Biology subjects

Monteiro, A.

Publications and source records attributed to Monteiro, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Spatial atlas of the ovary identifies molecular events in primordial follicle activation in humans

The human ovarian reserve is established prenatally, when granulosa cells encapsulate germ cells to form a pool of quiescent primordial follicles, that ultimately determines the female reproductive and endocrine lifespan. From birth to menopause, subsets of these quiescent follicles, located in the thin outer ovarian cortex, are activated and undergo a growth programme with progressive inward migration to the inner cortex, before either undergoing atresia or, during reproductive age, ovulation. Disruption of this process may lead to infertility, metabolic disorders and early menopause, yet early follicle development remains largely poorly understood. Here, we generate the most comprehensive single-cell and spatial multiomics atlas of the human pre- and postnatal ovarian cortex, integrating transcriptomic and chromatin profiles from over four million cells obtained from fetal and newly profiled pediatric and adult donors. We resolve the early granulosa cell trajectory at unprecedented resolution and identify a retinoic acid-associated regulatory switch accompanying follicle activation. We further show that stromal fibroblasts are not homogeneous, but instead form a dynamic scaffold establishing previously unrecognised morphogen and paracrine gradients that organise the cortex into functional niches supporting quiescent, growing, and atretic follicles. Finally, we identify ovarian lipid associated macrophages (oLAMs) that localise around follicles and are likely to support tissue remodelling during folliculogenesis. Together, this atlas provides a foundational blueprint for human ovarian development and homeostasis, and provides a framework for improving strategies in fertility preservation and in vitro follicle maturation.

cell biology↗

bioRxiv2026

Parallels in leg and wing proximal-distal patterning in Holometabola

The wings and legs of insects are both appendages that develop along a proximal-distal (PD) axis and likely share many underlying patterning mechanisms. Comparisons between the two appendages have been detailed in Drosophila melanogaster, a derived insect where both traits develop initially as imaginal discs. Here, we visualise and compare the expression of prominent PD developmental genes in the embryonic legs and larval wings of a lepidopteran species, Bicyclus anynana. We examine the domains of twelve leg gap genes that subdivide legs into distal, medial, or proximal domains, three morphogens, and two genes that refine the PD axis after initial specification. Our results reveal high spatial congruence in the order of PD gene expression between the two appendages. Notably, we observe a distinct loss of the medial domain in the Bicyclus wing compared to the leg, providing evidence for the evolutionary re-patterning of these structures. Comparisons with Drosophila further highlight conserved versus lineage-specific regulatory architectures. These findings suggest a deeply conserved PD patterning logic across Holometabola, while pointing to divergent mechanisms that likely facilitated the morphological innovation of butterfly wings.

developmental biology↗

bioRxiv2026

Modular fluidic automation platform with integrated thermal control for multi-step molecular imaging workflows

Automation of multi-step mRNA imaging protocols increases reproducibility and throughput in spatial biology, as many workflows require repeated buffer exchanges, precise timing, and controlled reaction conditions. Commercial automation platforms can be expensive, proprietary, and difficult to customise, limiting their use in most laboratories. Here, we present two open-source robots for the Rapid Amplified Multiplexed Fluorescent In-Situ Hybridization (RAM-FISH) workflow based on programmable delivery of fluids and integrated thermal control with no dedicated bubble trap requirement. The first robot is designed to perform the steps necessary for signal localization (Multiplexer), and the second performs signal removal (RemBot). Both robots function without manual supervision and conduct precise, repeatable buffer exchanges, temperature regulation, and timed reactions. Both can operate on free-floating and gel-embedded tissues and can be assembled using widely available components. The robots support iterative imaging workflows, enabling detection of multiple genes across sequential hybridization rounds within the same sample. By providing customizable and accessible robots, we lower the technical know-how barriers that need to be overcome to perform complex spatial imaging experiments and enable scalable, hands-free execution of multi-step multiplex-FISH.

bioengineering↗

bioRxiv2026

Intergenerational shifts in innate odour preferences upon odour injections in Bicyclus anynana butterfly larvae

How insects transmit food odour preferences acquired during the larval stage to their offspring is unknown. Bicyclus anynana butterfly larvae can learn to prefer a banana-smelling odour, isoamyl acetate (IAA), via feeding on coated leaves, or simply via haemolymph transfusions from an IAA-fed animal, and transmit this preference to their naive offspring. Here we explore how larvae respond to different concentrations of IAA using olfaction choice tests, and how injections of different concentrations of IAA directly into the haemolymph impact odour learning and transmission of learned preferences. We find that naive larvae showed a slight preference towards low concentrations of IAA, and a slight avoidance towards higher concentrations. Injections of IAA at low concentrations directly into the haemolymph led to an increase in preference for IAA, whereas higher concentrations led to an increase in avoidance. Naive offspring inherited the odour preferences of their parents. Finally, injections of IAA at different concentrations into embryos did not alter choices made by hatched larvae. We establish that the same molecule (IAA) can illicit both a preference as well as an aversive reaction when directly injected into the haemolymph, but IAA is not directly implicated in intergenerational inheritance.

animal behavior and cognition↗

bioRxiv2026

Parallel evolution of industrial melanism in the peppered moth: one locus, many alleles

The extent to which adaptation to environmental change occurs via single or multiple advantageous mutations remains an open question, which we examined by studying the spread of melanic forms of the peppered moth in Britain and continental Europe, in response to industrial coal pollution. In Britain, the darkest melanic form is due to the insertion of a transposable element (carb-TE) into a genomic region called ivory. Here, we characterize the spread of melanic forms in continental Europe from historical records and uncover the genetic basis of European melanism using genomic analyses of modern and museum specimens. We show that European melanism is also associated with variants at ivory, but with multiple alleles featuring structural variants, including several transposable elements, though not carb-TE. The primary central European melanic allele is a genetically dominant 805 base pair deletion (sollichau). Interestingly, melanic individuals with either sollichau (deletion) or carb-TE (insertion) alleles show elevated expression of ivory and its effector micro-RNA (mir-193) compared to non-melanic (typica) individuals, suggesting that contrasting structural variants in ivory have similar regulatory effects. A functional role for sollichau was corroborated, serendipitously, by its presence in a typica individual which also contains a linked deletion of mir-193, which is predicted to cancel the melanizing effect of the sollichau deletion. Our results support the idea that there can be many genetic origins of the same adaptive trait within a species, particularly one with a large effective population size and heterogeneous natural habitat, and when many mutations can give rise to the same phenotype.

evolutionary biology↗

bioRxiv2025

A novel Hox gene promoter fuels the evolution of adaptive phenotypic plasticity

Adaptive phenotypic plasticity allows organisms to display distinct phenotypes in response to variable environments, but little is known about the genomic changes that promote the evolution of plasticity on a macroevolutionary scale. Combining spatial transcriptomics, comparative genomics, and genome editing, we show that temperature-mediated plasticity in the size of eyespot wing patterns in satyrid butterflies, a derived seasonal adaptation evolved [~]80 million years ago in this subfamily, is associated with the taxon-specific evolution of a novel promoter in the Hox gene Antennapedia (Antp). The promoter activates Antp expression specifically in satyrid eyespot center cells, sensitizes these cells to environmental temperatures, and plays a critical role in regulating satyrid eyespot size plasticity. Our study demonstrates that a taxon-specific cis-regulatory innovation in an ancestral developmental gene fueled the evolution of adaptive phenotypic plasticity across a clade of animals.

evolutionary biology↗

bioRxiv2024

Spatial mRNA profiling using Rapid Amplified Multiplexed-FISH (RAM-FISH)

Spatial localization of multiple mRNAs in intact tissues provides vital insights into development and function, yet routine multiplexed imaging remains constrained by complex workflows, specialized equipment costs, or sample preparation requirements. Here, we present Rapid Amplified Multiplexed FISH (RAMFISH), an accessible, modular, benchtop workflow for targeted spatial mRNA profiling in intact tissues and whole-mount specimens. RAMFISH achieves reliable multiplexing of over 30 transcripts through iterative cycles of standard hybridization either manually or via open-source automation and imaging in shared confocal systems. To streamline analysis, we introduce an integrated, open-source pipeline automating signal normalization, rigid and non-rigid alignment, Laplacian of Gaussian spot calling, and 30-channel composite merging. We validate the platform by mapping multiple transcripts in developing Bicyclus anynana butterfly wings and intact 14 days-post-fertilization Danio rerio larvae, resolving both historically inferred domains and newly characterized gene expression. Overall, RAMFISH methodology offers an end-to-end, completely open-source ecosystem for robust, semi-quantitative, spatial mRNA mapping on a variety of sample types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/627193v4_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@14dc9a3org.highwire.dtl.DTLVardef@1f11e3dorg.highwire.dtl.DTLVardef@f85483org.highwire.dtl.DTLVardef@1746701_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Butterfly brains change in morphology and in gene splicing patterns after brief pheromone exposure

How insect brains differ between the sexes and respond to sex-specific pheromones is still not well understood. Here we briefly exposed female Bicyclus anynana butterflies to wild type (Wt) and modified male sex pheromone blends, previously shown to modify females sexual preferences, and examined how their brains were modified at the morphological and molecular levels, three days later. First, we 3D-reconstructed male and female brains of this species and documented sexual dimorphism in the size of seven of 67 glomeruli present in the olfactory lobe. Then we showed that several glomeruli changed in volume after blend exposures, implicating them in sex pheromone perception. Finally, we found that a few genes were differentially expressed but many more were differentially spliced between male and female naive brains, and between naive and pheromone blend-exposed brains. These are primarily calcium-binding channels and RNA-binding genes, respectively. A learned preference for changed levels in a single pheromone component was linked to variants of proteins involved in synaptic transmission. Our work shows that naive male and female brains differ primarily in gene splicing patterns and that a brief, 3-minute, exposure to pheromones produces slight changes in brain volume and large changes in the splicing of genes involved in neural development, that correlate with changes in sexual preferences in females. Significance statementHow brains differ between the sexes and respond to sex-specific cues is a hot research topic. Here we investigate how the brains of female butterflies differ from those of males and respond to male sex pheromones. We find that the sexes differ in the volume of a sub-set of olfactory lobe glomeruli, and the volume of some glomeruli also changes after exposure to pheromone blends. In addition, male and female brains differ primarily in hundreds of splice variants, both before and after pheromone exposure. These findings suggest that different proteins (splice variants) characterize male and female brains and that a brief exposure to pheromones can lead to changes in brain structure and in further gene splicing linked to altered sexual preferences in female butterflies.

animal behavior and cognition↗

bioRxiv2024

Calreticulin is required for cuticle deposition and trabeculae formation inside butterfly wing scale cells

Insect cuticle is normally deposited outside the plasma membrane of epidermal cells, making it unclear how cuticular pillars (trabeculae) are found inside butterfly wing scale cells. By co-labelling the cuticle and the plasma membrane, we found evidence that the plasma membrane invaginates towards the interior of the scale during development, and that chitin pillars form within these invaginations within the cell, but topologically outside it. Furthermore, we found that Calreticulin, a multifunctional protein, is essential for the formation of these trabeculae. This protein was found colocalized with chitin outside the cell membrane, as scales were developing, and its knockout led to loss of chitin pillars, disruption of other scale morphologies, and loss of pigmentation. Our results implicate this multifunctional protein in butterfly wing scale coloration and morphology.

developmental biology↗

bioRxiv2024

chrna3 modulates biphasic response to alcohol

Alcohol use disorders (AUDs) are complex phenomena governed by genetics, neurophysiology, environment, and societal structures. New methods to understand the underlying neurogenetics are valuable for designing personalised interventional strategies. Here, we used a two-choice self-administration zebrafish assay (SAZA) to isolate the function of nicotinic acetylcholine receptor (nAChR) subunit alpha3 (chrna3) in alcohol response. Juvenile zebrafish, prior to sex differentiation, were examined in this study. They exhibited a biphasic response when self-administering alcohol that transitioned from attraction to aversion within minutes, suggesting they can regulate exposure to alcohol. This inverted U-shaped self-administration mirrored the effect alcohol has on shoaling behaviour. Exposure to low concentration of alcohol reduced anxiety-like behaviours, while sedative effects became prominent at higher concentrations resulting in reduced locomotion and uncoordinated swimming. In contrast, these responses are blunted in chrna3 mutants. They exhibited prolonged alcohol self-administration, and increased gregariousness. Transcriptomic analyses suggest that glutamatergic and GABAergic neurotransmission alongside cholinergic signalling is impacted in the mutant brains. Our results thus suggest that chrna3 dysfunction has a systemic change with an increase in alcohol tolerance being one effect. These findings also highlight the use of non-rodent alternatives to understand the neurogenetics of development of AUD.

neuroscience↗

bioRxiv2024

Brain and gonadal genes are differentially expressed in Bicyclus anynana butterfly larvae that learned a heritable novel food odor preference

Bicyclus anynana butterfly larvae learn to prefer novel odors added to their plant food and transmit those learned preferences to the next generation. However, the molecular mechanisms regulating the inheritance of this acquired preference remain unexplored. Here we examined how larval diet affected gene expression patterns in the larval brain as well as the gonads of adults to explore a potential genetic basis of this inheritance. We fed B. anynana larvae leaves coated with a novel banana odor (isoamyl acetate), or with a control solution, and performed five choice assays on individual larvae during their development to identify individuals that showed a majority preference for the treatment odor they were fed with. We then dissected male and female larval brains, adult spermatophores, or adult oocytes from those individuals, and profiled mRNA in all tissues and micro-RNA (miRNA) expression in oocytes only. Our results show that there are 83 differentially expressed genes (DEGs) across all tissue types in odor and control groups, of which 23 play roles in metabolism, transcription, response to various chemical stimuli, and olfactory pathways. MiRNAs did not differ in expression across diet treatments, but we discovered 57 novel miRNAs in oocytes. The DEGs in gonads are potential epigenetic factors that can regulate the inheritance of a learned odor preference. Still, caution is required as there was no overlap between DEGs across male and female brains, as well as male and female gonads.

genomics↗

bioRxiv2024

Lack of Synergistic Outcomes with Roflumilast Combined with Levetiracetam or Adipose Stem Cell Secretome After Spinal Cord Injury

The Spinal Cord Injury (SCI) pathophysiology is highly complex, contributing to a poor prognosis and lack of effective treatments. Previously, we demonstrated that Roflumilast (Rof), leads to functional recovery when tested in a SCI contusion model. However, it is unlikely that Rof treatment on its own could fully restore the spinal cord. Therefore, our objective was to scrutinize the synergistic effects of combining Rof with neuroprotective approaches. Herein we tested two therapies, firstly, Rof combined with Levetiracetam (Lev), and in a second phase, the complementary interplay between Rof and Adipose Stem Cells secretome (Sec). We induced SCI using a weight drop trauma model at the T8 level. Functional recovery was assessed according to the Basso, Beattie, and Bresnahan scale, Activity Box Test, Motor Swimming Test, and Von Frei test. Results indicate that the unilateral use of Rof, Lev, or Sec was effective in promoting functional recovery. However, the combination of Rof + Lev or Rof + Sec did not lead to an improvement in functional outcomes when compared to standalone treatments. Moreover, the combination of Rof + Sec actually led to worst functional outcome than the single treatments. Further studies are needed to find a combinatorial treatment that can lead to superior therapeutic effects with potential clinical application.

neuroscience↗

bioRxiv2024

Ridge and crossrib height of butterfly wing scales is a toolbox for structural color diversity

The brightest and most vivid colors of butterflies usually originate from light reflecting off the cuticular scales that cover the wing membrane. These scales have an intricate architecture that consists of an upper layer, a grid of longitudinal ridges and transverse crossribs, connected to a lower lamina by pillars called trabeculae. Whereas the role of the lower lamina as a reflector has been well documented in simpler scales, this study unveils the role of the scales upper surface in generating or fine-tuning hue, brightness, and saturation. In the nymphalid Bicyclus anynana, we showed that changes in ridge and trabecula heights accompanied changes in hue of scales produced via artificial selection. We further found that this correlation between ridge height and hue can be generalized to 40 scale types from 35 species across butterfly families. By combining focused ion beam milling, microspectrophotometry, and optical modelling, we found that modifying the ridge height is sufficient to change ridge hue, notably in Morpho didius whose blue color was thought to be generated exclusively by lamella protruding from ridges, rather than ridge height. This study identifies the scales upper surface as a toolbox for structural color diversity in butterflies and proposes a geometrical model to predict color that unifies species with and without Morpho-type Christmas-tree ridges.

evolutionary biology↗

bioRxiv2024

A micro-RNA drives a 100-million-year adaptive evolution of melanic patterns in butterflies and moths

In Lepidoptera (butterflies and moths), the genomic region around the gene cortex is a hotspot locus, repeatedly used to generate intraspecific melanic wing color polymorphisms across 100-million-years of evolution. However, the identity of the effector gene regulating melanic wing color within this locus remains unknown. Here, we show that none of the four candidate protein-coding genes within this locus, including cortex, serve as major effectors. Instead, a micro-RNA (miRNA), mir-193, serves as the major effector across three deeply diverged lineages of butterflies, and its function is conserved in Drosophila. In Lepidoptera, mir-193 is derived from a gigantic long non-coding RNA, ivory, and it functions by directly repressing multiple pigmentation genes. We show that a miRNA can drive repeated instances of adaptive evolution in animals.

genetics↗

bioRxiv2024

Eyespots originated multiple times independently across the Lepidoptera

Eyespot colour patterns are an effective defence against predators and are found in many animal lineages. In nymphalid butterflies, eyespots have a single evolutionary origin close to the base of this clade, but eyespots are also present in many other lepidopteran lineages and may have multiple independent origins. Here we use phylogenetic comparative methods to investigate the evolution of eyespots across a multi-superfamily phylogeny of Lepidoptera, and to pinpoint lineages in which eyespots likely originated independently. We find a total of 32 separate origins of Discal eyespots (in the discal wing region) and 21 separate origins of Marginal eyespots (in the marginal wing region). In two instances, eyespots were preserved in most extant representatives of a subsequent species radiation. In one such instance, in the Nymphalidae, ancestral state reconstructions suggest that an ancestor with Marginal eyespots predated the origin of one or more lineages with Discal eyespots, while in the Saturniidae we observed the opposite. We conclude that eyespots do not appear to be homologous across the Lepidoptera. Our phylogenetic inference provides a roadmap for future developmental and functional studies that can address whether Discal and Marginal eyespots share the same gene-regulatory network. This study, therefore, has implications for our understanding of the evolution of serial homologues and of convergent evolution of visual signals in insects.

developmental biology↗

bioRxiv2023

Hemolymph transfusions transfer heritable learned novel odor preferences to naive larvae of Bicyclus anynana butterflies

The mechanisms whereby environmental experiences of parents are transmitted to their offspring to impact their behavior and fitness are poorly understood. Previously, we showed that naive Bicyclus anynana butterfly larvae, whose parents fed on a normal plant feed but coated with a novel odor, inherited a learned preference towards that odor, which had initially elicited avoidance in the naive parents. Here, we performed simple hemolymph transfusions from odor-fed and control-fed larvae to naive larval recipients. We found that larvae injected with hemolymph from odor-fed donors stopped avoiding the novel odor, and their naive offspring preferred the odor more, compared to the offspring of larvae injected with control hemolymph. These results indicate that factors in the hemolymph, potentially the odor molecule itself, play an important role in odor learning and preference transmission across generations. Furthermore, this mechanism of odor preference inheritance, mediated by the hemolymph, bypasses the peripheral odor-sensing mechanisms taking place in the antennae, mouthparts, or legs, and may mediate host plant switching and diversification in the Lepidoptera or more broadly across insects.

animal behavior and cognition↗

bioRxiv2023

The molecular basis of macrochaete diversification highlighted by a single-cell atlas of Bicyclus anynana butterfly pupal forewings

Butterfly wings display a large diversity of cell types, including scale cells of different colors, shapes, and sizes, yet the molecular basis of such diversity is poorly understood. Scales are single-cellular projections homologous to sensory bristles in other arthropods, and different scale types are often found intermingled on the wing, alongside other sensory and epidermal cell types, making the analysis of the complete transcriptomes of each scale type difficult to achieve. To explore scale cell diversity at a transcriptomic level we employed single-cell RNA sequencing of [~]5200 large cells (>6 {micro}m) from 22.5-25-hour male pupal forewings of the butterfly Bicyclus anynana. Using single-cell unsupervised, transcriptome clustering, based on shared and differentially expressed genes, followed by in situ hybridization, immunofluorescence, and CRISPR/Cas9 editing of candidate cell type specific genes, we annotate various cell types on the wing. We identify the molecular identities of non-innervated scale cells at various stages of differentiation, innervated sensory cell types, and pheromone producing glandular cells. We further show that senseless, a zinc finger transcription factor, and HR38, a hormone receptor, determine the identity, size, and color of different scale cell types and are important regulators of scale cell differentiation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/554425v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@10624org.highwire.dtl.DTLVardef@13a4963org.highwire.dtl.DTLVardef@1a83a9borg.highwire.dtl.DTLVardef@19f9d1e_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly eyespots

Wnt signaling is involved in the differentiation of eyespot color patterns on the wings of butterflies, but the identity and spatio-temporal regulation of specific Wnt pathway members remains unclear. Here we explore the localization and function of armadillo/{beta}-catenin dependent (canonical) and armadillo/{beta}- catenin independent (non-canonical) Wnt signaling in eyespot development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all seven wnt ligand and four frizzled receptor transcripts present in the genome of this species, and testing the function of arm and frizzled4 using CRISPR-Cas9. During mid to late larval wing development, Arm protein was present in cells at the center of the future eyespots, the foci, and the wing margin, but wnts expressed on the wing, wnt1 (wingless), wnt6, and wnt10 showed expression only some distance away from the foci, along the wing margin. The receptor frizzled9 was expressed in the wing margin and in finger-like projections leading to the foci during early larval wing development, overlapping in expression with Arm. At the same time, the receptor frizzled4 showed a novel expression pattern, anti-localized with Arm, where it is likely transducing non-canonical Wnt signaling. In the early pupal stage, wnt1 was newly expressed in the foci, as previously shown, along with Arm. In addition, frizzled4 and frizzled9-mediated Wnt signaling is likely repressing the expression of frizzled2, as these receptors have anti-colocalized expression domains. Arm had a conserved expression in three other nymphalid butterflies, and functional knockouts of arm and frizzled4 in B. anynana showed that both genes are essential for the differentiation of eyespots. These results show that distinct Wnt signaling pathways are essential for eyespot development in butterflies and are likely interacting to control their active domains.

developmental biology↗