Biology subjects

Casares, F.

Publications and source records attributed to Casares, F..

16 recordsLinked to original sources

bioRxiv2026

A sex-specific regulator expands the embryonic ocular field to generate a novel visual system

Morphological novelties often emerge through the redeployment of conserved developmental programs, but how such programs become activated in new developmental contexts remains poorly understood. We investigated the evolutionary origin of the turbanate eyes (TurbEyes) of mayflies, a male-specific additional visual system. Using single-cell transcriptomics, chromatin accessibility profiling and functional genetics, we show that this novelty develops through deployment of the ancestral retinal determination gene network (RDGN). We identify a novel, male-specific paralog of the sex determination transcription factor Doublesex, DsxM, that regulates the eye-specification gene eyeless/Pax6 and promotes the expansion of the embryonic ocular field. Functional assays impairing dsxM activity reduce this male-specific expansion and causes defects in TurbEye development. Our findings reveal a developmental mechanism by which a sex-specific regulator generates a new developmental territory, enabling the spatial redeployment of a conserved developmental program and to generate a novel male-specific visual system.

evolutionary biology↗

bioRxiv2025

A Natural Programmable Metamaterial Controls 3D Curvature of Compound Eyes

The panoramic vision of the convex compound eyes, common to insects and crustaceans, relies on micrometer-scale curvature variations1. These variations create specialized visual zones adapted to specific tasks, including detecting prey, mates, or predators2,3. However, the mechanisms by which such fine-scale curvature is encoded during development remain unknown. Here we show that the developing eye of Drosophila melanogaster functions as a natural metamaterial that programs the organs precise 3D curvature. We discover a supracellular triangular mesh in the basal retina with a specific pattern of triangles sizes. Computational simulations demonstrate its role directing the small scale curvature variations of the eye. Genetic disruption of this micropattern prevents local curvature establishment. Furthermore, the presence of a homologous mesh-curvature relationship in Drosophila mauritiana indicates evolutionary conservation of this mechanism. These results reveal a novel mechanism of morphogenesis control in which the supracellular 2D patterning give rise to a biological programmable metamaterial that encodes 3D curvature with great precision4. Our in vivo finding offers a novel framework for the design of shape-programmable 3D biological surfaces with broad implications from synthetic morphogenesis to clinical applications.

developmental biology↗

bioRxiv2025

Expanding the fly eye gene regulatory network: from Drosophila to the hoverfly Episyrphus balteatus.

Comparing developmental mechanisms across long evolutionary distances is challenging, especially when the level of description in the compared species is very disparate. This is the case for the gene regulatory network (GRN) that underlies the development of compound eyes in insects, where the very detailed level description of the network in the fruit fly Drosophila melanogaster contrasts with the knowledge of the network in other well studied insects, such as the flour beetle Tribolium or the desert locust Schistocerca. It is not even known to what extent the Drosophila eye GRN is typical of flies (diptera). Here we have introduced the marmalade fly Episyrphus balteatus (Syrphidae), separated from Drosophila approximately 90 MYrs, as a fly species to compare the mechanisms of compound eye development. The Episyrphus eye develops from an imaginal disc which is, in the overall, similar to that of Drosophila. By generating parallel data sets in Episyrphus and Drosophila to maximize comparability, our results show how the genes known to participate in the early specification of the Drosophila eye and the onset of its differentiation are likely participating in the eye GRN of Episyrphus, further expanding the set of conserved genes/nodes in this network. This gene set might be the basis for further explorations of the developmental mechanisms involved in compound eye development beyond flies. By combining RNAseq and ATACseq profiling with TF motifs we derive the first eye GRN in Episyrphus. When built using Episyrphus/Drosophila conserved nodes, the resulting GRN model exhibited abundant internal connectivity, suggesting that, as proposed in Drosophila, the GRN is knitted with many regulatory feedbacks. Using link conservation as a criterion for discovering new regulatory interactions, we find that the AML1/Runx transcription factor lozenge (lz) is a negative regulator of the retinal determination gene dachshund (dac) in Drosophila. However, although many of the predicted regulatory links are conserved, a large number of regulatory connections seem species-specific, even among conserved nodes. This suggests quite a significant regulatory rewiring during the 90Myr period separating Episyrphus and Drosophila.

developmental biology↗

bioRxiv2024

Gill regeneration in the mayfly Cloeon uncovers new molecular pathways in insect regeneration

The capacity to regenerate lost or damaged organs is widespread among animals, and yet, the species in which regeneration has been experimentally probed using molecular and functional assays is very small. This is also the case for insects, for which we still lack a complete picture of their regeneration mechanisms and the extent of conservation of these mechanisms. Here we contribute to filling this gap by investigating regeneration in the mayfly Cloeon dipterum. Mayflies, or Ephemeroptera, appeared early in the evolution of insects. We focus on the abdominal gills of Cloeon nymphs, which are critical for osmoregulation and gas exchange. After amputation, gills re-grow faster than they do during normal development. Direct cell count and EdU proliferation assays indicate that growth acceleration involves an uniform increase in cell proliferation throughout the gill, rather than a localized growth zone. Transcriptomic analysis reveals an early enrichment in cell cycle-related genes, in agreement with fast proliferation. Several other gene classes are also enriched in regenerating gills, including protein neddylation and other proteostatic processes. We then showed that protein neddylation, the activin signaling pathway or the mRNA-binding protein Lin28, among other genes and processes, are required for Drosophila larval/pupal wing regeneration, and that some of these genes may have a regeneration-specific function in the wing. Globally, our results contribute to elucidating regeneration mechanisms in mayflies and suggest a conservation of regeneration mechanisms across insects, as evidenced by the regenerative role of candidate genes identified in Cloeon in the distant Drosophila.

developmental biology↗

bioRxiv2023

Feedback control of organ size precision is mediated by BMP2-regulated apoptosis in the Drosophila eye

Biological processes are intrinsically noisy and yet, the result of development -like the species-specific size and shape of organs- is usually remarkably precise. This precision suggests the existence of mechanisms of feedback control that ensure that deviations from a target size are minimized. Still, we have very limited understanding of how these mechanisms operate. Here, we investigate the problem of organ size precision using the Drosophila eye. The size of the adult eye depends on the rates at which eye progenitor cells grow and differentiate. We first find that the progenitor net growth rate results from the balance between their proliferation and apoptosis, with this latter contributing to determining both final eye size and its variability. In turn, apoptosis of progenitor cells is hampered by Dpp, a BMP2/4 signaling molecule transiently produced by early differentiating retinal cells. Our genetic and computational experiments show how the status of retinal differentiation is communicated to progenitors through the differentiation-dependent production of Dpp which, by adjusting the rate of apoptosis, exerts a feedback control over the net growth of progenitors to reduce final eye size variability.

developmental biology↗

bioRxiv2021

Patterning on the move: the effects of Hh morphogen source movement on signaling dynamics

Morphogens of the Hh-family trigger gene expression changes of receiving cells in a concentration-dependent manner. The outputs of the pathway include regulation of cell identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its internal logic includes a negative feedback loop involving the Hh receptor Ptc. In this paper, we use experiments and computational models to study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distribution of Ptc and the activator form of the Gli transcription factor, CiA, in wing, antenna and ocellus show similar features, but markedly different from that in the compound eye (CE). We show that these two profile types represent two time points along the signaling dynamics, and that the interplay between the spatial displacement of the Hh source in the CE and the negative feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results indicate that the dynamics of the Hh source strongly influences the signaling profile Hh elicits in receiving cells, and show how the interaction between spatial and temporal dynamics of signaling and differentiation processes can contribute to the informational versatility of the conserved Hh signaling pathway.

developmental biology↗

bioRxiv2021

The mayfly subimago explained. The regulation of metamorphosis in Ephemeroptera

In the Paleozoic era, more than 400 million years ago, insects continued molting after forming functional wings. Today, however, all flying insects stop molting after metamorphosis when they become fully winged. The only exception is the mayflies (Ephemeroptera), which molt in the subimago, a flying intermediate stage between the nymph and the adult. However, the identity and homology of the subimago remains 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 do mayflies maintain 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. However, by combining morphological studies, hormonal treatments, and molecular analysis in the mayfly species Cloeon dipterum, we found new answers to these old questions. We observed that treatment with a juvenile hormone analog in the last nymphal instar stimulated the expression of Kr-h1 gene and reduced that of E93, which suppress and trigger metamorphosis, respectively. Consequently, the subimago is not formed in these treated mayflies. This indicates that metamorphosis is determined prior to the formation of the subimago, which must therefore be considered an instar of the adult stage. We also observed that the forelegs dramatically grow between the last nymphal instar, the subimago, and the adult. This necessary growth is spread over the last two stages, which could explain, at least in part, the adaptive sense of the subimago.

evolutionary biology↗

bioRxiv2021

Differences in orthodenticle expression promote ommatidial size variation between Drosophila species

The compound eyes of insects exhibit extensive variation in ommatidia number and size, which affects how they see and underlies adaptations in their vision to different environments and lifestyles. However, very little is known about the genetic and developmental bases of differences in compound eye size. We previously showed that the larger eyes of Drosophila mauritiana compared to D. simulans is generally caused by differences in ommatidia size rather than number. Furthermore, we identified an X-linked chromosomal region in D. mauritiana that results in larger eyes when introgressed into D. simulans. Here, we used a combination of fine-scale mapping and gene expression analysis to further investigate positional candidate genes on the X chromosome. We found earlier expression of orthodenticle (otd) during ommatidial maturation in third instar larvae in D. mauritiana than in D. simulans, and we show that this gene is required for the correct organisation and size of ommatidia in D. melanogaster. We discovered that the activity of an otd eye enhancer is consistent with the difference in the expression of this gene between species, with the D. mauritiana enhancer sequence driving earlier expression than that of D. simulans. We also identified potential direct targets of Otd that are differentially expressed between D. mauritiana and D. simulans during ommatidial maturation. Taken together, our results suggest that differential timing of otd expression contributes to natural variation in ommatidia size between D. mauritiana and D. simulans, which provides new insights into the mechanisms underlying the regulation and evolution of compound eye size in insects.

evolutionary biology↗

bioRxiv2020

Variation in a pleiotropic regulatory module drives evolution of head shape and eye size in Drosophila

Revealing the mechanisms underlying the breath-taking 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 rather simple 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 complex morphological traits, such as the size and shape of organs. Since 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 that differences in both traits are established late during head development and we applied comparative transcriptomics and chromatin accessibility data to identify the GATA transcription factor Pannier (Pnr) as central factor regulating 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 co-evolution of expression of pnr and its co-factor u-shaped (ush). We propose that natural variation in 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 suggests that evolutionary hotspots do not contribute to the repeated evolution of eye size and head shape in Drosophila.

evolutionary biology↗

bioRxiv2019

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

The first winged insects underwent profound morphological and functional transformations leading to the most successful animal radiations in the history of earth. Despite this, we still have a very incomplete picture of the changes in their genomes that underlay this radiation. Mayflies (Ephemeroptera) are one of the extant sister groups of all other winged insects and therefore are at a key phylogenetic position to understand this radiation. Here, we describe the genome of the cosmopolitan mayfly Cloeon dipterum and study its expression along development and in specific organs. We discover an expansion of odorant-binding proteins, some expressed specifically in the breathing gills of aquatic nymphs, suggesting a novel sensory role for gills. In contrast, as flying adults, mayflies make use of an enlarged set of opsins and utilise these visual genes in a sexually dimorphic manner, with some opsins expressed only in males. Finally, to illuminate the origin of wings, we identify a core set of deeply conserved wing-specific genes at the root of the pterygote insects. Globally, this is the first comprehensive study of the structure and expression of the genome of a paleopteran insect and shows how its genome has kept a record of its functional adaptations.

evolutionary biology↗

bioRxiv2019

Minimisation of surface energy drives apical epithelial organisation and gives rise to Lewis’ law

The packing of cells in epithelia exhibits striking regularities, regardless of the organism and organ. One of these regularities is expressed in Lewis law, which states that the average apical cell area is linearly related to the number of neighbours, such that cells with larger apical area have on average more neighbours. The driving forces behind the almost 100-year old Lewis law have remained elusive. We now provide evidence that the observed apical epithelial packing minimizes surface energy at the intercellular apical adhesion belt. Lewis law emerges because the apical cell surfaces then assume the most regular polygonal shapes within a contiguous lattice, thus minimising the average perimeter per cell, and thereby surface energy. We predict that the linear Lewis law generalizes to a quadratic law if the variability in apical areas is increased beyond what is normally found in epithelia. We confirm this prediction experimentally by generating heterogeneity in cell growth in Drosophila epithelia. Our discovery provides a link between epithelial organisation, cell division and growth and has implications for the general understanding of epithelial dynamics.

biophysics↗

bioRxiv2019

Aboave-Weaire’s law in epithelia results from an angle constraint in contiguous polygonal lattices

It has long been noted that the cell arrangements in epithelia, regardless of their origin, exhibit some striking regularities: first, the average number of cell neighbours at the apical side is (close to) six. Second, the average apical cell area is linearly related to the number of neighbours, such that cells with larger apical area have on average more neighbours, a relation termed Lewis law. Third, Aboav-Weaires (AW) law relates the number of neighbours that a cell has to that of its direct neighbours. While the first rule can be explained with topological constraints in contiguous polygonal lattices, and the second rule (Lewis law) with the minimisation of the lateral contact surface energy, the driving forces behind the AW law have remained elusive. We now show that also the AW law emerges to minimise the lateral contact surface energy in polygonal lattices by driving cells to the most regular polygonal shape, but while Lewis law regulates the side lengths, the AW law controls the angles. We conclude that global apical epithelial organization is the result of energy minimisation under topological constraints.

biophysics↗

bioRxiv2019

A new image segmentation algorithm with applications in confocal microscopy analysis

Gene regulatory networks (GRNs) represent the molecular interactions that govern the behavior of cells in tissues during development. The building and analysis of GRNs require quantitative information on gene expression from tissues. Laser Scanning Confocal Microscopy (LSCM) is commonly used to obtain such information, where immunofluorescence signal can be used as a correlate of gene expression or protein levels. However, a critical step for the extraction of this information is the segmentation of LSCM digital images. Popular segmentation algorithms are frequently based on watershed methods. Here we present an algorithm for the 3D segmentation of nuclei from LSCM (x,y,z) image stacks based on regional merging and graph contractions. This algorithm outperforms watershed methods, especially when the density of images along the z-axis is low and there is a high nuclear signal crowding. In addition, it reduces the parameterization since no filter is needed in order to prevent signal noise side effects (e.g. oversegmentation). Based on this algorithm, we developed an application (iFLIC, immunoFLuorescence Imaging Cytometry tool) for the Java Virtual Machine (JVM). The application supports basic operations for reading, writing and filtering 8-bit depth multicolor TIFF image formats, including indexed file directories (IFD), which are provided by the Java Advanced Imaging (JAI) library. It also provides with basic 3D-rendering and ROI specification that make extensive use of the Java3D library. iFLIC is also a plugin based application powered by the Java Plugin Platform (JPF), so each specific operation is declared as a unique command associated to one plugin and linked to a common interface. Results from segmentation can be exported both as TIFF images and a descriptive file format (iFLIC format)

systems biology↗

bioRxiv2018

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 established 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.

evolutionary biology↗

bioRxiv2018

Dynamic Hh signaling can generate temporal information during tissue patterning

The differentiation of tissues and organs requires that cells exchange information in space and time. Spatial information is often conveyed by morphogens, molecules that disperse across receiving cells generating signaling gradients. Cells translate such concentration gradients into space-dependent patterns of gene expression and cellular behavior [1, 2]. But could morphogen gradients also convey developmental time? Here, investigating the developmental role of Hh on a component of the Drosophila visual system, the ocellar retina, we discovered that ocellar cells use the non-linear gradient of Hh as a temporal cue, collectively performing the biological equivalent of a mathematical logarithmic transformation. In this way, a morphogen diffusing from a non-moving source is decoded as a wave of differentiating photoreceptors that travels at constant speed throughout the retinal epithelium.

developmental biology↗

bioRxiv2016

Specific expression and function of the Six3 optix in serially homologous organs

Organ size and pattern results from the integration of two positional information systems. One global, encoded by the Hox genes, links organ type with position along the main body axis. Within specific organs, local information is conveyed by signaling molecules that regulate organ growth and pattern. The mesothoracic (T2) wing and the metathoracic (T3) haltere of Drosophila represent a paradigmatic example of this coordination. The Hox gene Ultrabithorax (Ubx), expressed in the developing T3, selects haltere identity by, among other processes, modulating the production and signaling efficiency of Dpp, a BMP2-like molecule that acts as a major regulator of size and pattern. Still, the mechanisms of the Hox-signal integration even in this well-studied system, are incomplete. Here we have investigated this issue by studying the expression and function of the Six3 transcription factor optix during the development of the Drosophila wing and haltere development. We find that in both organs Dpp defines the expression domain of optix through repression, and that the specific position of this domain in wing and haltere seems to reflect the differential signaling profile among these organs. We show that optix expression in wing and haltere primordia is conserved beyond Drosophila in other higher diptera. Despite the similar expression pattern, optix plays different roles in wing and haltere. In the wing, optix is required for the growth of the most anterior/proximal region (the \"marginal cell\") and for the correct formation of sensory structures along the proximal anterior wing margin. In contrast, in the haltere optix is necessary for the suppression of sensory bristles without any noticeable effect on organ growth. Therefore, optix shows an organ-specific function. Beyond dipterans, optix expression in the anterior wing has been shown also in butterflies. We propose that the ancestral role of optix might have been structural in the anterior wing. Once expressed in the wing, optix expression had been re-deployed for wing spot formation in other parts of the wing of Heliconius butterflies.

developmental biology↗