The Mexican Tetra, Astyanax mexicanus, as a Model System in Cell and Developmental Biology.

Our understanding of cell and developmental biology has been greatly aided by a focus on a small number of model organisms. However, we are now in an era where techniques to investigate gene function can be applied across phyla, allowing scientists to explore the diversity and flexibility of developmental mechanisms and gain a deeper understanding of life. Researchers comparing the eyeless cave-adapted Mexican tetra, Astyanax mexicanus, with its river-dwelling counterpart are revealing how the development of the eyes, pigment, brain, cranium, blood, and digestive system evolves as animals adapt to new environments. Breakthroughs in our understanding of the genetic and developmental basis of regressive and constructive trait evolution have come from A. mexicanus research. They include understanding the types of mutations that alter traits, which cellular and developmental processes they affect, and how they lead to pleiotropy. We review recent progress in the field and highlight areas for future investigations that include evolution of sex differentiation, neural crest development, and metabolic regulation of embryogenesis.

orco Mutagenesis Causes Loss of Antennal Lobe Glomeruli and Impaired Social Behavior in Ants.

Life inside ant colonies is orchestrated with diverse pheromones, but it is not clear how ants perceive these social signals. It has been proposed that pheromone perception in ants evolved via expansions in the numbers of odorant receptors (ORs) and antennal lobe glomeruli. Here, we generate the first mutant lines in the clonal raider ant, Ooceraea biroi, by disrupting orco, a gene required for the function of all ORs. We find that orco mutants exhibit severe deficiencies in social behavior and fitness, suggesting they are unable to perceive pheromones. Surprisingly, unlike in Drosophila melanogaster, orco mutant ants also lack most of the ∼500 antennal lobe glomeruli found in wild-type ants. These results illustrate that ORs are essential for ant social organization and raise the possibility that, similar to mammals, receptor function is required for the development and/or maintenance of the highly complex olfactory processing areas in the ant brain. VIDEO ABSTRACT.

Progressive Loss of Function in a Limb Enhancer during Snake Evolution.

The evolution of body shape is thought to be tightly coupled to changes in regulatory sequences, but specific molecular events associated with major morphological transitions in vertebrates have remained elusive. We identified snake-specific sequence changes within an otherwise highly conserved long-range limb enhancer of Sonic hedgehog (Shh). Transgenic mouse reporter assays revealed that the in vivo activity pattern of the enhancer is conserved across a wide range of vertebrates, including fish, but not in snakes. Genomic substitution of the mouse enhancer with its human or fish ortholog results in normal limb development. In contrast, replacement with snake orthologs caused severe limb reduction. Synthetic restoration of a single transcription factor binding site lost in the snake lineage reinstated full in vivo function to the snake enhancer. Our results demonstrate changes in a regulatory sequence associated with a major body plan transition and highlight the role of enhancers in morphological evolution. PAPERCLIP.

The 3D-Evo Space: Evolution of Gene Expression and Alternative Splicing Regulation.

Animal species present relatively high levels of gene conservation, and yet they display a great variety of cell type and tissue phenotypes. These diverse phenotypes are mainly specified through differential gene usage, which relies on several mechanisms. Two of the most relevant mechanisms are regulated gene transcription, usually referred to as gene expression (rGE), and regulated alternative splicing (rAS). Several works have addressed how either rGE or rAS contributes to phenotypic diversity throughout evolution, but a back-to-back comparison between the two molecular mechanisms, specifically highlighting both their common regulatory principles and unique properties, is still missing. In this review, we propose an innovative framework for the unified comparison between rGE and rAS from different perspectives: the three-dimensional (3D)-evo space. We use the 3D-evo space to comprehensively (a) review the molecular basis of rGE and rAS (i.e., the molecular axis), (b) depict the tissue-specific phenotypes they contribute to (i.e., the tissue axis), and (c) describe the determinants that drive the evolution of rGE and rAS programs (i.e., the evolution axis). Finally, we unify the perspectives emerging from the three axes by discussing general trends and specific examples of rGE and rAS tissue program evolution.

Toward a synthesis of developmental biology with evolutionary theory and ecology.

The evolutionary conservation of developmental mechanisms is a truism in biology, but few attempts have been made to integrate development with evolutionary theory and ecology. To work toward such a synthesis, we summarize studies in the nematode model Pristionchus pacificus, focusing on the development of the dauer, a stress-resistant, alternative larval stage. Integrative approaches combining molecular and genetic principles of development with natural variation and ecological studies in wild populations have identified a key role for a developmental switch mechanism in dauer development and evolution, one that involves the nuclear hormone receptor DAF-12. DAF-12 is a crucial regulator and convergence point for different signaling inputs, and its function is conserved among free-living and parasitic nematodes. Furthermore, DAF-12 is the target of regulatory loops that rely on novel or fast-evolving components to control the intraspecific competition of dauer larvae. We propose developmental switches as paradigms for understanding the integration of development, evolution, and ecology at the molecular level.

Mechanotransduction's impact on animal development, evolution, and tumorigenesis.

Mechanotransduction translates mechanical signals into biochemical signals. It is based on the soft-matter properties of biomolecules or membranes that deform in response to mechanical loads to trigger activation of biochemical reactions. The study of mechanotransductive processes in cell-structure organization has been initiated in vitro in many biological contexts, such as examining cells' response to substrate rigidity increases associated with tumor fibrosis and to blood flow pressure. In vivo, the study of mechanotransduction in regulating physiological processes has focused primarily on the context of embryogenesis, with an increasing number of examples demonstrating its importance for both differentiation and morphogenesis. The conservation across species of mechanical induction in early embryonic patterning now suggests that major animal transitions, such as mesoderm emergence, may have been based on mechanotransduction pathways. In adult animal tissues, permanent stiffness and tissue growth pressure contribute to tumorigenesis and appear to reactivate such conserved embryonic mechanosensitive pathways.

A new dawn for the study of cell type evolution.

Animal evolution is influenced by the emergence of new cell types, yet our understanding of this process remains elusive. This prompts the need for a broader exploration across diverse research organisms, facilitated by recent breakthroughs, such as gene editing tools and single-cell genomics. Essential to our understanding of cell type evolution is the accurate identification of homologous cells. We delve into the significance of considering developmental ontogeny and potential pitfalls when drawing conclusions about cell type homology. Additionally, we highlight recent discoveries in the study of cell type evolution through the application of single-cell transcriptomics and pinpoint areas ripe for further exploration.

Topological analysis of 3D digital ovules identifies cellular patterns associated with ovule shape diversity.

Tissue morphogenesis remains poorly understood. In plants, a central problem is how the 3D cellular architecture of a developing organ contributes to its final shape. We address this question through a comparative analysis of ovule morphogenesis, taking advantage of the diversity in ovule shape across angiosperms. Here, we provide a 3D digital atlas of Cardamine hirsuta ovule development at single cell resolution and compare it with an equivalent atlas of Arabidopsis thaliana. We introduce nerve-based topological analysis as a tool for unbiased detection of differences in cellular architectures and corroborate identified topological differences between two homologous tissues by comparative morphometrics and visual inspection. We find that differences in topology, cell volume variation and tissue growth patterns in the sheet-like integuments and the bulbous chalaza are associated with differences in ovule curvature. In contrast, the radialized conical ovule primordia and nucelli exhibit similar shapes, despite differences in internal cellular topology and tissue growth patterns. Our results support the notion that the structural organization of a tissue is associated with its susceptibility to shape changes during evolutionary shifts in 3D cellular architecture.

Utilizing developmental dynamics for evolutionary prediction and control.

Understanding, predicting, and controlling the phenotypic consequences of genetic and environmental change is essential to many areas of fundamental and applied biology. In evolutionary biology, the generative process of development is a major source of organismal evolvability that constrains or facilitates adaptive change by shaping the distribution of phenotypic variation that selection can act upon. While the complex interactions between genetic and environmental factors during development may appear to make it impossible to infer the consequences of perturbations, the persistent observation that many perturbations result in similar phenotypes indicates that there is a logic to what variation is generated. Here, we show that a general representation of development as a dynamical system can reveal this logic. We build a framework that allows predicting the phenotypic effects of perturbations, and conditions for when the effects of perturbations of different origins are concordant. We find that this concordance is explained by two generic features of development, namely the dynamical dependence of the phenotype on itself and the fact that all perturbations must affect the developmental process to have an effect on the phenotype. We apply our theoretical framework to classical models of development and show that it can be used to predict the evolutionary response to selection using information of plasticity and to accelerate evolution in a desired direction. The framework we introduce provides a way to quantitatively interchange perturbations, opening an avenue of perturbation design to control the generation of variation.

A CUC1/auxin genetic module links cell polarity to patterned tissue growth and leaf shape diversity in crucifer plants.

How tissue-level information encoded by fields of regulatory gene activity is translated into the patterns of cell polarity and growth that generate the diverse shapes of different species remains poorly understood. Here, we investigate this problem in the case of leaf shape differences between Arabidopsis thaliana, which has simple leaves, and its relative Cardamine hirsuta that has complex leaves divided into leaflets. We show that patterned expression of the transcription factor CUP-SHAPED COTYLEDON1 in C. hirsuta (ChCUC1) is a key determinant of leaf shape differences between the two species. Through inducible genetic perturbations, time-lapse imaging of growth, and computational modeling, we find that ChCUC1 provides instructive input into auxin-based leaf margin patterning. This input arises via transcriptional regulation of multiple auxin homeostasis components, including direct activation of WAG kinases that are known to regulate the polarity of PIN-FORMED auxin transporters. Thus, we have uncovered a mechanism that bridges biological scales by linking spatially distributed and species-specific transcription factor expression to cell-level polarity and growth, to shape diverse leaf forms.

Parasitic fish embryos do a "front-flip" on the yolk to resist expulsion from the host.

Embryonic development is often considered shielded from the effects of natural selection, being selected primarily for reliable development. However, embryos sometimes represent virulent parasites, triggering a coevolutionary "arms race" with their host. We have examined embryonic adaptations to a parasitic lifestyle in the bitterling fish. Bitterlings are brood parasites that lay their eggs in the gill chamber of host mussels. Bitterling eggs and embryos have adaptations to resist being flushed out by the mussel. These include a pair of projections from the yolk sac that act as an anchor. Furthermore, bitterling eggs all adopt a head-down position in the mussel gills which further increases their chances of survival. To examine these adaptations in detail, we have studied development in the rosy bitterling (Rhodeus ocellatus) using molecular markers, X-ray tomography, and time-lapse imaging. We describe a suite of developmental adaptations to brood parasitism in this species. We show that the mechanism underlying these adaptions is a modified pattern of blastokinesis-a process unique, among fish, to bitterlings. Tissue movements during blastokinesis cause the embryo to do an extraordinary "front-flip" on the yolk. We suggest that this movement determines the spatial orientation of the other developmental adaptations to parasitism, ensuring that they are optimally positioned to help resist the ejection of the embryo from the mussel. Our study supports the notion that natural selection can drive the evolution of a suite of adaptations, both embryonic and extra-embryonic, via modifications in early development.

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus.

The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.

Modulation of tooth regeneration through opposing responses to Wnt and BMP signals in teleosts.

Most vertebrate species undergo tooth replacement throughout adult life. This process is marked by the shedding of existing teeth and the regeneration of tooth organs. However, little is known about the genetic circuitry regulating tooth replacement. Here, we tested whether fish orthologs of genes known to regulate mammalian hair regeneration have effects on tooth replacement. Using two fish species that demonstrate distinct modes of tooth regeneration, threespine stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio), we found that transgenic overexpression of four different genes changed tooth replacement rates in the direction predicted by a hair regeneration model: Wnt10a and Grem2a increased tooth replacement rate, whereas Bmp6 and Dkk2 strongly inhibited tooth formation. Thus, similar to known roles in hair regeneration, Wnt and BMP signals promote and inhibit regeneration, respectively. Regulation of total tooth number was separable from regulation of replacement rates. RNA sequencing of stickleback dental tissue showed that Bmp6 overexpression resulted in an upregulation of Wnt inhibitors. Together, these data support a model in which different epithelial organs, such as teeth and hair, share genetic circuitry driving organ regeneration.

Diverse branching forms regulated by a core auxin transport mechanism in plants.

Diverse branching forms have evolved multiple times across the tree of life to facilitate resource acquisition and exchange with the environment. In the vascular plant group, the ancestral pattern of branching involves dichotomy of a parent shoot apex to form two new daughter apices. The molecular basis of axillary branching in Arabidopsis is well understood, but few regulators of dichotomous branching are known. Through analyses of dichotomous branching in the lycophyte, Selaginella kraussiana, we identify PIN-mediated auxin transport as an ancestral branch regulator of vascular plants. We show that short-range auxin transport out of the apices promotes dichotomy and that branch dominance is globally coordinated by long-range auxin transport. Uniquely in Selaginella, angle meristems initiate at each dichotomy, and these can develop into rhizophores or branching angle shoots. We show that long-range auxin transport and a transitory drop in PIN expression are involved in angle shoot development. We conclude that PIN-mediated auxin transport is an ancestral mechanism for vascular plant branching that was independently recruited into Selaginella angle shoot development and seed plant axillary branching during evolution.

Frizzled2 receives WntA signaling during butterfly wing pattern formation.

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes. Although the secreted ligand WntA has been shown to instruct the color pattern formation in butterflies, its mode of reception remains elusive. Butterfly genomes encode four homologs of the Frizzled-family of Wnt receptors. Here, we show that CRISPR mosaic knockouts of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss of function in multiple nymphalids. Whereas WntA mosaic clones result in intermediate patterns of reduced size, fz2 clones are cell-autonomous, consistent with a morphogen function. Shifts in expression of WntA and fz2 in WntA crispant pupae show that they are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity in the wing epithelium. Fz3 and Fz4 show phenotypes consistent with Wnt competitive-antagonist functions in vein formation (Fz3 and Fz4), wing margin specification (Fz3), and color patterning in the Discalis and Marginal Band Systems (Fz4). Overall, these data show that the WntA/Frizzled2 morphogen-receptor pair forms a signaling axis that instructs butterfly color patterning and shed light on the functional diversity of insect Frizzled receptors.

A shark-inspired general model of tooth morphogenesis unveils developmental asymmetries in phenotype transitions.

Developmental complexity stemming from the dynamic interplay between genetic and biomechanic factors canalizes the ways genotypes and phenotypes can change in evolution. As a paradigmatic system, we explore how changes in developmental factors generate typical tooth shape transitions. Since tooth development has mainly been researched in mammals, we contribute to a more general understanding by studying the development of tooth diversity in sharks. To this end, we build a general, but realistic, mathematical model of odontogenesis. We show that it reproduces key shark-specific features of tooth development as well as real tooth shape variation in small-spotted catsharks Scyliorhinus canicula. We validate our model by comparison with experiments in vivo. Strikingly, we observe that developmental transitions between tooth shapes tend to be highly degenerate, even for complex phenotypes. We also discover that the sets of developmental parameters involved in tooth shape transitions tend to depend asymmetrically on the direction of that transition. Together, our findings provide a valuable base for furthering our understanding of how developmental changes can lead to both adaptive phenotypic change and trait convergence in complex, phenotypically highly diverse, structures.

GRASSY TILLERS1 (GT1) and SIX-ROWED SPIKE1 (VRS1) homologs share conserved roles in growth repression.

Crop engineering and de novo domestication using gene editing are new frontiers in agriculture. However, outside of well-studied crops and model systems, prioritizing engineering targets remains challenging. Evolution can guide us, revealing genes with deeply conserved roles that have repeatedly been selected in the evolution of plant form. Homologs of the transcription factor genes GRASSY TILLERS1 (GT1) and SIX-ROWED SPIKE1 (VRS1) have repeatedly been targets of selection in domestication and evolution, where they repress growth in many developmental contexts. This suggests a conserved role for these genes in regulating growth repression. To test this, we determined the roles of GT1 and VRS1 homologs in maize (Zea mays) and the distantly related grass brachypodium (Brachypodium distachyon) using gene editing and mutant analysis. In maize, gt1; vrs1-like1 (vrl1) mutants have derepressed growth of floral organs. In addition, gt1; vrl1 mutants bore more ears and more branches, indicating broad roles in growth repression. In brachypodium, Bdgt1; Bdvrl1 mutants have more branches, spikelets, and flowers than wild-type plants, indicating conserved roles for GT1 and VRS1 homologs in growth suppression over ca. 59 My of grass evolution. Importantly, many of these traits influence crop productivity. Notably, maize GT1 can suppress growth in arabidopsis (Arabidopsis thaliana) floral organs, despite ca. 160 My of evolution separating the grasses and arabidopsis. Thus, GT1 and VRS1 maintain their potency as growth regulators across vast timescales and in distinct developmental contexts. This work highlights the power of evolution to inform gene editing in crop improvement.

Deep conservation and co-option of programmed cell death facilitates evolution of alternative phenotypes at multiple biological levels.

Alternative phenotypes, such as polyphenisms and sexual dimorphisms, are widespread in nature and appear at all levels of biological organization, from genes and cells to morphology and behavior. Yet, our understanding of the mechanisms through which alternative phenotypes develop and how they evolve remains understudied. In this review, we explore the association between alternative phenotypes and programmed cell death, a mechanism responsible for the elimination of superfluous cells during development. We discuss the ancient origins and deep conservation of programmed cell death (its function, forms and underlying core regulatory gene networks), and propose that it was co-opted repeatedly to generate alternative phenotypes at the level of cells, tissues, organs, external morphology, and even individuals. We review several examples from across the tree of life to explore the conditions under which programmed cell death is likely to facilitate the evolution of alternative phenotypes.

Sox9 is associated with two distinct patterning events during snake lung morphogenesis.

The evolutionary forces that allowed species adaptation to different terrestrial environments and led to great diversity in body shape and size required acquisition of innovative strategies of pattern formation during organogenesis. An extreme example is the formation of highly elongated viscera in snakes. What developmental patterning strategies allowed to overcome the space constraints of the snake's body to meet physiological demands? Here we show that the corn snake uses a Sox2-Sox9 developmental tool kit common to other species to generate and shape the lung in two phases. Initially Sox9 was found at low levels at the tip of the primary lung bud during outgrowth and elongation of the bronchial bud, without driving branching programs characteristic of mammalian lungs. Later, Sox9 induction is recapitulated in the formation of an extensive network of radial septae emerging along the elongated bronchial bud that generates the respiratory region. We propose that altogether these represent key patterning events for formation of both the respiratory faveolar and non-respiratory posterior compartments of the snake's lung.

TF-High-Evolutionary: In Vivo Mutagenesis of Gene Regulatory Networks for the Study of the Genetics and Evolution of the Drosophila Regulatory Genome.

Understanding the evolutionary potential of mutations in gene regulatory networks is essential to furthering the study of evolution and development. However, in multicellular systems, genetic manipulation of regulatory networks in a targeted and high-throughput way remains challenging. In this study, we designed TF-High-Evolutionary (HighEvo), a transcription factor (TF) fused with a base editor (activation-induced deaminase), to continuously induce germline mutations at TF-binding sites across regulatory networks in Drosophila. Populations of flies expressing TF-HighEvo in their germlines accumulated mutations at rates an order of magnitude higher than natural populations. Importantly, these mutations accumulated around the targeted TF-binding sites across the genome, leading to distinct morphological phenotypes consistent with the developmental roles of the tagged TFs. As such, this TF-HighEvo method allows the interrogation of the mutational space of gene regulatory networks at scale and can serve as a powerful reagent for experimental evolution and genetic screens focused on the regulatory genome.