-spots, and substantial differences in gene expression were also found between the two types of blotches. A similar gene expression profile between the egg-spots of derived haplochromines and the blotch pattern in the basal haplochromine P. philander would be indicative of a common origin for both traits, whereas similar expression profiles between the haplochromine egg-spots and the blotch of C. macrops would suggest that convergent evolution of this trait involved the same genetic pathways. Our study reveals the opposite for the genes under investigation, i.e. egg-spots and blotches show different expression profiles and also the two types of blotches differ in gene expression profiles, suggesting that egg-spots and blotches do not share a genetic basis and that convergent phenotypic evolution does not correspond to parallelism at the genetic level.

Results and discussion
Transcript profile in anal fin and egg-spot tissue In order to identify genes involved in egg-spot morphogenesis we quantified differences in gene expression patterns between egg-spots and the surrounding nonpigmented anal fin of six Astatotilapia burtoni males (Fig. 1). Illumina RNAseq (RNA sequencing) provided a total of 193,054,988 high quality reads from the six eggspot tissue samples and 194,099,061 reads from anal fin tissue samples of the same individuals. The replicates for each tissue were sequenced separately and the average number of reads per sample was 3,226,2837.42 (2,750,960.2-3,226,2837.42). We mapped the reads from each replicate to a reference A. burtoni embryonic library, which is a transcript collection from several different embryonic and larval developmental stages, and therefore probably the most comprehensive available representation of the entire gene set from A. burtoni [29]. In total we identified 1229 genes that were DE between the two types of tissues, with 620 genes being over-expressed in the egg-spot tissue, whilst 609 were under-expressed ( Table 1). The DE transcripts, their identification using tBLASTx and BLASTx searches (against the NCBI non-redundant database [30]), together with the respective expression levels, are provided in Additional file 1. A first inspection of those DE genes between egg-spot and non-egg-spot tissue revealed that our experiment retrieved many genes with a known function in pigment formation and patterning in different model organisms including paired box 7 (pax7), endothelin receptor b1 (ednrb1), microphthalmia-associated transcription factor a (mitfa), Agouti signaling protein 1 (asip1), sex determining region Y box 10 (sox10) and anaplastic lymphoma receptor tyrosine kinase (alk) [31], suggesting that our strategy is a valid approach to identify candidate genes for egg-spot morphogenesis.

Functional annotation of the DE genes
The reference A. burtoni transcriptome was annotated by performing a BLASTx search against NCBI's Danio rerio protein database [30]. From the 1229 DE genes, 58.6 % (720) had significant BLAST hits against the database (annotated datasets can be found in Additional file 2), while 41.4 % (509) of the DE contigs were nonidentified. From the 720 contigs with a BLAST hit we could functionally annotate 495 using BLAST2GO [32]. We further described the Gene Ontology (GO) term composition for egg-spot over-expression and egg-spot under-expression in comparison to the reference transcriptome GO representation (Fig. 2). Overall, the GO terms representation was similar between the two tissues. However, there were several GO terms for "Molecular function" and "Cellular component" that differed significantly between the two data-sets, suggesting, as expected, that the two tissues are functionally different (Fig. 2).
To narrow down the list of relevant GO terms, and to use them as a tool to find candidates, we used a twosided Fisher's exact test (false discovery rate (FDR) <0.05) to determine which functional GO categories were enriched in the genes over-expressed in the egg-spot in comparison to the total embryonic transcriptome. Five categories were significantly enriched in our overexpression gene dataset: 'Pigmentation' (GO:0043473), 'Developmental pigmentation' (GO:0048066), 'G-protein coupled peptide receptor activity' (GO:0008528), 'Peptide receptor activity' (GO:0001653) and 'Cell adhesion molecule binding' (GO:0050839) (Fig. 3). These are GO functional categories known to play a role in the development of pigmentation patterns. Neural crest cells are precursors of pigment cells and migrate from their original location to the anal fin where they will form the egg-spots [33][34][35], therefore genes playing a role in cell migration, cell adhesion and pigmentation development are relevant to the formation of this trait. Egg-spot formation relies on pigment production, which in turn is often activated via membrane receptor activity [36][37][38]. In Table 2 we present the list of genes belonging to these enriched functional categories that are potentially good candidates for egg-spot morphogenesis. The genes belonging to the GO term 'Developmental pigmentation' were overlapping with the ones included in the 'Pigmentation' category and the same is true for the two receptor GO term categories, therefore we only show three of the five enriched functional GO categories. This method of functional description of a gene dataset to extract candidates represents a supervised search, meaning that we might bias our findings towards what is already known. We note, however, that there are many other non-described genes, or known genes with incomplete GO term annotations, which could play a role in egg-spot morphogenesis.

Potential lineage specific genes are DE in the egg-spot
How novel traits emerge and are modified is one of the many unresolved problems in evolutionary biology [39][40][41]. It has long been advocated that new traits can emerge via the co-option of conserved regulators [42]. More recently, however, evidence is accumulating that new, i.e. lineage specific, genes can also play an important role in the development of novel traits [43][44][45]. Around 41 % of our candidate contigs did not have a BLAST hit against the D. rerio protein database. This could be due to the incompleteness of this database or to the lack of homologs in this species. To control for these factors we performed BLASTx and tBLASTx searches against the NCBI non-redundant (nr) protein and nucleotide databases [30]. Around 15.5 % (191/ 1229) of the DE contigs could not be assigned to a specific gene present in either nr database (Additional file 1). The contigs without positive BLAST hits could represent non-coding RNAs, partial sequences of known genes that could not be identified, or lineage specific genes (new or fast evolving genes) [46]. These results add to previous work on comparative transcriptomics of East African cichlids reporting that only 51 % of the total transcriptomes of the species studied (A. burtoni and Ophthalmotilapia ventralis) have hits on the NCBI nr nucleotide database [46]. In our case, the reduction in percentage of non-identified contigs is, most probably, due to the recent availability of five cichlid genomes [29].
It has previously been shown that lineage specific genes might play a role in the emergence and development of novel traits. In cnidarians 15 % of the transcripts expressed in a phylum specific cell type are lineage-specific, though the functional role of these transcripts was not tested [45]. The relative contribution of novel genes to the evolution of new morphologies, when compared to the co-option of conserved genes, is still under debate and further studies are needed to clarify their role on the evolution of such traits. Therefore, it would be interesting to identify the unknown DE transcripts and assess their role in the development and evolution of egg-spots.

Rates of evolution of the egg-spot DE genes
Changes in gene function can result either from modification in a cis-regulatory element that changes gene expression pattern and timing, and/or from a modification in the protein sequence that alters its function [47][48][49][50]. To test for protein sequence evolution in the egg-spot DE genes we calculated the rates of evolution in the form of dN/dS (ratio of non-synonymous substitutions over synonymous substitutions) of this gene dataset and compared the values obtained with a previously Fig. 3 Enrichment of functional GO terms in the egg-spot over-expressed genes (yellow bar) when compared to the total transcriptome of A. burtoni (blue bar). Those were calculated with a two-tailed Fisher exact test (FDR < 0.05) published dataset that estimated transcriptome-wide dN/dS values between cichlid species [46]. We were able to estimate dN/dS values (averages across species pairwise dN/dS) for 196 out of the 1229 contigs (see Additional file 1). As expected, the majority of the genes were under purifying selection (dN/dS < 1) and there was no significant difference in the rates of evolution between the over and under-expressed genes (Fig. 4). However, for both the over-and under-expressed genes, the average dN/dS values were significantly higher than those of the entire transcriptome (Fisher's exact test, pvalue <0.05), which means that, on average, the genes that are DE between the egg-spot and the anal fin are evolving at a faster rate. The haplochromine egg-spot is a male ornamental trait and, hence, most likely under sexual selection, either directly via female choice or via male-male competition [17][18][19][20][21]. Our results thus provide support to the general finding that genes underlying sexually selected traits evolve more rapidly [51][52][53][54].
We found seven genes to be under positive selection (dN/dS > 1), four of which were over-expressed in the egg-spot tissue (Table 3). Among them there are genes that play a role in neural crest differentiation (tenascin) and in cell migration (tenascin, mucin and family with sequence similarity 110c (fam110c)), which are important processes in pigmentation development [55][56][57][58]. The other genes have no a priori functional link with egg-spot formation. Nonetheless, due to their difference in expression and their signature of adaptive sequence evolution, they should be considered as good candidates and their functional roles in egg-spot development should be tested in the future.

Comparative gene expression via quantitative real time PCR
To confirm the results obtained via RNAseq, we examined a subset of 46 of the 1229 DE genes and tested their expression in egg-spot versus non-egg-spot tissue via quantitative real-time PCR (qPCR) in a second haplochromine species with a different egg-spot arrangement on the anal fin, Cynotilapia pulpican from Lake Malawi (Fig. 1). Half of these genes were over-expressed and half under-expressed in the egg-spot (Tables 4 and 5, respectively). These candidate genes were chosen randomly across the spectrum of the different levels of expression (from 1.3 to 5 fold differences in gene expression). Under-expressed genes were included as they might be acting as pigmentation inhibitors, thus preventing the appearance of egg-spots in other regions of the anal fin when over-expressed. Overall, there was no obvious trend with respect to functional GO categories associated with the top DE genes (see Additional file 2).
Note that six out of the 46 candidates remained unidentified after tBLASTx searches against a non-redundant NCBI database. While the egg-spots of A. burtoni are located in the proximal region of the anal fin, C. pulpican has its eggspots in the distal region of the anal fin. By measuring the expression of these genes in this species, we effectively control for positional effects in gene expression along the proximal-distal axis.
We also aimed to determine whether egg-spots and blotches share a conserved gene expression profile, which would indicate a common origin of these two traits. We thus tested if the candidate genes identified in A. burtoni had similar expression levels in the blotches of a basal haplochromine species (Pseudocrenilabrus philander) and in the blotches of a member of a distinct cichlid tribe, an ectodine species (Callochromis macrops), where this trait has likely evolved independently.

Comparative gene expression in haplochromine egg-spots
The qPCR gene expression analysis in the second haplochromine species revealed that 14 of the 23 genes that were over-expressed in the egg-spots of A. burtoni showed a similar expression pattern in C. pulpican (Fig. 5a), suggesting they are egg-spot specific and not simply involved in fin patterning. Among them are the previously identified egg-spot gene fhl2a [15], two transcription factors well known for their involvement in patterning and cell fate specification (homeobox C12a (hoxC12a) and heart and neural crest derivatives expressed 2 (hand2)), and an important growth morphogen (bone morphogenetic protein 3b (bmp3b)) [59][60][61]. The detection of fhl2a, in particular, suggests that our results are robust, since the gene was recently shown to be over-expressed across egg-spot development [15]. Included in the list are five of the unidentified contigs.
The remaining nine genes that were over-expressed in the egg-spots of A. burtoni either showed no difference in expression (4) or were under-expressed (5) in the eggspots of C. pulpican (Fig. 5a). These genes are most likely involved in fin rather than egg-spot patterning, as suggested by the fact that three of these of genes are known to participate in fin development (retinol binding protein 7 (rbp7), retinol binding protein 4 (rbp4) and insulin-like growth factor 1 (igf1)) [62][63][64]. Overall, we confirmed the over-expression of 14 genes in the adult egg-spots from both A. burtoni and C. pulpican making them strong candidates genes for egg-spot formation that deserve further investigation. Among the 23 under-expressed genes in A. burtoni, 15 were also consistently under-expressed in the egg-spots of C. pulpican (Fig. 5b), including one unidentified contig. Again, this suggests that these genes are egg-spot related. Among them is aristaless 3 (Axl3), a gene belonging to the homeobox gene family, known for its patterning effects [65]. Axl3 displays the highest expression differences among all genes (under-and over-expression included) and might putatively represent an inhibitor of the pigmentation/egg-spot pattern, although no role in pigmentation has been reported yet. The remaining eight genes showed no differences in gene expression between egg-spot and anal fin tissue on C. pulpican, and could therefore be involved in fin patterning. Thus far, none of these eight genes have been related to a function in pigmentation.
We cannot rule out that the genes that did not show the same pattern in both species do not have a function in egg-spots. Although egg-spots in A. burtoni and C. pulpican are homologous they do not necessarily have to share the exact same genetic network. It is thus possible that the DE genes might be responsible for interspecific differences of the egg-spot phenotype acting in a lineage-specific manner as has been shown in other taxa. For instance, the eyespots (concentric wing pigmentation patterns) of nymphalid butterflies, which are arranged along the distal half of the wing, are considered homologous [43,66]. Nevertheless, there is a great flexibility in the expression patterns of four genes involved in the development of these structures in the different species studied: antennapedia was the only gene where there was a gain of expression associated with the origin of the eyespot phenotype, whereas there were many gain or loss events for notch, distalless and spalt in the different species [67]. Overall, the genetic network underlying the nymphalid eyespot pattern appears to be highly variable, suggesting that homologous structures are not necessarily controlled by the same set of genes. Perhaps the same is true for cichlid egg-spots, which might initially have been under the control of the same set of genes followed by diversification in the recruitment of different genes. A broader phylogenetic sampling of egg-spot phenotypes would be necessary to clarify this question. The 29 genes that were consistently over-or underexpressed in the adult egg-spots in both haplochromine species are nevertheless strong candidates genes for egg-spot development and merit further investigation to understand their role in the origin and diversification of this trait. These genes should be studied in detail throughout development and their function should be tested, not only in one species but also across several species of egg-spot bearing haplochromines with variable egg-spot phenotypes. With this approach we will be able to distinguish between a functional role in the evolution of the trait and or merely a function in the development and/or physiology of the trait.

Comparative gene expression between egg-spots and haplochromine blotches
We then measured gene expression of our set of 46 candidate genes in a basal haplochromine species, Pseudocrenilabrus philander, which displays a blotch rather than an egg-spot on its anal fin (Fig. 1). It is not known whether the blotches found in basal haplochromines are ancestral to the egg-spots found in 'modern haplochomines' [13,25]. Homology inferences are typically made according to shared phenotypic criteria between traits and also according to parallelism at the developmental and genetic level [68] Therefore, if egg-spots and blotches are homologous we might expect that the gene expression patterns in both traits are, at least, partially conserved.
According to our results, haplochromine blotch and egg-spots differ substantially in their expression profiles (Fig. 5a, b). None of the 14 genes that were overexpressed in both A. burtoni and C. pulpican egg-spots were over-expressed in the blotch of P. philander (Fig. 5a), and only four of the 15 genes under-expressed in the two modern haplochromines were also underexpressed in P. philander (Fig. 5b). Although not conclusive, the poorly conserved expression pattern between the two traits suggests that the haplochromines' egg-spots and the blotches have emerged independently within the Haplochromini lineage. These results have to be taken with caution, though, as haplochromine egg-spots could have evolved from Fig. 5 Gene expression results for 46 DE genes as measured by qPCR. qPCR was performed for C. pulpican, P. philander and C. macrops (Relative position of the egg-spot/blotch on the fin are shown on top of each panel). Expression of these genes was quantified in the egg-spots and blotches relative to the anal fin tissue. Blue box denotes over-expression, red denotes under-expression and grey denotes no significant difference. Instances where it was not possible to measure gene expression are colored white with NA. ***: p < 0.001, **: p < 0.01, *: p < 0.05, • p < 0.1 (for more details please see Additional files 4, 5 and 6). a Results for egg-spot over-expression dataset (Table 4). In the first column are the RNAseq results for A. burtoni. In the second, third and fourth column are the results for C. pulpican, P. philander and C. macrops respectively. b Results for egg-spot under-expression dataset ( Table 5). Details of the statistical analyses used are found in Additional file 4 (P.pulpican), Additional file 5 (P. philander) and Additional file 6 (C. macrops) c Distance tree calculated using the gene expression results (over-expression, under-expression and no difference of expression) as characters blotches by up-regulation of different effector genes within the same genetic network. This has been observed in Drosophila, where the phenotypically diverse wing pigmentation patterns are controlled by the key regulator distalless (dll) [49]. The emergence of this wing spot phenotype was brought by the evolution of regulatory links between dll and multiple downstream pigmentation genes, which resulted in their up-regulation in the wing [49].

Comparative gene expression between eggs-spots, haplochromine and ectodine blotches
The blotch phenotype evolved more than once and is also found in some ectodine cichlids from Lake Tanganyika [12]. Ectodine anal fin blotches are similar to the ones found in basal haplochromines (Fig. 1), but apparently have an independent origin [25]. Although nonhomologous, ectodine blotches might still share the same genetic network with both haplochromine eggspots and blotches, as has previously been shown for other convergent traits [69].
In this study, we measured gene expression of our set of 46 candidate genes in the blotch of Callochromis macrops. Our gene expression assays revealed that only four of the genes that were over-expressed in A. burtoni and C. pulpican egg-spots were also over-expressed in the blotch of C. macrops (Fig. 5a). They encode transcription factors (cat eye syndrome critical region 5 (cecr5)), co-factors (fhl2a) [70], cytoskeleton components and kinases (a-kinase anchoring protein 2 (akap2)) and a non-identified transcript. These genes could be related to the pigmentation patterning or production of pigment in all three species. Furthermore, C. macrops also shares with A. burtoni and C. pulpican four genes that are consistently under-expressed in both species (Fig. 5b). One gene (vitronectin [71]) was over-expressed in C. macrops blotch and A. burtoni egg-spots, but not in C. pulpican egg-spots. These two species (A. burtoni and C. macrops) have in common that their egg-spots and blotches, respectively, contain orange pigments, while the egg-spot of C. pulpican is yellow. These genes might therefore correlate with patterning or production of orange pigment, although no such role has been previously described.
The comparison of expression profiles between the blotch bearing P. philander and C. macrops revealed that the underlying gene expression patterns are different indicating that there is probably no parallel evolution at the genetic level determining the phenotypic resemblance of the blotches. Curiously there are six genes that are under-expressed in the A. burtoni egg-spots that show no difference in expression in C. pulpican, but are over-expressed in blotches of both P. philander and C. macrops. The expression pattern of those six genes could be correlated to the blotch phenotype, but the most probable explanation is that they are involved in fin morphogenesis, since the non-pigmented region of A. burtoni matches the pigmented one in the two species with blotches.

Gene expression clustering
To determine the relationship between the pigmented anal fin tissues (egg-spots and blotches), we coded the gene expression results of the 46 genes in the four different species into a matrix of discrete data points (0under-expression, 1no difference, 2over-expression) and constructed a distance genealogy (Fig. 5c). The resulting tree diagram shows a clear separation between eggspot and blotch phenotype. The different species clearly cluster by gene expression phenotype (bootstrap of 100 %) and the observed similarities do not correspond to the species phylogeny (Fig. 5c, Table 6). The character distance matrix also shows that of the two blotches, C. macrops blotch is more similar to the haplochromine eggspots in terms of gene expression (Fig. 5c, Table 6). Our results suggest that egg-spots, haplocromine blotches and ectodine blotches are not regulated by the same genetic components.
Overall our results suggest that haplochromine eggspots, haplochromine blotches and ectodine blotches are novel pigmentation traits that evolved independently by re-using a limited number of common genes ( Fig. 5 and Table 6). The genes in common seem to be related to the cellular composition of the trait, which is re-used every time a new pigmentation pattern emerges, and not with the pigmentation pattern per se. Therefore, a thorough comparison of the different fin phenotypes should be done to assess what are the cellular components of each of the pigmentation phenotypes to better understand and interpret the gene expression underlying it.
These homology inferences have to be taken with caution, as we have only studied a subset of candidate genes (46/1229) derived from the egg-spot versus non-egg-spot tissue transcriptomic comparison in A. burtoni. An indepth comparison of the blotch tissue will certainly require comparative transcriptomics in the blotched species.

Conclusions and future perspectives
Understanding the genetic and molecular basis of both evolutionary innovation and phenotypic variation is a major challenge in evolutionary biology. Using nextgeneration sequencing we here present a transcriptional survey of egg-spot tissue in the haplochromine cichlid Astatotilapia burtoni. This collection of DE transcripts represents the largest set of egg-spot candidate genes available and will greatly contribute to the understanding of the genetics underlying this trait. We provide a list of 1229 genes that are DE between egg-spots and non-eggspots fin tissues, many of which are fast evolving genes that might be involved in the genetic network determining the egg-spot phenotype.
A closer look at the expression profiles of 46 of the DE genes shows that the expression profiles are not conserved between egg-spots and blotches, which suggests that haplochromine egg-spots, haplochromine blotches and ectodine blotches do not share the same genetic basis. This result indicates that these traits emerged independently in the evolution of this group of fishes. It has been hypothesized that egg-spots are modifications of the "Perlfleckmuster" (pearly spot) pattern that is present in fins of many cichlid species [12,14]. In the future it will be interesting to determine if the same genes that underlie the egg-spots of haplochromines are also expressed in the "Perlfleckmuster".
With our current approach, we identified 29 genes whose expression patterns are egg-spot specific in two distinct cichlid species, strongly pointing to a role in the formation of this trait. These genes definitely deserve further investigation; in particular, their expression dynamics should be examined during egg-spot development and their function should be assessed with transgenic experiments, now available for cichlids [72]. The functional characterization of these genes during egg-spot development and in a broader phylogenetic context will inform us about the origin and diversification of this innovation in the most species rich vertebrate lineagethe haplochromine cichlid fishesthus leading to major advances in the understanding of the emergence and diversification of novel traits.

Samples
Astatotilapia burtoni and Cynotilapia pulpican bred laboratory strains were kept at the University of Basel (Switzerland) under standard conditions (12 h light/12 h dark; 26°C, pH7). All individuals were euthanized with MS222 (Sigma-Aldrich, USA), following approved procedures (permit number 2317 issued by the Basel cantonal veterinary office) before tissue dissections. Callochromis macrops individuals were captured at Lake Tanganyika, Mpulungu (Zambia), P. philander were captured in a river near Mpulungu (both under a research permit issued by the Department of Fisheries, Republic of Zambia). Dissections were carried out in situ, tissues were stored in RNAlater (Ambion, USA) and shipped to the University of Basel.

RNA extractions
Isolation of RNA was performed using TRIzol® (Invitrogen, USA). All dissected tissues were incubated in 750 μl of TRIzol and left at 4°C overnight (or 8-16 hours). The tissues were homogenized with a BeadBeater (Fas-tPrep-24; MP, Biomedicals, USA). Extractions proceeded according to manufacturer's instructions and DNase treatment was performed with DNA-Free™ (Ambion, USA). RNA quantity and quality was determined with a NanoDrop 1000 spectrophotometer (Thermo Scientific, USA). cDNA was synthetized using the High Capacity RNA-to-cDNA kit (Applied Biosystems, USA).

Differential gene expression analysis using RNAseq -Illumina
The anal fins of six Astatotilapia burtoni male juveniles were dissected and RNA was extracted from egg-spot and anal fin tissue for each individual. One microgram of RNA per sample was sent for library construction and Illumina sequencing at the Department of Biosystems Science and Engineering (D-BSSE), University of Basel and ETH Zurich. Samples were run in two lanes of an Illumina Genome Analyzer IIx (maximum read length was 50 base pairs (bp)).
The reads from each sample were mapped against a reference A. burtoni embryonic transcriptome that contains 171,136 reference transcripts. We mapped the reads from each library against the reference transcriptome using Bowtie2 as aligner [73] and RSEM (RNA-Seq by Expectation-Maximization) [74] as the method to estimate gene abundance. The individual RSEM files were concatenated into one single dataset and analyzed using the Bioconductor R package EdgeR [75]. Transcripts that had less than one count per million in one of the samples were discarded. We tested for differential expression between egg-spot and anal fin samples, using anal fin as reference. Since the samples were paired (each replicate of the egg-spot and anal fin belong to one individual fish), we included the individual information in the statistical model. For that we used a negative binomial generalized linear model (GLM) based on common dispersion using the individual as the blocking factor, i.e. we tested for consistent differences in expression between egg-spot and anal fin within individuals. Transcripts were considered as DE if, after correction for multiple testing, the false discovery rate (FDR) was lower than 0.05 [76].

Functional annotation of differential expressed transcripts
Gene ontology (GO) [77] annotation of the differential expressed transcripts was conducted with Blast2GO version 2.5.0 [32]. BLASTx searches were done against the Danio rerio protein database using a threshold of e −5 and maximum number of hits of 20. These GO terms were used to estimate transcript function. A table with the list of the differential expressed transcripts, their respective values of expression, and their GO terms is provided in Additional file 1. Between dataset differences in the proportion of genes for individual level 2 GO terms were tested by means of chi-squared tests with p-values adjusted for multiple tests using Bonferroni corrections [78]. The enrichment of functional GO terms in the eggspot over-expressed gene dataset was calculated with a two-sided Fisher's exact test with a FDR of 0.05.

Rates of evolution for the differential expressed transcripts
Transcriptome data from the five available cichlid species (Pundamila nyererei, Neolamprologus brichardi, Oreochromis niloticus, Maylandia zebra, and Astatotilapia burtoni) were downloaded from Broad Institute [29]. Each species' transcriptome consisted of multiple libraries that were concatenated. The 1229 DE genes from A. burtoni were compared using a BLASTn search (threshold: e −50 ) against each species' transcriptome and DE genes with a hit in all cichlid species were retained (599). The 599 DE genes were then compared using BLASTx (threshold: e −20 ) against the tilapia (Oreochromis niloticus) proteome from the ENSEMBL database and corresponding coding sequences (cds) retrieved (378). Finally, the database of 378 tilapia cds was queried against the individual cichlid transcriptomes using BLASTn (threshold: e −35 ). BLAST outputs were parsed and filtered to retain hits with identity >90 %, length >200 bp and bit score >200. We obtained 298 tilapia cds that have at least a hit on all cichlid transcriptomes. A concatenated fasta file was built to include the ten top hits from each cichlid transcriptome and the 298 tilapia cds. Sequences were then aligned using MAFFT v7.245 [79] with einsi -adjustdirection options (einsi is suitable for sequences containing large unalignable regions, as expected with the presence of UTRs (untranslated regions) and splicing variants in our transcriptome data). Alignments were trimmed using the tilapia cds as a reference and visually inspected. Alignments with paralogous sequences resulting from recent duplications were discarded. Within each individual alignment a consensus was built across transcripts from each cichlid species with 'cons' from EMBOSS [80] (−plurality 1.5, indicating the cut-off for the number of positive matches below which there is no consensus). Alignments were then translated to proteins and checked for all sequences being in the corresponding tilapia reading frame (no stop codons). The whole pipeline was run with customized R and Unix scripts. We obtained 196 good alignments, 74 % of which comprised of all five cichlid species sequences, while the remaining included at least three species each. Average alignment length was 1716 bp, ranging from 270 to 7794 bp. Alignments are available from the author upon request. dN/dS estimates were calculated using the script kaks.pl in Bioperl [81] which computes the dN/dS for all sequence pairs, using the Nei-Gojobori method [82].

Gene expression analysis using qPCR
The expression of 46 genes (23 over-expressed genes in the egg-spot region and 23 under-expressed genes in the egg-spot) was further studied in three other species -Cynotilapia pulpican, Pseudocrenilabrus philander and Callochromis macrops. Primers were designed with GenScript Real-time PCR (TaqMan) Primer Design software available at https://www.genscript.com/ssl-bin/app/primer. Where possible, primers were designed in exon spanning regions to avoid effects of gDNA contamination. Primers were tested in all species and in cases where primers pairs did not work we designed new species-specific primers. Genes studied and primer sequences are available in Additional file 3.
Three qPCR experiments were carried out: qPCR experiment 1: Gene expression was compared between the non-egg-spot anal fin tissue and the egg-spot tissue of C. pulpican. This species has its egg-spot in a different position in the fin compared to A. burtoni (Fig. 1, n = 4-5). qPCR experiment 2: Gene expression was compared between the non-blotch anal fin tissue and blotch tissue of P. philander (Fig. 1, n = 6). In this experiment six individuals were used. qPCR experiment 3: Gene expression was compared between the non-blotch anal fin tissue and blotch tissue of C. macrops (Fig. 1, n = 4-7). In this experiment 4 to 7 individuals were used. For all experiments each individual was an independent replicate meaning that there was no pooling of samples.
The reactions were run on the StepOnePlus™ Real-Time PCR system (Applied Biosystems, USA) with Fas-tStart Universal SYBR Green Master mix (Roche, Switzerland), following the manufacturer's protocols. All reactions were performed with an annealing temperature of 58°C, a final concentration of cDNA of 1 ng/μl and a final primer concentration of 200 ng/μl. The comparative threshold cycle (CT) method [83] was used to calculate the relative concentrations between tissues, where anal fin was taken as the reference tissue and Ribosomal protein L7 (rpl7) or the Ribosomal protein SA3 (rpsa3) genes as endogenous controls. Primer efficiencies were calculated using standard curves. Efficiency values of test primers were comparable to the efficiency of endogenous control primers (rpl7, rspa3) and are available in Additional file 3.


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

text:
Beyond VEGF—The Weisenfeld Lecture

Purpose To review advances made in the treatment of age-related macular degeneration (AMD) and share perspectives on the future of AMD treatment. Methods Review of published clinical and experimental studies. Results Inhibitors of vascular endothelial growth factor (VEGF) truly revolutionized the treatment of AMD. However, available results from longer-term studies suggest that a degenerative process is unveiled, and continues to occur, even when neovascularization is controlled. Furthermore, anti-VEGF therapy may play a role in the development of atrophic changes. We have proposed using neuroprotection to prevent atrophy, and multiple models of retinal degeneration have shown that it is necessary to block both apoptotic and necrotic cell death pathways. Despite the success of anti-VEGF therapy and the promise of neuroprotection, neither addresses the underlying cause of AMD. It has been postulated that in early AMD, the retention and abnormal accumulation of lipids in Bruch's membrane and below the retinal pigmented epithelium (RPE) lead to drusen. Thus, it is conceivable to target the retained lipoproteins and seek to remove them. In a case study and pilot multicenter clinical trial, we observed significant regression of drusen and an improvement in visual acuity in patients taking high-dose statin therapy. These results, though preliminary, warrant further investigation. Conclusion Future treatment of AMD should be based on biology, which will require continued elucidation of the pathogenic mechanisms of AMD development. Neuroprotection represents a potential therapeutic approach, and other promising targets include immune pathways (e.g., inflammation, complement, and inflammasomes) and lipid/lipoprotein accumulation. Finally, due to the heterogeneity of AMD, future progress in therapy will benefit from improved phenotyping and classification.

I t is a real honor to deliver the Weisenfeld Lecture-especially to be the first woman to do so. Mildred Weisenfeld was diagnosed with retinitis pigmentosa at age 15, and lost all of her vision by age 23. She decided that patients with blinding diseases needed more than vision aids-a dog, a cane, and Braille texts-and she thought that we should provide hope through eye research. In 1946 she founded the nonprofit that became Fight for Sight, and she campaigned for the founding of the National Eye Institute.

ADVANCES IN AGE-RELATED MACULAR DEGENERATION THERAPY
In this lecture, I will review some of the advances we have made in the treatment of age-related macular degeneration (AMD), and share some of my perspectives on where I think we should be headed next. Age-related macular degeneration remains an important cause of blindness throughout the world. According to the World Health Organization, it is the third leading cause of blindness worldwide (after cataract and glaucoma) and the leading cause of blindness in industrialized countries. 1 As clinicians, we recognize AMD by looking into the eye, and seeing deposits (drusen) in the macula, pigmentary changes, or, in the more advanced forms, geographic atrophy or neovascular AMD (Fig. 1). We have made some advances in the treatment of AMD-a little progress in the early and interme-diate stages, with vitamin and mineral supplementation based on studies such as the Age-Related Eye Disease Study (AREDS)-but we placed more focus on late neovascular AMD. This began with laser photocoagulation, followed by photodynamic therapy, with a brief foray into surgical treatments, such as removal and translocation of choroidal neovascularization (CNV), and also intravitreal steroids.

ANTI-VEGF THERAPY
However, AMD treatment was truly revolutionized with the development of inhibitors of vascular endothelial growth factor (VEGF). We are familiar with the Phase III findings of the anti-VEGF therapy ranibizumab (Lucentis) for AMD-indeed, many retina surgeons have the vision data imprinted in their brainsbut it was truly remarkable to achieve sustained improvement of vision in patients with neovascular AMD compared to treatments available at the time. 2,3 With anti-VEGF therapy, more than 90% of patients avoid moderate vision loss, and approximately one-third achieve vision of 20/40 or better. 2 With the CATT, 4,5 IVAN, 6 and other trials, [7][8][9][10] we have demonstrated that we can achieve good results, and very similar vision outcomes, with a variety of anti-VEGF agents. Today, multiple anti-VEGF therapies are available to the clinician-pegaptanib (Macugen), ranibizumab (Luceniovs.arvojournals.org j ISSN: 1552-5783 tis), bevacizumab (Avastin), and aflibercept (Eylea)-and we can offer different treatments as needed.

LONGER-TERM RESULTS OF ANTI-VEGF THERAPY
But what about the longer-term results of anti-VEGF therapy? Prospective randomized controlled clinical trials (RCTs) have largely analyzed treatment outcomes up to 24 months at most. 2,5,11 Although there have been some extension studies, such as SEVEN-UP, 12 and retrospective studies from retina practice groups like Peden and colleagues 13 and work at Massachusetts Eye and Ear (Roh M, et al. IOVS 2002;43:ARVO E-Abstract 1415), these have been limited by patient numbers and variation in treatment protocols and drugs utilized. Still, it is worth reviewing the information that we have available. In the current economic and health care environment, it seems unlikely that we will have large, prospective trials that will give us definitive answers on the longer-term results of anti-VEGF therapy. On the other hand, we may obtain important information from registries that are in development, which may provide actual long-term outcomes of treatment in large populations.

UNVEILING OF THE DEGENERATIVE PROCESS
So what happens when the neovascular process is controlled? I would postulate that the major event is an unveiling of the degenerative process that we know is occurring in AMD-and hence the development of geographic atrophy. There may also be progression of atrophic changes secondary to poor perfusion, and anti-VEGF therapy may actually play a role.
Regarding the degenerative process, loss of cones, rods, and retinal pigment epithelium (RPE) occurs in the atrophic (dry) form of AMD in early and late stages (Fig. 2). Presumably, this occurs in the neovascular form of AMD as well-particularly when neovascularization is controlled.
Indeed, in earlier studies, Green 15 demonstrated photoreceptor and RPE atrophy in CNV lesions and disciform scars (Fig. 3). More recently, results from the CATT showed that geographic atrophy growth rates in treated neovascular AMD were similar to the rates in nonneovascular AMD. 16 Clearly, this degeneration is a pathologic process that needs to be targeted.
It is also possible that the progression of atrophy is due to poor perfusion. As seen in Figure 4, as CNV grows, there is loss of normal choriocapillaris. 17 As clinicians, we treat the CNV and initiate its regression-but in doing so, we actually may be eliminating the only remaining blood supply for the outer retina. Therefore, it would not be surprising that anti-VEGF therapy would have secondary atrophic effects. Finally, VEGF has known neurotrophic effects, and blocking it may accelerate atrophy. 18 While this is scientifically plausible, the clinical evidence is currently lacking.

NEUROPROTECTION
We have proposed that we might intervene in the degenerative process using neuroprotection, and thus prevent these atrophic changes. We have suggested neuroprotective adjuvant therapy along with anti-VEGF therapy to prevent photoreceptor cell death. Through this approach, we believe that we can improve vision outcomes, both short-and long-term. We and others have studied neuroprotection using a model of retinal detachment, 19,20 and while this may seem distantly related to AMD, separation of photoreceptors from RPE, or a retinal detachment, occurs in various retinal disorders-including neovascular AMD, diabetic retinopathy, and rhegmatogenous retinal detachment. 21 Moreover, retinal detachment can be readily modeled in small animals. 19 Using this retinal detachment model, we and others found evidence that apoptosis is involved, with activation of caspases and known upstream ligands, including TNF-alpha and Fas ligand (FasL). 19,[22][23][24] However, inhibition of caspase activation with a pan-caspase inhibitor was insufficient in preventing photoreceptor cell death. 19 Therefore, we investigated whether photoreceptor cell death might involve other cell death pathways.
In published literature from the 1970s, three cell death modes were described based on morphology. 25 Type I cell death shows cellular condensation and fragmentation. Type II is associated with the formation of numerous autophagic vacuoles, and type III exhibits a cellular and organelle swelling and plasma membrane rupture. These cell death types are now referred as apoptosis, autophagy, and necrosis, respectively. Apoptosis is the best characterized programmed cell death, and caspases have been established as a central regulator of apoptosis. Recent studies also have identified that autophagyrelated proteins (ATG) play a key role in the induction of autophagy. Necrosis was believed to be an unregulated form of cell death. However, recent studies indicate that necrosis can be regulated, induced by regulated signal transduction pathways such as receptor-interacting protein (RIP) kinases. 26,27 We looked for evidence of programmed necrosis, or necroptosis, in photoreceptor cell death. In the retinal   detachment model, in situ hybridization showed that Rip3 expression increased after retinal detachment, especially in the outer nuclear layer. 28 We tried to inhibit photoreceptor cell death using either a necrosis inhibitor (Nec-1) or an apoptosis inhibitor (ZVAD). Treatment with Nec-1 or ZVAD alone showed no effect on photoreceptor loss after experimental retinal detachment. In contrast, combined treatment with Nec-1 and ZVAD significantly reduced the photoreceptor death. Using electron microscopy to examine modes of cell death in the retinal detachment model, we observed that caspase inhibition alone led to increased necrotic cell death. Thus, cells have alternative death pathways through RIP kinase activation; in order to prevent photoreceptor cell death after retinal detachment, it is necessary to block both apoptotic and necrotic pathways.
We wondered whether similar mechanisms might occur in other models of AMD, such as the dsRNA model of retinal degeneration; double-stranded RNA (dsRNA) is a component of drusen and a ligand for Toll-like receptor 3 (TLR3), which mediates innate immune response and cell death. 29 Subretinal injection of polyinosinic-polycytidylic acid [poly(I:C)], a synthetic analogue of dsRNA, induced TLR3-dependent retinal degeneration, resulting in areas of subretinal atrophy, loss of RPE cells, and TUNEL-positive cells in the outer nuclear layer and inner nuclear layer. 29 Thus, we used this model to examine the mechanism of cell death in the RPE and photoreceptors, as well as to investigate the role of inflammation in AMD. We found that photoreceptor cell death occurred predominantly by apoptosis, whereas RPE death occurred mainly by necrosis, showing almost exclusively necrotic features. 30 Thus, apoptosis and necroptosis are indeed active in a dsRNA model of AMD, and a combination of apoptosis and necrosis inhibition is effective in preventing photoreceptor and RPE cell degeneration.
More recently, we found further evidence linking immune responses, necroptosis, and photoreceptor cell death. In patients with photoreceptor injury associated with retinal detachment, we found increased levels of cleaved interleukin 1 beta (IL1b), a downstream product of inflammasome activation. 31 In rodents with experimental retinal detachment, infiltrating macrophages were the primary source of IL1b, and photoreceptor cell death led to inflammasome activation in a macrophage-and RIP3-dependent manner. 31 Additionally, we found that resident microglia and infiltrating macrophages express FasL, which triggered photoreceptor death in the membrane-bound form (mFasL) yet had neuroprotective properties in the soluble form (sFasL). 32 We thus believe that neuroprotection may provide a broad-based treatment approach for a wide variety of retinal disorders, including AMD. It could be conceived as an adjuvant therapy with anti-VEGF for neovascular AMD. It could also be initiated sooner, to treat early and intermediate AMD; this will require long-term delivery, with gene therapy as a potential delivery platform.

TREATMENT OF AMD: BIOLOGY BASED
Despite its promise, neuroprotection still does not address the underlying cause of AMD-and if the goal is to intervene early in the disease, we will need to attack a key pathway. It is worth emphasizing that the success in treating neovascular AMD was based on such a strategy: targeting the VEGF pathway as a key mediator of angiogenesis and permeability. Targeted therapies for early AMD will require better understanding of AMD pathogenesis. Historically, insights into AMD pathogenesis have been derived from clinical observation and imaging, epidemiology, and histopathology-and more recently from genetics and molecular biology. Considering the available evidence, the pathogenesis of AMD may be narrowed down to six major pathways (Table). 33 Angiogenesis has been treated effectively with anti-VEGF agents, and neuroprotection would potentially address the last pathway: cellular stress and toxicity, which leads to cell death. However, even if introduced early, neuroprotection does not address the underlying cause of AMD. Therefore, early biologically based treatment would ideally interfere with an upstream pathway in AMD pathogenesis to limit disease development and progression.

Lipid and Lipoprotein Metabolism and Transport
Regarding lipid transport and metabolism, similarities have been observed between AMD and atherosclerosis, with Bruch's membrane acting like vascular endothelium. Curcio and colleagues 34 have postulated that lipoproteins, such as apolipoprotein B, deliver cholesterol to tissues and become ''retained'' in Bruch's membrane and sub-RPE space. These retained lipids lead to a lipid wall, basal linear deposits (BlinD), and drusen (Fig. 5A).
The RPE plays a key role in metabolizing lipoproteins that originate from the photoreceptor outer segments and the systemic circulation. The RPE itself synthesizes lipoproteins as well. With age, the RPE starts to accumulate lipofuscin, and a lipid wall develops along Bruch's membrane in the sub-RPE space, resulting in formation of BlinD, basal laminar deposits (BlamD), and ultimately drusen, which are visible upon clinical observation. Considering attacking this abnormal lipid accumulation, obvious targets would be the processes of lipid transport or metabolism, as well as associated genes, many of which have been identified. However, it is also conceivable to target the retained lipoproteins and seek to remove them.

Inflammation and Immunity
Inflammation and immunity are attractive targets because they appear to be central to all stages of AMD-not only in its development, but also in progression to the intermediate and advanced stages. It appears that early in the disease process, lipoprotein accumulation results in a smoldering and chronic inflammatory response that is directed to the RPE, choriocapillaris, and Bruch's membrane. This includes deposition of complement components (Fig. 5B), as well as recruitment and activation of inflammatory cells (including circulating leukocytes, resident microglia, and infiltrating macrophages). Additionally, inflammasome activation is a growing area of research and therapeutic development, although groups differ in their approach to this target. Important gene associations have been identified for complement and inflammatory pathways. 33

PROGRESSION TO ADVANCED AMD
As AMD progresses, lipoprotein retention and inflammation can lead to angiogenesis, which is associated with dissolution of Bruch's membrane and disturbances in the extracellular matrix. This, in turn, leads to the advanced neovascular form of AMD (Fig. 6A). Alternatively, RPE injury and subsequent RPE and photoreceptor cell death result in atrophic changes, which underlie the pathogenesis of advanced atrophic AMD or geographic atrophy (Fig. 6B).

STATIN THERAPY FOR AMD
Numerous investigations over the past few decades have explored the therapeutic potential of statins for AMD, not only for their well-known lipid-lowering effects but also for their potential anti-inflammatory effects. Previous investigations on whether statins could affect AMD status or alter progression showed mixed results, and a 2015 Cochrane systematic review and meta-analysis concluded that the available evidence is insufficient to support a role for statins in preventing or delaying onset of AMD, or in progression of AMD. 35 Indeed, some studies show effectiveness for statins in AMD therapy, while others do not. Recently, Guymer and colleagues 36 conducted a prospective, randomized, placebo-controlled study with 114 subjects, and found that treatment with oral simvastatin (Zocor) 40 mg daily may slow progression of nonadvanced AMD, especially in those with the complement factor H (CFH) risk allele. Regarding the association between serum lipids and AMD risk, VanderBeek and colleagues 37 showed that increased serum low-density lipoprotein (LDL), increased serum triglycerides, and more than 1 year of statin use led to increased risk of neovascular AMD; while one might conclude that statins might promote AMD, the authors postulated that study patients had lipid profiles that were resistant to statin treatment and thus were at increased risk of AMD. 37 Cougnard-Gregoire et al. 38 demonstrated that increased serum high-density lipoprotein (HDL) increases AMD risk in the ALIENOR study. Conversely, a meta-analysis of three cohorts by Klein and colleagues 39 showed no association of AMD incidence or progression with serum lipids, statin use, or lipid pathway genes.
The variability in these studies may be explained in part by the intrinsic heterogeneity of AMD. Even the term ''intermediate AMD'' covers a wide spectrum from large drusen to confluent soft drusen and a variety of atrophic changes. Moreover, studies conducted to date have involved not only variable statin dosing, but also variable activity among different statins; for example, 40-mg simvastatin (Zocor) is approxi-  mately equivalent to 20-mg atorvastatin (Lipitor), 40 and researchers should be aware of this variability when surveying the literature. Finally, the wide use of statins in the general population complicates research in the AMD patient population.
Nonetheless, the cardiovascular disease literature may inform the potential use of statins for AMD. Pitt et al. 41 demonstrated that in patients with coronary artery disease, high-dose (80 mg daily) atorvastatin (Lipitor) prevented restenosis, and additional studies by Nissen et al., 42 Zhao et al., 43 and others using high statin doses have confirmed protective effects and even demonstrated resorption of atherosclerotic plaque. These studies suggest that intensive statin therapy may reverse the ''retained'' lipid in early AMD. In a single patient treated by Demetrios Vavvas, we observed complete disappearance of large, soft, confluent macular drusen (without accompanying atrophy of the RPE) and gains in visual acuity in a patient with AMD after 6 months of 80 mg atorvastatin daily (Fig. 7). 44 Based on the promising results of this case study, we initiated a pilot prospective interventional study in two centers of high-dose atorvastatin therapy in AMD patients with bilateral large soft drusen/drusenoid pigment epithelial detachments (PEDs) in both eyes, without significant geographic atrophy or CNV in either eye. 44 Of 23 patients who completed follow-up of at least 12 months, 10 showed drusen regression without atrophy; of these patients, visual acuity improved by an average of three letters, and regression was nearly complete in eight patients. No patients progressed to neovascular AMD.
When we have seen drusen regression previously, it is accompanied by atrophy and vision loss. However, the evidence in our study suggests that drusen regression with high-dose atorvastatin occurs without any development of atrophy or neovascularization. Possible mechanisms include altering RPE metabolism or creating a gradient to allow efflux of lipids from the outer retina and/or the infiltrating macrophages. In addition, statins have anti-inflammatory and antiangiogenic effects. While this is a pilot study, high-dose atorvastatin is a tantalizing prospect for treating AMD, and warrants further investigation.

FUTURE TREATMENT OF AMD
Future treatment of AMD should be based on biology, and this will require continuing to elucidate the interconnections between the pathogenic mechanisms in AMD development (Table). Therapeutic targets include inflammation, the complement pathway, and inflammasomes; accordingly, there are many clinical trials under way in this space, so we will be learning if these are effective therapeutic strategies. Neuroprotection represents another promising area of research and therapeutic development.
Because the heterogeneity of AMD creates challenges to developing effective treatments for early and intermediate disease, future progress in therapy will benefit from improvements in phenotyping and classification. We need to use our findings from imaging and dark adaptation and perhaps combine that with metabolomics and genotyping in order to tease out the subtypes within this heterogeneous patient population.