Biology subjects

Banerjee, T. D.

Publications and source records attributed to Banerjee, T. D..

11 recordsLinked to original sources

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↗

bioRxiv2025

Role of chrna5 in multi-substance preference and phenotypes comorbid with the development of substance dependence

Addiction to nicotine and alcohol continues to be a leading cause of death and loss of productivity as measured in disability-adjusted life years. Polymorphisms in the nicotinic acetylcholine receptor subunit 5 (CHRNA5) have been identified as risk factors associated with nicotine dependence in human genetic studies and rodent models. Whether the chrna5 function is independently relevant to phenotypes associated with disorders comorbid with substance use, and if genetic factors influence subsequent outcomes when exposure to psychoactive substances happens at an early age, are questions of interest. We generated a stable mutant line in zebrafish using the CRISPR-Cas9 technique. We found that the chrna5 mutant fish exhibit an increased acute preference to both nicotine and alcohol in the Self-Administration Zebrafish Assay (SAZA). When subjected to multi-day exposures to either, chrna5 mutants exhibited greater behavioural change, but reduced transcriptomic changes compared to WT siblings, suggesting an impaired homeostatic regulation following drug exposure. Further, chrna5 mutants exhibited drug-independent changes in appetite and circadian rhythms, suggesting a genetic predisposition to disorders often comorbid with substance dependence. We expect these results to give new insights into the operation of genes whose normal function modulates vulnerability to multi-substance use and comorbid disorders.

neuroscience↗

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

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

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↗

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↗

bioRxiv2021

Conserved roles of Distal-less and spalt in regulating butterfly melanic patterns

Two genes, Distal-less (Dll) and spalt (sal), are known to be involved in establishing nymphalid butterfly wing patterns. They function in several ways: in the differentiation of the eyespots central signaling cells, or foci; in the differentiation of the surrounding black disc; in overall scale melanisation (Dll); and in elaborating marginal patterns, such as parafocal elements. However, little is known about the functions of these genes in the development of wing patterns in other butterfly families. Here, we study the expression and function of Dll and sal in the development of spots and other melanic wing patterns of the Indian cabbage white, Pieris canidia, a pierid butterfly. In P. canidia, both Dll and Sal proteins are expressed in the scale-building cells at the wing tips, in chevron patterns along the pupal wing margins, and in areas of future scale melanisation. Additionally, Sal alone is expressed in the future black spots. CRISPR knockouts of Dll and sal showed that each gene is required for the development of melanic wing pattern elements, and repressing pteridine granule formation, in the areas where they are expressed. We conclude that both genes likely play ancestral roles in organising distal butterfly wing patterns, across pierid and nymphalid butterflies, but are unlikely to be differentiating signalling centers in pierids black spots. The genetic and developmental mechanisms that set up the location of spots and eyespots are likely distinct in each lineage.

evolutionary biology↗

bioRxiv2021

optix is involved in eyespot development via a possible positional information mechanism

Novel organismal traits might reuse ancestral gene-regulatory networks (GRNs) in their development, but data supporting this mechanism are still sparse. Here we show the reuse of an ancestral insect venation gene regulatory subnetwork patterning the sharp and distinct rings of color in butterfly eyespots. Using laser microdissection followed by RNA-Seq we first obtained transcriptional profiles of the anterior and posterior compartment of larval wings, and eyespot and adjacent control tissue in pupal wings of Bicyclus anynana butterflies. We identified key venation patterning genes such as Mothers against dpp 6 (Mad6), thickveins, Optix, spalt, optomotor-blind (omb), aristaless, cubitus interruptus, and patched differentially expressed (DE) across compartments, and a sub-set of these genes also DE across eyespot and non-eyespot samples. Fluorescent in-situ hybridization (HCR3.0) on the jointly DE genes Mad6, Optix, and spalt, as well as dpp showed clear eyespot-center, eyespot-rings, and compartment-specific expression. Knocking out dpp resulted in an individual with venation defects and loss of eyespots, whereas knockouts of Optix and spalt resulted in the loss of orange scales and black scales, respectively. Furthermore, using CRISPR-Cas9 followed by immunostainings, we showed that Spalt represses Optix in the central region of the eyespot, limiting Optix expression to a more peripheral ring, which parallels the regulatory interaction found in venation patterning in the anterior compartment of fly larval wings. These network similarities suggest that part of the venation GRN was co-opted to aid in the differentiation of the eyespot rings. One-sentence summaryWe showed the reuse of an ancestral insect wing venation GRN in patterning a novel complex trait in butterflies.

developmental biology↗

bioRxiv2020

Molecular mechanism underlying venation patterning in butterflies

The mechanism of wing vein differentiation in Drosophila is a classic text-book example of pattern formation using a system of positional-information, yet very little is known about how this mechanism differs in species with a different number of veins and how insect venation patterns evolved. Here, we examine the expression patterns of genes previously implicated in vein differentiation in Drosophila in two butterfly species with more complex venation, the African squinting bush brown Bicyclus anynana and the Asian cabbage white, Pieris canidia. We also test the function of one of these genes, spalt (sal), with CRISPR-Cas9 in B. anynana. We identify both conserved as well as new domains of decapentaplegic (dpp), engrailed (en), invected (inv) and sal gene expression in B. anynana, and propose how the simplified venation in Drosophila might have evolved via loss of dpp and sal gene expression domains, silencing of vein inducing programs at Sal-expression boundaries, and changes in gene expression of vein maintenance genes. Summary statementThe paper describes new domains of venation patterning genes in butterflies and proposes how simplified venation in other insect lineages might have evolved.

developmental biology↗