From whole bodies to single cells: A guide to transcriptomic approaches for ecology and evolutionary biology.

RNA sequencing (RNAseq) methodology has experienced a burst of technological developments in the last decade, which has opened up opportunities for studying the mechanisms of adaptation to environmental factors at both the organismal and cellular level. Selecting the most suitable experimental approach for specific research questions and model systems can, however, be a challenge and researchers in ecology and evolution are commonly faced with the choice of whether to study gene expression variation in whole bodies, specific tissues, and/or single cells. A wide range of sometimes polarised opinions exists over which approach is best. Here, we highlight the advantages and disadvantages of each of these approaches to provide a guide to help researchers make informed decisions and maximise the power of their study. Using illustrative examples of various ecological and evolutionary research questions, we guide the readers through the different RNAseq approaches and help them identify the most suitable design for their own projects.

Regulation of metamorphosis in neopteran insects is conserved in the paleopteran Cloeon dipterum (Ephemeroptera).

In the Paleozoic era, more than 400 Ma, a number of insect groups continued molting after forming functional wings. Today, however, flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Paleoptera, Ephemeroptera), which molt in the subimago, a flying stage between the nymph and the adult. However, the identity and homology of the subimago still is underexplored. Debate remains regarding whether this stage represents a modified nymph, an adult, or a pupa like that of butterflies. Another relevant question is why mayflies have the subimago stage despite the risk of molting fragile membranous wings. These questions have intrigued numerous authors, but nonetheless, clear answers have not yet been found. By combining morphological studies, hormonal treatments, and molecular analysis in the mayfly Cloeon dipterum, we found answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of the Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. The regulation of metamorphosis thus follows the MEKRE93 pathway, as in neopteran insects. Moreover, the treatment prevented the formation of the subimago. These findings suggest that the subimago must be considered an instar of the adult mayfly. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth spread over the last two stages could explain, at least in part, the adaptive sense of the subimago.

Variation in Pleiotropic Hub Gene Expression Is Associated with Interspecific Differences in Head Shape and Eye Size in Drosophila.

Revealing the mechanisms underlying the breathtaking morphological diversity observed in nature is a major challenge in Biology. It has been established that recurrent mutations in hotspot genes cause the repeated evolution of morphological traits, such as body pigmentation or the gain and loss of structures. To date, however, it remains elusive whether hotspot genes contribute to natural variation in the size and shape of organs. As natural variation in head morphology is pervasive in Drosophila, we studied the molecular and developmental basis of differences in compound eye size and head shape in two closely related Drosophila species. We show differences in the progression of retinal differentiation between species and we applied comparative transcriptomics and chromatin accessibility data to identify the GATA transcription factor Pannier (Pnr) as central factor associated with these differences. Although the genetic manipulation of Pnr affected multiple aspects of dorsal head development, the effect of natural variation is restricted to a subset of the phenotypic space. We present data suggesting that this developmental constraint is caused by the coevolution of expression of pnr and its cofactor u-shaped (ush). We propose that natural variation in expression or function of highly connected developmental regulators with pleiotropic functions is a major driver for morphological evolution and we discuss implications on gene regulatory network evolution. In comparison to previous findings, our data strongly suggest that evolutionary hotspots are not the only contributors to the repeated evolution of eye size and head shape in Drosophila.

Gene regulatory network architecture in different developmental contexts influences the genetic basis of morphological evolution.

Convergent phenotypic evolution is often caused by recurrent changes at particular nodes in the underlying gene regulatory networks (GRNs). The genes at such evolutionary 'hotspots' are thought to maximally affect the phenotype with minimal pleiotropic consequences. This has led to the suggestion that if a GRN is understood in sufficient detail, the path of evolution may be predictable. The repeated evolutionary loss of larval trichomes among Drosophila species is caused by the loss of shavenbaby (svb) expression. svb is also required for development of leg trichomes, but the evolutionary gain of trichomes in the 'naked valley' on T2 femurs in Drosophila melanogaster is caused by reduced microRNA-92a (miR-92a) expression rather than changes in svb. We compared the expression and function of components between the larval and leg trichome GRNs to investigate why the genetic basis of trichome pattern evolution differs in these developmental contexts. We found key differences between the two networks in both the genes employed, and in the regulation and function of common genes. These differences in the GRNs reveal why mutations in svb are unlikely to contribute to leg trichome evolution and how instead miR-92a represents the key evolutionary switch in this context. Our work shows that variability in GRNs across different developmental contexts, as well as whether a morphological feature is lost versus gained, influence the nodes at which a GRN evolves to cause morphological change. Therefore, our findings have important implications for understanding the pathways and predictability of evolution.

Human eye conditions: insights from the fly eye.

The fruit fly Drosophila melanogaster has served as an excellent model to study and understand the genetics of many human diseases from cancer to neurodegeneration. Studying the regulation of growth, determination and differentiation of the compound eyes of this fly, in particular, have provided key insights into a wide range of diseases. Here we review the regulation of the development of fly eyes in light of shared aspects with human eye development. We also show how understanding conserved regulatory pathways in eye development together with the application of tools for genetic screening and functional analyses makes Drosophila a powerful model to diagnose and characterize the genetics underlying many human eye conditions, such as aniridia and retinitis pigmentosa. This further emphasizes the importance and vast potential of basic research to underpin applied research including identifying and treating the genetic basis of human diseases.

A robust (re-)annotation approach to generate unbiased mapping references for RNA-seq-based analyses of differential expression across closely related species.

BACKGROUND: RNA-seq based on short reads generated by next generation sequencing technologies has become the main approach to study differential gene expression. Until now, the main applications of this technique have been to study the variation of gene expression in a whole organism, tissue or cell type under different conditions or at different developmental stages. However, RNA-seq also has a great potential to be used in evolutionary studies to investigate gene expression divergence in closely related species. RESULTS: We show that the published genomes and annotations of the three closely related Drosophila species D. melanogaster, D. simulans and D. mauritiana have limitations for inter-specific gene expression studies. This is due to missing gene models in at least one of the genome annotations, unclear orthology assignments and significant gene length differences in the different species. A comprehensive evaluation of four statistical frameworks (DESeq2, DESeq2 with length correction, RPKM-limma and RPKM-voom-limma) shows that none of these methods sufficiently accounts for inter-specific gene length differences, which inevitably results in false positive candidate genes. We propose that published reference genomes should be re-annotated before using them as references for RNA-seq experiments to include as many genes as possible and to account for a potential length bias. We present a straight-forward reciprocal re-annotation pipeline that allows to reliably compare the expression for nearly all genes annotated in D. melanogaster. CONCLUSIONS: We conclude that our reciprocal re-annotation of previously published genomes facilitates the analysis of significantly more genes in an inter-specific differential gene expression study. We propose that the established pipeline can easily be applied to re-annotate other genomes of closely related animals and plants to improve comparative expression analyses.

ß amyloid protein precursor-like (Appl) is a Ras1/MAPK-regulated gene required for axonal targeting in Drosophila photoreceptor neurons.

In a genome-wide expression profile search for genes required for Drosophila R7 photoreceptor development we found ß amyloid protein precursor-like (Appl), the ortholog of human APP, which is a key factor in the pathogenesis of Alzheimer's disease. We analyzed Appl expression in the eye imaginal disc and found that is highly accumulated in R7 photoreceptor cells. The R7 photoreceptor is responsible for UV light detection. To explore the link between high expression of Appl and R7 function, we have analyzed Appl null mutants and found reduced preference for UV light, probably because of mistargeted R7 axons. Moreover, axon mistargeting and inappropriate light discrimination are enhanced in combination with neurotactin mutants. R7 differentiation is triggered by the inductive interaction between R8 and R7 precursors, which results in a burst of Ras1/MAPK, activated by the tyrosine kinase receptor Sevenless. Therefore, we examined whether Ras1/MAPK is responsible for the high Appl expression. Inhibition of Ras1 signaling leads to reduced Appl expression, whereas constitutive activation drives ectopic Appl expression. We show that Appl is directly regulated by the Ras/MAPK pathway through a mechanism mediated by PntP2, an ETS transcription factor that specifically binds ETS sites in the Appl regulatory region. We also found that zebrafish appb expression increased after ectopic fgfr activation in the neural tube of zebrafish embryos, suggesting a conserved regulatory mechanism.

Sexual dimorphism and natural variation within and among species in the Drosophila retinal mosaic.

BACKGROUND: Insect compound eyes are composed of ommatidia, which contain photoreceptor cells that are sensitive to different wavelengths of light defined by the specific rhodopsin proteins that they express. The fruit fly Drosophila melanogaster has several different ommatidium types that can be localised to specific retinal regions, such as the dorsal rim area (DRA), or distributed stochastically in a mosaic across the retina, like the 'pale' and 'yellow' types. Variation in these ommatidia patterns very likely has important implications for the vision of insects and could underlie behavioural and environmental adaptations. However, despite the detailed understanding of ommatidia specification in D. melanogaster, the extent to which the frequency and distribution of the different ommatidium types vary between sexes, strains and species of Drosophila is not known. RESULTS: We investigated the frequency and distribution of ommatidium types based on rhodopsin protein expression, and the expression levels of rhodopsin transcripts in the eyes of both sexes of different strains of D. melanogaster, D. simulans and D. mauritiana. We found that while the number of DRA ommatidia was invariant, Rh3 expressing ommatidia were more frequent in the larger eyes of females compared to the males of all species analysed. The frequency and distribution of ommatidium types also differed between strains and species. The D. simulans strain ZOM4 has the highest frequency of Rh3 expressing ommatidia, which is associated with a non-stochastic patch of pale and odd-coupled ommatidia in the dorsal-posterior of their eyes. CONCLUSIONS: Our results show that there is striking variation in the frequency and distribution of ommatidium types between sexes, strains and species of Drosophila. This suggests that evolutionary changes in the underlying regulatory mechanisms can alter the distribution of ommatidium types to promote or restrict their expression in specific regions of the eye within and between species, and that this could cause differences in vision among these flies.

Evolution of tissue-specific expression of ancestral genes across vertebrates and insects.

Regulation of gene expression is arguably the main mechanism underlying the phenotypic diversity of tissues within and between species. Here we assembled an extensive transcriptomic dataset covering 8 tissues across 20 bilaterian species and performed analyses using a symmetric phylogeny that allowed the combined and parallel investigation of gene expression evolution between vertebrates and insects. We specifically focused on widely conserved ancestral genes, identifying strong cores of pan-bilaterian tissue-specific genes and even larger groups that diverged to define vertebrate and insect tissues. Systematic inferences of tissue-specificity gains and losses show that nearly half of all ancestral genes have been recruited into tissue-specific transcriptomes. This occurred during both ancient and, especially, recent bilaterian evolution, with several gains being associated with the emergence of unique phenotypes (for example, novel cell types). Such pervasive evolution of tissue specificity was linked to gene duplication coupled with expression specialization of one of the copies, revealing an unappreciated prolonged effect of whole-genome duplications on recent vertebrate evolution.

Genetic and developmental analysis of differences in eye and face morphology between Drosophila simulans and Drosophila mauritiana.

Eye and head morphology vary considerably among insects and even between closely related species of Drosophila. Species of the D. melanogaster subgroup, and other Drosophila species, exhibit a negative correlation between eye size and face width (FW); for example, D. mauritiana generally has bigger eyes composed of larger ommatidia and conversely a narrower face than its sibling species. To better understand the evolution of eye and head morphology, we investigated the genetic and developmental basis of differences in eye size and FW between male D. mauritiana and D. simulans. QTL mapping of eye size and FW showed that the major loci responsible for the interspecific variation in these traits are localized to different genomic regions. Introgression of the largest effect QTL underlying the difference in eye size resulted in flies with larger eyes but no significant difference in FW. Moreover,introgression of a QTL region on the third chromosome that contributes to the FW difference between these species affected FW, but not eye size. We also observed that this difference in FW is detectable earlier in the development of the eye­antennal disc than the difference in the size of the retinal field. Our results suggest that different loci that act at different developmental stages underlie changes in eye size and FW. Therefore, while there is a negative correlation between these traits in Drosophila, we show genetically that they also have the potential to evolve independently and this may help to explain the evolution of these traits in other insects.

The Lnk/SH2B adaptor provides a fail-safe mechanism to establish the Insulin receptor-Chico interaction.

BACKGROUND: Insulin/insulin-like growth factor signalling (IIS) has been described as one of the major pathways involved in growth control and homeostasis in multicellular organisms. Whereas its core components are well established, less is known about the molecular functions of IIS regulators. The adaptor molecule Lnk/SH2B has been implicated in IIS but the mechanism by which it promotes IIS activity has remained enigmatic. RESULTS: In this study, we analyse genetic and physical interactions among InR, Chico and Lnk in Drosophila tissues. FRET analysis reveals in vivo binding between all three molecules. Genetically, Lnk acts upstream of Chico. We demonstrate that Chico's plasma membrane localisation is ensured by both its PH domain and by the interaction with Lnk. Furthermore, Lnk is able to recruit an intracellular InR fragment to the membrane. CONCLUSIONS: Thus, by acting as a scaffolding molecule that ensures InR and Chico enrichment at the membrane, Lnk provides a fail-safe mechanism for IIS activation.

Interlocking of co-opted developmental gene networks in Drosophila and the evolution of pre-adaptive novelty.

The re-use of genes in new organs forms the base of many evolutionary novelties. A well-characterised case is the recruitment of the posterior spiracle gene network to the Drosophila male genitalia. Here we find that this network has also been co-opted to the testis mesoderm where is required for sperm liberation, providing an example of sequentially repeated developmental co-options. Associated to this co-option event, an evolutionary expression novelty appeared, the activation of the posterior segment determinant Engrailed to the anterior A8 segment controlled by common testis and spiracle regulatory elements. Enhancer deletion shows that A8 anterior Engrailed activation is not required for spiracle development but only necessary in the testis. Our study presents an example of pre-adaptive developmental novelty: the activation of the Engrailed transcription factor in the anterior compartment of the A8 segment where, despite having no specific function, opens the possibility of this developmental factor acquiring one. We propose that recently co-opted networks become interlocked, so that any change to the network because of its function in one organ, will be mirrored by other organs even if it provides no selective advantage to them.

Competition between SOCS36E and Drk modulates Sevenless receptor tyrosine kinase activity.

Modulation of signalling pathways can trigger different cellular responses, including differences in cell fate. This modulation can be achieved by controlling the pathway activity with great precision to ensure robustness and reproducibility of the specification of cell fate. The development of the photoreceptor R7 in the Drosophila melanogaster retina has become a model in which to investigate the control of cell signalling. During R7 specification, a burst of Ras small GTPase (Ras) and mitogen-activated protein kinase (MAPK) controlled by Sevenless receptor tyrosine kinase (Sev) is required. Several cells in each ommatidium express sev. However, the spatiotemporal expression of the boss ligand and the action of negative regulators of the Sev pathway will restrict the R7 fate to a single cell. The Drosophila suppressor of cytokine signalling 36E (SOCS36E) protein contains an SH2 domain and acts as a Sev signalling attenuator. By contrast, downstream of receptor kinase (Drk), the fly homolog of the mammalian Grb2 adaptor protein, which also contains an SH2 domain, acts as a positive activator of the pathway. Here, we apply the Förster resonance energy transfer (FRET) assay to transfected Drosophila S2 cells and demonstrate that Sev binds directly to either the suppressor protein SOCS36E or the adaptor protein Drk. We propose a mechanistic model in which the competition between these two proteins for binding to the same docking site results in either attenuation of the Sev transduction in cells that should not develop R7 photoreceptors or amplification of the Ras-MAPK signal only in the R7 precursor.

Parallel evolution of a splicing program controlling neuronal excitability in flies and mammals.

Alternative splicing increases neuronal transcriptomic complexity throughout animal phylogeny. To delve into the mechanisms controlling the assembly and evolution of this regulatory layer, we characterized the neuronal microexon program in Drosophila and compared it with that of mammals. In nonvertebrate bilaterians, this splicing program is restricted to neurons by the posttranscriptional processing of the enhancer of microexons (eMIC) domain in Srrm234. In Drosophila, this processing is dependent on regulation by Elav/Fne. eMIC deficiency or misexpression leads to widespread neurological alterations largely emerging from impaired neuronal activity, as revealed by a combination of neuronal imaging experiments and cell type-specific rescues. These defects are associated with the genome-wide skipping of short neural exons, which are strongly enriched in ion channels. We found no overlap of eMIC-regulated exons between flies and mice, illustrating how ancient posttranscriptional programs can evolve independently in different phyla to affect distinct cellular modules while maintaining cell-type specificity.

Virtual meeting, real and sound science: report of the 17th Meeting of the Spanish Society for Developmental Biology (SEBD-2020).

The Spanish Society for Developmental Biology (SEBD) organized its 17th meeting in November 2020 (herein referred to as SEBD2020). This meeting, originally programmed to take place in the city of Bilbao, was forced onto an online format due to the SARS-CoV2, COVID-19 pandemic. Although, we missed the live personal interactions and missed out on the Bilbao social scene, we were able to meet online to present our work and discuss our latest results. An overview of the activities that took place around the meeting, the different scientific sessions and the speakers involved are presented here. The pros and cons of virtual meetings are discussed.

SOCS36E specifically interferes with Sevenless signaling during Drosophila eye development.

During the development of multicellular organisms the fate of individual cells is specified with great precision and reproducibility. Although classical genetic approaches led to the identification of many of the signaling pathways contributing to cell fate specification, they have provided little insight into the mechanisms that ensure robustness and reproducibility. We have used the specification of the R7 photoreceptor cells controlled by the Sevenless receptor tyrosine kinase (Sev) pathway to screen for modulators of pathway activity and to uncover the mechanisms underlying the robustness of cell fate decisions. Here we provide genetic evidence that the Drosophila SOCS36E adaptor protein containing an SH2 domain and a SOCS box acts as an attenuator of Sev signaling. Overexpression of Socs36E strongly suppresses the specification of extra R7 photoreceptor cells in response to constitutive activation of Sev, and loss of Socs36E function suppresses the loss of R7 cells when Sev activity is impaired. In a wild-type background, however, loss and gain of Socs36E function exhibits little effect on R7 specification. We also show that SH2 domain of SOCS36E is essential for this function in inhibiting Sev action and that Socs36E expression is suppressed by high Sev pathway activity. In our model, only the cell able to activate high levels of receptor tyrosine kinase signaling will repress SOCS36E expression, reduce the negative effect on Sev signaling and allow this cell to differentiate into R7. In contrast, the remaining cells fail to receive high signaling, and thus maintain high levels of SOCS36E. This represses residual Sev activity and blocks R7 development. Therefore, Socs36E constitutes a novel partially redundant feedback mechanism that contributes to the robustness of R7 specification. The SOCS family of adaptor proteins may have evolved as modulators of specific signaling pathways that contribute to the robustness and precision of cell fate specification.

Genomic adaptations to aquatic and aerial life in mayflies and the origin of insect wings.

The evolution of winged insects revolutionized terrestrial ecosystems and led to the largest animal radiation on Earth. However, we still have an incomplete picture of the genomic changes that underlay this diversification. Mayflies, as one of the sister groups of all other winged insects, are key to understanding this radiation. Here, we describe the genome of the mayfly Cloeon dipterum and its gene expression throughout its aquatic and aerial life cycle and specific organs. We discover an expansion of odorant-binding-protein genes, some expressed specifically in breathing gills of aquatic nymphs, suggesting a novel sensory role for this organ. In contrast, flying adults use an enlarged opsin set in a sexually dimorphic manner, with some expressed only in males. Finally, we identify a set of wing-associated genes deeply conserved in the pterygote insects and find transcriptomic similarities between gills and wings, suggesting a common genetic program. Globally, this comprehensive genomic and transcriptomic study uncovers the genetic basis of key evolutionary adaptations in mayflies and winged insects.

Sensory Organ Size Evolution: A View from Drosophila.

In this issue of Developmental Cell, Ramaekers et al. (2019) show that changes in eyeless/Pax6 expression cause differences in compound eye size within and between Drosophila species. These findings reveal how changes in the underlying gene regulatory network facilitate eye size evolution and provide insights into organ size regulation.

Establishment of the mayfly Cloeon dipterum as a new model system to investigate insect evolution.

The great capability of insects to adapt to new environments promoted their extraordinary diversification, resulting in the group of Metazoa with the largest number of species distributed worldwide. To understand this enormous diversity, it is essential to investigate lineages that would allow the reconstruction of the early events in the evolution of insects. However, research on insect ecology, physiology, development and evolution has mostly focused on few well-established model species. The key phylogenetic position of mayflies within Paleoptera as the sister group of the rest of winged insects and life history traits of mayflies make them an essential order to understand insect evolution. Here, we describe the establishment of a continuous culture system of the mayfly Cloeon dipterum and a series of experimental protocols and omics resources that allow the study of its development and its great regenerative capability. Thus, the establishment of Cloeon as an experimental platform paves the way to understand genomic and morphogenetic events that occurred at the origin of winged insects.

A novel protein domain in an ancestral splicing factor drove the evolution of neural microexons.

The mechanisms by which entire programmes of gene regulation emerged during evolution are poorly understood. Neuronal microexons represent the most conserved class of alternative splicing in vertebrates, and are critical for proper brain development and function. Here, we discover neural microexon programmes in non-vertebrate species and trace their origin to bilaterian ancestors through the emergence of a previously uncharacterized 'enhancer of microexons' (eMIC) protein domain. The eMIC domain originated as an alternative, neural-enriched splice isoform of the pan-eukaryotic Srrm2/SRm300 splicing factor gene, and subsequently became fixed in the vertebrate and neuronal-specific splicing regulator Srrm4/nSR100 and its paralogue Srrm3. Remarkably, the eMIC domain is necessary and sufficient for microexon splicing, and functions by interacting with the earliest components required for exon recognition. The emergence of a novel domain with restricted expression in the nervous system thus resulted in the evolution of splicing programmes that qualitatively expanded the neuronal molecular complexity in bilaterians.