A Rhodnius prolixus catalytically inactive Calpain protease patterns the insect embryonic dorsal-ventral axis.

The calcium dependent Calpain proteases are modulatory enzymes with important roles in cell cycle control, development and immunity. In the fly model Drosophila melanogaster Calpain A cleaves Cactus/IkappaB and consequently modifies Toll signals during embryonic dorsal-ventral (DV) patterning. Here we explore the role of Calpains in the hemiptera Rhodnius prolixus, an intermediate germband insect where the Bone Morphogenetic Protein (BMP) instead of the Toll pathway plays a major role in DV patterning. Phylogenetic analysis of Calpains in species ranging from Isoptera to Diptera indicates an increase of Calpain sequences in the R. prolixus genome and other hemimetabolous species. One locus encoding each of the CalpC, CalpD and Calp7 families, and seven Calpain A/B loci are present in the R. prolixus genome. Several predicted R. prolixus Calpains display a unique architecture, such as loss of Calcium-binding EF-hand domains and loss of catalytic residues in the active site CysPc domain, yielding catalytically dead Calpains A/B. Knockdown for one of these inactive Calpains results in embryonic DV patterning defects, with expansion of ventral and lateral gene expression domains and consequent failure of germ band elongation. In conclusion, our results reveal that Calpains may exert a conserved function in insect DV patterning, despite the changing role of the Toll and BMP pathways in defining gene expression territories along the insect DV axis.

5th Argentinean Meeting on Evolutionary Biology (RABE V): Report on the "Evo-Devo" Extended Symposium.

Evolutionary developmental biology (Evo-Devo) is flourishing in Latin America, particularly Argentina, where researchers are leveraging this integrative field to unlock the secrets of the region's remarkable biodiversity. A recent symposium held at the 5th Argentinean Meeting on Evolutionary Biology (RABE V) showcased a vibrant Evo-Devo community and the diversity of its research endeavors. The symposium included 3 plenary talks, 3 short talks, and 12 posters, and spanned a range of organisms and approaches. Interestingly, the symposium highlighted a prevalence of "top-down" Evo-Devo studies in the region, where researchers first analyze existing diversity and then propose potential developmental mechanisms. This approach, driven in part by financial constraints and the region's historical focus on natural history, presents a unique opportunity to bridge disciplines like comparative biology, paleontology, and botany. The symposium's success underscores the vital role of Evo-Devo in Latin America, not only for advancing our understanding of evolution but also for providing valuable tools to conserve and manage the region's irreplaceable biodiversity. As Evo-Devo continues to grow in Latin America, fostering collaboration and knowledge exchange within the region and beyond will be crucial for realizing the full potential of this transformative field.

Musculoskeletal and tendinous details of selected anomalies in the locomotor system of anurans.

Previous studies on anuran anomalies predominantly examine isolated cases or focus on external and skeletal features. Our study analyzes a comprehensive sample collected from 1991 to 2017, examining the muscle-tendon system in 24 anuran species across adult, juvenile, and metamorphic stages. This extensive sample size allows us to investigate consistent anomaly patterns across different developmental stages and anuran families, exploring potential common developmental or genetic factors. Our detailed anatomical examination, encompassing musculature, tendons, and skeletal structures, revealed that 21% of the specimens displayed anomalies, a noteworthy finding considering the extensive sample size and duration of the studied sample. Of these anomalies, 17% affected the locomotor system, predominantly in the upper limbs. Key anomalies included, forelimbs and hindlimbs brachydactyly, rotation in forelimbs, partial kyphotic lordosis, and scoliosis. Notably, the digit 4 in the forelimbs and digits 4 and 5 in the hindlimbs were particularly susceptible to teratogenic effects, indicating possible prolonged exposure during development. Our study also uncovered combinations of anomalies and identified a phenotype similar to Poland syndrome. The findings validate the "Logic of Monsters" (LoMo theory) by Alberch, although the name itself may not be deemed appropriate, showing that developmental disruptions in tetrapods are not random but follow distinct sequences and patterns. The name, while unfortunate, accurately reflects the unusual nature of these developmental anomalies. This contributes to the evolving "Evo-Devo-Path" framework, highlighting the study's importance in understanding developmental disruptions in tetrapods.

Jaw size variation is associated with a novel craniofacial function for galanin receptor 2 in an adaptive radiation of pupfishes.

Understanding the genetic basis of novel adaptations in new species is a fundamental question in biology. Here we demonstrate a new role for galr2 in vertebrate craniofacial development using an adaptive radiation of trophic specialist pupfishes endemic to San Salvador Island, Bahamas. We confirmed the loss of a putative Sry transcription factor binding site upstream of galr2 in scale-eating pupfish and found significant spatial differences in galr2 expression among pupfish species in Meckel's cartilage using in situ hybridization chain reaction (HCR). We then experimentally demonstrated a novel role for Galr2 in craniofacial development by exposing embryos to Garl2-inhibiting drugs. Galr2-inhibition reduced Meckel's cartilage length and increased chondrocyte density in both trophic specialists but not in the generalist genetic background. We propose a mechanism for jaw elongation in scale-eaters based on the reduced expression of galr2 due to the loss of a putative Sry binding site. Fewer Galr2 receptors in the scale-eater Meckel's cartilage may result in their enlarged jaw lengths as adults by limiting opportunities for a circulating Galr2 agonist to bind to these receptors during development. Our findings illustrate the growing utility of linking candidate adaptive SNPs in non-model systems with highly divergent phenotypes to novel vertebrate gene functions.

Spatio-temporal expression of candidate genes for nectar spur development in Tropaeolum (Tropaeolaceae: Brassicales).

BACKGROUND AND AIMS: Tropaeolaceae (Brassicales) comprise ~100 species native to South and Central America. Tropaeolaceae flowers have a nectar spur, formed by a late expansion and evagination of the fused proximal region of the perianth (i.e. the floral tube). This spur is formed in the domain of the tube oriented towards the inflorescence axis, which corresponds to the adaxial floral region. However, little is known about the molecular mechanisms responsible for the evolution of spurs in Tropaeolaceae. METHODS: In this study, we examined the spatio-temporal expression of genes putatively responsible for differential patterns of cell division between the adaxial and abaxial floral regions in Tropaeolaceae. These genes include previously identified TCP and KNOX transcription factors and the cell division marker HISTONE H4 (HIS4). KEY RESULTS: We found a TCP4 homologue concomitantly expressed with spur initiation and elaboration. Tropaeolaceae possess two TCP4-like (TCP4L) copies, as a result of a Tropaeolaceae-specific duplication. The two copies (TCP4L1 and TCP4L2) in Tropaeolum longifolium show overlapping expression in the epidermis of reproductive apices (inflorescence meristems) and young floral buds, but only TlTCP4L2 shows differential expression in the floral tube at early stages of spur formation, restricted to the adaxial region. This adaxial expression of TlTCP4L2 overlaps with the expression of TlHIS4. Later in development, only TlTCP4L2 is expressed in the nectariferous tissue of the spur. CONCLUSIONS: Based on these results, we hypothesize that Tropaeolaceae TCP4L genes had a plesiomorphic role in epidermal development and that, after gene duplication, TCP4L2 acquired a new function in spur initiation and elaboration. To better understand spur evolution in Tropaeolaceae, it is critical to expand developmental genetic studies to their sister group, the Akaniaceae, which possess simultaneously an independent duplication of TCP4L genes and a spurless floral tube.

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.

The ring-legged earwig Euborellia annulipes as a new model for oogenesis and development studies in insects.

Insects are the dominant group of animals on Earth. Despite this abundance, most of our knowledge about many aspects of their biology and development come from a unique model, the vinegar fly, Drosophila melanogaster. Nevertheless, in the last years, the advances in molecular tools and imaging techniques have allowed the emergence of new insect models, adding valuable information to decipher the morphogenetic bases behind the formation and evolution of the vast diversity of shapes, sizes, and patterns that characterize them. Earwigs belong to Dermaptera which is a small order clustered in the Polyneopteran group. They are hemimetabolous insects with a flattened body, characteristic abdominal pincers, and maternal care behavior. This last feature and their role in agroecosystems have been studied in cosmopolitan species such as Forficula auricularia and Euborellia annulipes; however, their reproduction and embryonic development have been poorly addressed in laboratory conditions. In response, here we describe the ring-legged earwig Euborellia annulipes embryogenesis and life cycle from nymphal to adult stages, its reproduction, and essential morphological and behavioral characters. Additionally, using confocal and transmission electron microscopy we analyzed in detail the morphogenesis of its peculiar meroistic polytrophic ovary. Our aim is to provide an emerging model system to perform comparative studies on insect oogenesis, development, and morphological evolution.

Predicted landscape of RETINOBLASTOMA-RELATED LxCxE-mediated interactions across the Chloroplastida.

The colonization of land by a single streptophyte algae lineage some 450 million years ago has been linked to multiple key innovations such as three-dimensional growth, alternation of generations, the presence of stomata, as well as innovations inherent to the birth of major plant lineages, such as the origins of vascular tissues, roots, seeds and flowers. Multicellularity, which evolved multiple times in the Chloroplastida coupled with precise spatiotemporal control of proliferation and differentiation were instrumental for the evolution of these traits. RETINOBLASTOMA-RELATED (RBR), the plant homolog of the metazoan Retinoblastoma protein (pRB), is a highly conserved and multifunctional core cell cycle regulator that has been implicated in the evolution of multicellularity in the green lineage as well as in plant multicellularity-related processes such as proliferation, differentiation, stem cell regulation and asymmetric cell division. RBR fulfills these roles through context-specific protein-protein interactions with proteins containing the Leu-x-Cys-x-Glu (LxCxE) short-linear motif (SLiM); however, how RBR-LxCxE interactions have changed throughout major innovations in the Viridiplantae kingdom is a question that remains unexplored. Here, we employ an in silico evo-devo approach to predict and analyze potential RBR-LxCxE interactions in different representative species of key Chloroplastida lineages, providing a valuable resource for deciphering RBR-LxCxE multiple functions. Furthermore, our analyses suggest that RBR-LxCxE interactions are an important component of RBR functions and that interactions with chromatin modifiers/remodelers, DNA replication and repair machinery are highly conserved throughout the Viridiplantae, while LxCxE interactions with transcriptional regulators likely diversified throughout the water-to-land transition.

Genome of the Rio Pearlfish (Nematolebias whitei), a bi-annual killifish model for Eco-Evo-Devo in extreme environments.

The Rio Pearlfish, Nematolebias whitei, is a bi-annual killifish species inhabiting seasonal pools in the Rio de Janeiro region of Brazil that dry twice per year. Embryos enter dormant diapause stages in the soil, waiting for the inundation of the habitat which triggers hatching and commencement of a new life cycle. Rio Pearlfish represents a convergent, independent origin of annualism from other emerging killifish model species. While some transcriptomic datasets are available for Rio Pearlfish, thus far, a sequenced genome has been unavailable. Here, we present a high quality, 1.2 Gb chromosome-level genome assembly, genome annotations, and a comparative genomic investigation of the Rio Pearlfish as representative of a vertebrate clade that evolved environmentally cued hatching. We show conservation of 3D genome structure across teleost fish evolution, developmental stages, tissues, and cell types. Our analysis of mobile DNA shows that Rio Pearlfish, like other annual killifishes, possesses an expanded transposable element profile with implications for rapid aging and adaptation to harsh conditions. We use the Rio Pearlfish genome to identify its hatching enzyme gene repertoire and the location of the hatching gland, a key first step in understanding the developmental genetic control of hatching. The Rio Pearlfish genome expands the comparative genomic toolkit available to study convergent origins of seasonal life histories, diapause, and rapid aging phenotypes. We present the first set of genomic resources for this emerging model organism, critical for future functional genetic, and multiomic explorations of "Eco-Evo-Devo" phenotypes of resilience and adaptation to extreme environments.

Growth Patterns in Seedling Roots of the Pincushion Cactus Mammillaria Reveal Trends of Intra- and Inter-Specific Variation.

Genetic mechanisms controlling root development are well-understood in plant model species, and emerging frontier research is currently dissecting how some of these mechanisms control root development in cacti. Here we show the patterns of root architecture development in a gradient of divergent lineages, from populations to species in Mammillaria. First, we show the patterns of variation in natural variants of the species Mammillaria haageana. Then we compare this variation to closely related species within the Series Supertexta in Mammillaria (diverging for the last 2.1 million years) in which M. haageana is inserted. Finally, we compared these patterns of variation to what is found in a set of Mammillaria species belonging to different Series (diverging for the last 8 million years). When plants were grown in controlled environments, we found that the variation in root architecture observed at the intra-specific level, partially recapitulates the variation observed at the inter-specific level. These phenotypic outcomes at different evolutionary time-scales can be interpreted as macroevolution being the cumulative outcome of microevolutionary phenotypic divergence, such as the one observed in Mammillaria accessions and species.

Eye morphogenesis in the blind Mexican cavefish.

The morphogenesis of the vertebrate eye consists of a complex choreography of cell movements, tightly coupled to axial regionalization and cell type specification processes. Disturbances in these events can lead to developmental defects and blindness. Here, we have deciphered the sequence of defective events leading to coloboma in the embryonic eye of the blind cavefish of the species Astyanax mexicanus. Using comparative live imaging on targeted enhancer-trap Zic1:hsp70:GFP reporter lines of both the normal, river-dwelling morph and the cave morph of the species, we identified defects in migratory cell behaviours during evagination that participate in the reduced optic vesicle size in cavefish, without proliferation defect. Further, impaired optic cup invagination shifts the relative position of the lens and contributes to coloboma in cavefish. Based on these results, we propose a developmental scenario to explain the cavefish phenotype and discuss developmental constraints to morphological evolution. The cavefish eye appears as an outstanding natural mutant model to study molecular and cellular processes involved in optic region morphogenesis.

Blurring the Boundaries between a Branch and a Flower: Potential Developmental Venues in CACTACEAE.

Flowers are defined as short shoots that carry reproductive organs. In Cactaceae, this term acquires another meaning, since the flower is interpreted as a branch with a perianth at the tip, with all reproductive organs embedded within the branch, thus giving way to a structure that has been called a "flower shoot". These organs have long attracted the attention of botanists and cactologists; however, the understanding of the morphogenetic processes during the development of these structures is far from clear. In this review, we present and discuss some classic flower concepts used to define floral structures in Cactaceae in the context of current advances in flower developmental genetics and evolution. Finally, we propose several hypotheses to explain the origin of these floral shoot structures in cacti, and we suggest future research approaches and methods that could be used to fill the gaps in our knowledge regarding the ontogenetic origin of the "flower" in the cactus family.

Pescoids and Chimeras to Probe Early Evo-Devo in the Fish Astyanax mexicanus.

The fish species Astyanax mexicanus with its sighted and blind eco-morphotypes has become an original model to challenge vertebrate developmental evolution. Recently, we demonstrated that phenotypic evolution can be impacted by early developmental events starting from the production of oocytes in the fish ovaries. A. mexicanus offers an amenable model to test the influence of maternal determinants on cell fate decisions during early development, yet the mechanisms by which the information contained in the eggs is translated into specific developmental programs remain obscure due to the lack of specific tools in this emergent model. Here we describe methods for the generation of pescoids from yolkless-blastoderm explants to test the influence of embryonic and extraembryonic tissues on cell fate decisions, as well as the production of chimeric embryos obtained by intermorph cell transplantations to probe cell autonomous or non-autonomous processes. We show that Astyanax pescoids have the potential to recapitulate the main ontogenetic events observed in intact embryos, including the internalization of mesodermal progenitors and eye development, as followed with zic:GFP reporter lines. In addition, intermorph cell grafts resulted in proper integration of exogenous cells into the embryonic tissues, with lineages becoming more restricted from mid-blastula to gastrula. The implementation of these approaches in A. mexicanus will bring new light on the cascades of events, from the maternal pre-patterning of the early embryo to the evolution of brain regionalization.

Molecular mechanisms involved in functional macroevolution of plant transcription factors.

Transcription factors (TFs) are key components of the transcriptional regulation machinery. In plants, they accompanied the evolution from unicellular aquatic algae to complex flowering plants that dominate the land environment. The adaptations of the body plan and physiological responses required changes in the biological functions of TFs. Some ancestral gene regulatory networks are highly conserved, while others evolved more recently and only exist in particular lineages. The recent emergence of novel model organisms provided the opportunity for comparative studies, producing new insights to infer these evolutionary trajectories. In this review, we comprehensively revisit the recent literature on TFs of nonseed plants and algae, focusing on the molecular mechanisms driving their functional evolution. We discuss the particular contribution of changes in DNA-binding specificity, protein-protein interactions and cis-regulatory elements to gene regulatory networks. Current advances have shown that these evolutionary processes were shaped by changes in TF expression pattern, not through great innovation in TF protein sequences. We propose that the role of TFs associated with environmental and developmental regulation was unevenly conserved during land plant evolution.

From Devo to Evo: patterning, fusion and evolution of the zebrafish terminal vertebra.

BACKGROUND: With more than 30,000 species, teleosts comprise about half of today's living vertebrates, enriched with a wide set of adaptations to all aquatic systems. Their evolution was marked by modifications of their tail, that involved major rearrangements of the metameric organization of the axial skeleton. The most posterior or ural caudal skeleton, primitively included more than 10 vertebrae and, through a series of fusions and losses, became reduced to a single vertebra in modern ostariophysans, one of the largest clades of teleosts. The ontogeny of the ostariophysan Danio rerio recapitulates this process by forming two or three separate vertebrae that become a single vertebra in adults. We characterize the developmental sequence of this change by describing the processes of patterning, fusion and differential growth on each of the constitutive elements that sculpt the adult terminal vertebra. RESULTS: The ontogenetic changes of the terminal vertebra were characterized, highlighting their shared and derived characters in comparison with other teleosts. In zebrafish, there is: i) a loss of the preural centrum 1, ii) the formation of an hourglass-shaped autocentrum only in the anterior but not the posterior border of the compound centrum, iii) the formation of a vestigial posterior centrum that does not form an autocentrum and becomes incorporated beneath the compound centrum during development, and iv) the elongated dorso-posterior process of the compound centrum or pleurostyle appears as an independent element posterior to the compound centrum, before fusing to the ural neural arches and the anterior portion of the compound centrum. CONCLUSIONS: The unique features of the formation of the terminal vertebra in Danio rerio reflect the remarkable changes that occurred during the evolution of teleosts, with potential shared derived characteristics for some of the major lineages of modern teleosts. A new ontogenetic model is proposed to illustrate the development of the terminal vertebra, and the phylogenetic implications for the evolution of caudal skeleton consolidation in ostariophysans are discussed.

Diversity, distribution, development, and evolution of medullary bundles in Nyctaginaceae.

PREMISE: Medullary bundles, i.e., vascular units in the pith, have evolved multiple times in vascular plants. However, no study has ever explored their anatomical diversity and evolution within a phylogenetic framework. Here, we investigated the development of the primary vascular system within Nyctaginaceae showing how medullary bundles diversified within the family. METHODS: Development of 62 species from 25 of the 31 genera of Nyctaginaceae in stem samples was thoroughly studied with light microscopy and micro-computed tomography. Ancestral states were reconstructed using a maximum likelihood approach. RESULTS: Two subtypes of eusteles were found, the regular eustele, lacking medullary bundles, observed exclusively in representatives of Leucastereae, and the polycyclic eustele, containing medullary bundles, found in all the remaining taxa. Medullary bundles had the same origin and development, but the organization was variable and independent of phyllotaxy. Within the polycyclic eustele, medullary bundles developed first, followed by the formation of a continuous concentric procambium, which forms a ring of vascular bundles enclosing the initially formed medullary bundles. The regular eustele emerged as a synapomorphy of Leucastereae, while the medullary bundles were shown to be a symplesiomorphy for Nyctaginaceae. CONCLUSIONS: Medullary bundles in Nyctaginaceae developed by a single shared pathway, that involved the departure of vascular traces from lateral organs toward the pith. These medullary bundles were encircled by a continuous concentric procambium that also constituted the polycyclic eustele, which was likely a symplesiomorphy for Nyctaginaceae with one single reversion to the regular eustele.

Copulatory function and development shape modular architecture of genitalia differently in males and females.

Genitalia are multitasking structures whose development is mediated by numerous regulatory pathways. This multifactorial nature provides an avenue for multiple sources of selection. As a result, genitalia tend to evolve as modular systems comprising semi-independent subsets of structures, yet the processes that give rise to those patterns are still poorly understood. Here, we ask what are the relative roles of development and function in shaping modular patterns of genitalia within populations and across species of stink-bugs. We found that male genitalia are less integrated, more modular, and primarily shaped by functional demands. In contrast, females show higher integration, lower modularity, and a predominant role of developmental processes. Further, interactions among parts of each sex are more determinant to modularity than those between the sexes, and patterns of modularity are equivalent between and within species. Our results strongly indicate that genitalia have been subjected to sex-specific selection, although male and female genitalia are homologous and functionally associated. Moreover, modular patterns are seemingly constant in the evolutionary history of stink-bugs, suggesting a scenario of multivariate stabilizing selection within each sex. Our study demonstrates that interactions among genital parts of the same sex may be more fundamental to genital evolution than previously thought.

The uropygial gland of the monk parakeet Myiopsitta monachus: Histology, morphogenesis, and evolution within Psittaciformes (Aves).

We describe the morphology, histology, and histochemical characteristics of the uropygial gland (UG) of the monk parakeet Myiopsitta monachus. The UG has a heart-shape external appearance and adenomers extensively branched with a convoluted path, covered by a stratified epithelium formed by different cellular strata and divided into three zones (based on the epithelial height and lumen width), a cylindrical papilla with an internal structure of delicate type and two excretory pores surrounded by a feather tuft. Histochemical and lectin-histochemical techniques performed showed positivity against PAS, AB pH 2.5, AB-PAS, and some lectines, likely related to the granivorous feeding habits. Also, we describe the morphogenesis of the UG of the monk parakeet, which appears at embryological stage 34 as a pair of ectodermal invaginations. Heterochronic events in the onset development of the UG when compared with other birds could be recognized. Finally, to examine the phylogenetic occurrence of the UG within the Psittaciformes and infer its evolutionary history, we mapped its presence/absence over a molecular phylogeny. The reconstruction of the characters states at ancestral nodes revealed that the presence of the UG was the plesiomorphic feature for Psittaciformes and its loss evolved independently more than once.