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.

The effects of Hh morphogen source movement on signaling dynamics.

Morphogens of the Hh family trigger gene expression changes in receiving cells in a concentration-dependent manner to regulate their identity, proliferation, death or metabolism, depending on the tissue or organ. This variety of responses relies on a conserved signaling pathway. Its logic includes a negative-feedback loop involving the Hh receptor Ptc. Here, using experiments and computational models we study and compare the different spatial signaling profiles downstream of Hh in several developing Drosophila organs. We show that the spatial distributions of Ptc and the activator transcription factor CiA in wing, antenna and ocellus show similar features, but are markedly different from that in the compound eye. We propose 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 compound eye and the negative-feedback loop maintains the receiving cells effectively in an earlier stage of signaling. These results show how the interaction between spatial and temporal dynamics of signaling and differentiation processes may contribute to the informational versatility of the conserved Hh signaling pathway.

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.

Shaping an optical dome: The size and shape of the insect compound eye.

The insect compound eye is the most abundant eye architecture on earth. It comes in a wide variety of shapes and sizes, which are exquisitely adapted to specific ecosystems. Here, we explore the organisational principles and pathways, from molecular to tissular, that underpin the building of this organ and highlight why it is an excellent model system to investigate the relationship between genes and tissue form. The compound eye offers wide fields of view, high sensitivity in motion detection and infinite depth of field. It is made of an array of visual units called ommatidia, which are precisely tiled in 3D to shape the retinal tissue as a dome-like structure. The eye starts off as a 2D epithelium, and it acquires its 3D organisation as ommatidia get into shape. Each ommatidium is made of a complement of retinal cells, including light-detecting photoreceptors and lens-secreting cells. The lens cells generate the typical hexagonal facet lens that lies atop the photoreceptors so that the eye surface consists of a quasi-crystalline array of these hexagonal facet-lenses. This array is curved to various degree, depending on the size and shape of the eye, and on the region of the retina. This curvature sets the resolution and visual field of the eye and is determined by i) the number and size of the facet lens - large ommatidial lenses can be used to generate flat, higher resolution areas, while smaller facets allow for stronger curvature of the eye, and ii) precise control of the inter facet-lens angle, which determines the optical axis of the each ommatidium. In this review we discuss how combinatorial variation in eye primordium shape, ommatidial number, facet lens size and inter facet-lens angle underpins the wide variety of insect eye shapes, and we explore what is known about the mechanisms that might control these parameters.

Disorder in cellular packing can alter proliferation dynamics to regulate growth.

The mechanisms by which an organ regulates its growth are not yet fully understood, especially when the cells are closely packed as in epithelial tissues. We explain growth arrest as a collective dynamical transition in coupled oscillators on disordered lattices. As the cellular morphologies become homogeneous over the course of development, the signals induced by cell-cell contact increase beyond a critical value that triggers coordinated cessation of the cell-cycle oscillators driving cell division. Thus, control of cell proliferation is causally related to the geometry of cellular packing.

Quantitative Relationships Between Growth, Differentiation, and Shape That Control Drosophila Eye Development and Its Variation.

The size of organs is critical for their function and often a defining trait of a species. Still, how organs reach a species-specific size or how this size varies during evolution are problems not yet solved. Here, we have investigated the conditions that ensure growth termination, variation of final size and the stability of the process for developmental systems that grow and differentiate simultaneously. Specifically, we present a theoretical model for the development of the Drosophila eye, a system where a wave of differentiation sweeps across a growing primordium. This model, which describes the system in a simplified form, predicts universal relationships linking final eye size and developmental time to a single parameter which integrates genetically-controlled variables, the rates of cell proliferation and differentiation, with geometrical factors. We find that the predictions of the theoretical model show good agreement with previously published experimental results. We also develop a new computational model that recapitulates the process more realistically and find concordance between this model and theory as well, but only when the primordium is circular. However, when the primordium is elliptical both models show discrepancies. We explain this difference by the mechanical interactions between cells, an aspect that is not included in the theoretical model. Globally, our work defines the quantitative relationships between rates of growth and differentiation and organ primordium size that ensure growth termination (and, thereby, specify final eye size) and determine the duration of the process; identifies geometrical dependencies of both size and developmental time; and uncovers potential instabilities of the system which might constraint developmental strategies to evolve eyes of different size.

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.

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.

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.

The evolution and development of eye size in flies.

The compound eyes of flies exhibit striking variation in size, which has contributed to the adaptation of these animals to different habitats and their evolution of specialist behaviors. These differences in size are caused by differences in the number and/or size of ommatidia, which are specified during the development of the retinal field in the eye imaginal disc. While the genes and developmental mechanisms that regulate the formation of compound eyes are understood in great detail in the fruit fly Drosophila melanogaster, we know very little about the genetic changes and mechanistic alterations that lead to natural variation in ommatidia number and/or size, and thus overall eye size, within and between fly species. Understanding the genetic and developmental bases for this natural variation in eye size not only has great potential to help us understand adaptations in fly vision but also determine how eye size and organ size more generally are regulated. Here we explore the genetic and developmental mechanisms that could underlie natural differences in compound eye size within and among fly species based on our knowledge of eye development in D. melanogaster and the few cases where the causative genes and mechanisms have already been identified. We suggest that the fly eye provides an evolutionary and developmental framework to better understand the regulation and diversification of this crucial sensory organ globally at a systems level as well as the gene regulatory networks and mechanisms acting at the tissue, cellular and molecular levels. This article is categorized under: Establishment of Spatial and Temporal Patterns > Regulation of Size, Proportion, and Timing Invertebrate Organogenesis > Flies Comparative Development and Evolution > Regulation of Organ Diversity.

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.

Control of size, fate and time by the Hh morphogen in the eyes of flies.

Molecules of the hedgehog (hh) family are involved in the specification and patterning of eyes in vertebrates and invertebrates. These organs, though, are of very different sizes, raising the question of how Hh molecules operate at such different scales. In this paper we discuss the strategies used by Hh to control the development of the two eye types in Drosophila: the large compound eye and the small ocellus. We first describe the distinct ways in which these two eyes develop and the evidence for the key role played by Hh in both; then we consider the potential for variation in the range of action of a "typical" morphogen and measure this range ("characteristic length") for Hh in different organs, including the compound eye and the ocellus. Finally, we describe how different feedback mechanisms are used to extend the Hh range of action to pattern the large and even the small eye. In the ocellus, the basic Hh signaling pathway adds to its dynamics the attenuation of its receptor as cell differentiate. This sole regulatory change can result in the decoding of the Hh gradient by receiving cells as a wave of constant speed. Therefore, in the fly ocellus, the Hh morphogen adds to its spatial patterning role a novel one: patterning along a time axis.

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.

Dynamic Hh signalling 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 to generate signalling gradients. Cells translate such concentration gradients into space-dependent patterns of gene expression and cellular behaviour. But could morphogen gradients also convey developmental time? Here, by investigating the developmental role of Hh on a component of the Drosophila visual system, the ocellar retina, we have 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.

Growth and size control during development.

The size and shape of organs are characteristic for each species. Even when organisms develop to different sizes due to varying environmental conditions, such as nutrition, organ size follows species-specific rules of proportionality to the rest of the body, a phenomenon referred to as allometry. Therefore, for a given environment, organs stop growth at a predictable size set by the species's genotype. How do organs stop growth? How can related species give rise to organs of strikingly different size? No definitive answer has been given to date. One of the major models for the studies of growth termination is the vinegar fly Drosophila melanogaster. Therefore, this review will focus mostly on work carried out in Drosophila to try to tease apart potential mechanisms and identify routes for further investigation. One general rule, found across the animal kingdom, is that the rate of growth declines with developmental time. Therefore, answers to the problem of growth termination should explain this seemingly universal fact. In addition, growth termination is intimately related to the problems of robustness (i.e. precision) and plasticity in organ size, symmetric and asymmetric organ development, and of how the 'target' size depends on extrinsic, environmental factors.

Nuclear receptors connect progenitor transcription factors to cell cycle control.

The specification and growth of organs is controlled simultaneously by networks of transcription factors. While the connection between these transcription factors with fate determinants is increasingly clear, how they establish the link with the cell cycle is far less understood. Here we investigate this link in the developing Drosophila eye, where two transcription factors, the MEIS1 homologue hth and the Zn-finger tsh, synergize to stimulate the proliferation of naïve eye progenitors. Experiments combining transcriptomics, open-chromatin profiling, motif analysis and functional assays indicate that these progenitor transcription factors exert a global regulation of the proliferation program. Rather than directly regulating cell cycle genes, they control proliferation through an intermediary layer of nuclear receptors of the ecdysone/estrogen-signaling pathway. This regulatory subnetwork between hth, tsh and nuclear receptors might be conserved from Drosophila to mammals, as we find a significant co-overexpression of their human homologues in specific cancer types.

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

Organ size and pattern results from the integration of two positional information systems. One global information system, 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. However, the mechanisms of the Hox-signal integration 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 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. In Drosophila, optix is necessary for the growth of 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; the halteres of optix mutants are also significantly reduced. In addition, in the haltere, optix is necessary for the suppression of sensory bristles.

Growth control in the Drosophila eye disc by the cytokine Unpaired.

A fundamental question in developmental biology is how organ size is controlled. We have previously shown that the area growth rate in the Drosophila eye primordium declines inversely proportionally to the increase in its area. How the observed reduction in the growth rate is achieved is unknown. Here, we explore the dilution of the cytokine Unpaired (Upd) as a possible candidate mechanism. In the developing eye, upd expression is transient, ceasing at the time when the morphogenetic furrow first emerges. We confirm experimentally that the diffusion and stability of the JAK/STAT ligand Upd are sufficient to control eye disc growth via a dilution mechanism. We further show that sequestration of Upd by ectopic expression of an inactive form of the receptor Domeless (Dome) results in a substantially lower growth rate, but the area growth rate still declines inversely proportionally to the area increase. This growth rate-to-area relationship is no longer observed when Upd dilution is prevented by the continuous, ectopic expression of Upd. We conclude that a mechanism based on the dilution of the growth modulator Upd can explain how growth termination is controlled in the eye disc.

TGFß/Activin signalling is required for ribosome biogenesis and cell growth in Drosophila salivary glands.

Signalling by TGFß superfamily factors plays an important role in tissue growth and cell proliferation. In Drosophila, the activity of the TGFß/Activin signalling branch has been linked to the regulation of cell growth and proliferation, but the cellular and molecular basis for these functions are not fully understood. In this study, we show that both the RII receptor Punt (Put) and the R-Smad Smad2 are strongly required for cell and tissue growth. Knocking down the expression of Put or Smad2 in salivary glands causes alterations in nucleolar structure and functions. Cells with decreased TGFß/Activin signalling accumulate intermediate pre-rRNA transcripts containing internal transcribed spacer 1 regions accompanied by the nucleolar retention of ribosomal proteins. Thus, our results show that TGFß/Activin signalling is required for ribosomal biogenesis, a key aspect of cellular growth control. Importantly, overexpression of Put enhanced cell growth induced by Drosophila Myc, a well-characterized inducer of nucleolar hypertrophy and ribosome biogenesis.