Biology subjects

Loh, L. S.

Publications and source records attributed to Loh, L. S..

10 recordsLinked to original sources

bioRxiv2026

The gene ivory:mir-193 controls scale type differentiation in Heliconius butterflies

The ivory:mir-193 locus is a genetic hotspot underlying melanic wing pattern variation across Lepidoptera, acting as a master regulator of melanic scale fate. This includes the genus Heliconius, where aposematic mimicry is driven by three scale cell types: light Type I, dark-melanic Type II, and red Type III scales. We tested functions of ivory:mir-193 in Heliconius using CRISPR-induced somatic mosaic knockouts of ivory and mir-193 across multiple pattern morphs. Knockouts converted Type II scales to Type I scales. Effects on Type III scales varied among and within individuals, indicating that ivory:mir-193 is permissive for Type III development. Using single-nucleus RNA-seq, we profiled the transcriptional landscape of H. melpomene-{Delta}78k mutants lacking mir-193. Loss of mir-193 produced readthrough transcription, consistent with a model where miR-193 acts as a co-transcriptional terminator. These data show that ivory drives wing pattern variation, while mir-193 mediates downstream diversity in scale fate across Lepidoptera.

evolutionary biology↗

bioRxiv2024

Genetic basis of an adaptive polymorphism controlling butterfly silver iridescence

Identifying the genes and mutations that drive phenotypic variation and which are subject to selection is crucial for understanding evolutionary processes. Mormon Fritillary butterflies (Speyeria mormonia) exhibit a striking wing color polymorphism throughout their range: typical morphs bear silver spots on their ventral surfaces, and can co-occur with unsilvered morphs displaying a dull coloration1. Through genome-wide association studies in two polymorphic populations, we fine-map this difference in silvering to the 3 region of the transcription factor gene optix. The expression of optix is confined to the unsilvered regions that surround the spots, and these patterns are transformed to a silver identity upon optix RNAi knockdown, implicating optix as a repressor of silver scales in this butterfly. We show that the unsilvered optix haplotype shows signatures of recent selective sweeps, and that this allele is shared with the monomorphic, unsilvered species Speyeria hydaspe, suggesting that introgressions facilitate the exchange of variants of adaptive potential across species. Remarkably, these findings parallel the role of introgressions and cis-regulatory modulation of optix in shaping the aposematic red patterns of Heliconius butterflies2-7, a lineage that separated from Speyeria 45 million years ago8. The genetic basis of adaptive variation can thus be more predictable than often presumed, even for traits that appear divergent across large evolutionary distances. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=199 SRC="FIGDIR/small/628425v1_ufig1.gif" ALT="Figure 1"> View larger version (112K): org.highwire.dtl.DTLVardef@5d2a9borg.highwire.dtl.DTLVardef@982c74org.highwire.dtl.DTLVardef@8e90b3org.highwire.dtl.DTLVardef@1bddf3c_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

evolutionary biology↗

bioRxiv2024

Single-nucleus transcriptomics of wing sexual dimorphism and scale cell specialization in sulphur butterflies

The evolution of sexual secondary characteristics necessitates regulatory factors that confer sexual identity to differentiating tissues and cells. In Colias eurytheme butterflies, males exhibit two specialized wing scale types -- UV-iridescent (UVI) and lanceolate scales -- absent in females and likely integral to male courtship behavior. This study investigates the regulatory mechanisms and single-nucleus transcriptomics underlying these two sexually dimorphic cell types during wing development. We show that Doublesex (Dsx) expression is itself dimorphic and required to repress the UVI cell state in females, while unexpectedly, UVI activation in males is independent from Dsx. In the melanic marginal band, Dsx is required in each sex to enforce the presence of lanceolate scales in males, and their absence in females. Single-nucleus RNAseq reveals that UV-iridescent and lanceolate scale cell precursors each show distinctive gene expression profiles at 40% of pupal development, with marker genes that include regulators of transcription, cell signaling, cytoskeletal patterning, and chitin secretion. Both male-specific cell types share a low expression of the Bric-a-brac (Bab) transcription factor, a key repressor of the UVI fate. Bab ChIP-seq profiling suggests that Bab binds the cis-regulatory regions of gene markers associated to UVI fate, including potential effector genes involved in the regulation of cytoskeletal processes and chitin secretion, and loci showing signatures of recent selective sweeps in an UVI-polymorphic population. These findings open new avenues for exploring wing patterning and scale development, shedding light on the mechanisms driving the specification of sex-specific cell states and the differentiation of specialized cell ultrastructures.

developmental biology↗

bioRxiv2024

Lepidopteran scale cells derive from sensory organ precursors through a canonical lineage

The success of butterflies and moths is tightly linked to the origin of scales within the group. A long-standing hypothesis postulates that scales are homologous to the well-described mechanosensory bristles found in the fruit fly Drosophila melanogaster, where both derive from an epithelial precursor specified by lateral inhibition that then undergoes multiple rounds of division. Previous histological examination and candidate gene approaches identified parallels in genes involved in scale and bristle development. Here, we provide definitive developmental and transcriptomic evidence that the differentiation of lepidopteran scales derives from the canonical cell lineage, known as the Sensory Organ Precursor (SOP). Live imaging in moth and butterfly pupae shows that SOP cells undergo two rounds of asymmetric divisions that first abrogate the neurogenic lineage, and then lead to a differentiated scale precursor and its associated socket cell. Single-nucleus RNA sequencing across a time-series of early pupal development revealed differential gene expression patterns that mirror canonical lineage development, including Notch-Delta signalling components, cell adhesion molecules, cell cycling factors, and terminal cell differentiation markers, suggesting a shared origin of the SOP developmental program. Additionally, we recovered a novel gene, the POU-domain transcription factor pdm3, involved in the proper differentiation of butterfly wing scales. Altogether, these data open up avenues for understanding scale type specification and development, and illustrate how single-cell transcriptomics provide a powerful platform for understanding the evolution of cell types.

developmental biology↗

bioRxiv2024

A long non-coding RNA at the cortex locus controls adaptive colouration in butterflies

Evolutionary variation in the wing pigmentation of butterflies and moths offers striking examples of adaptation by crypsis and mimicry. The cortex locus has been independently mapped as the locus controlling colour polymorphisms in 14 lepidopteran species, suggesting it acts as a genomic hotspot for the diversification of wing patterns, but functional validation through protein-coding knockouts has proven difficult to obtain. Our study unveils the role of a novel long non-coding RNA (lncRNA) which we name ivory, transcribed from the cortex locus, in modulating colour patterning in butterflies. Strikingly, ivory expression prefigures most melanic patterns during pupal development, suggesting an early developmental role in specifying scale identity. To test this, we generated CRISPR mosaic knock-outs in five nymphalid butterfly species and show that ivory mutagenesis yields transformations of dark pigmented scales into white or light-coloured scales. Genotyping of Vanessa cardui germline mutants associates these phenotypes to small on-target deletions at the conserved first exon of ivory. In contrast, cortex germline mutant butterflies with confirmed null alleles lack any wing phenotype, and exclude a colour patterning role for this adjacent gene. Overall, these results show that a lncRNA acts as a master switch of colour pattern specification, and played key roles in the adaptive diversification of colour patterns in butterflies. Significance statementDeciphering the genetic underpinnings of adaptive variation is fundamental for a comprehensive understanding of evolutionary processes. Long non-coding RNAs (lncRNAs) represent an emerging category of genetic modulators within the genome, yet they have been overlooked as a source of phenotypic diversity. In this study, we unveil the pivotal role of a lncRNA in orchestrating colour transitions between dark and light patterns during butterfly wing development. Remarkably, this lncRNA gene is nested within the cortex locus, a genetic region known to control multiple cases of adaptive variation in butterflies and moths, including iconic examples of natural selection. These findings highlight the significant influence of lncRNAs in developmental regulation, and also underscore their potential as key genetic players in the evolutionary process itself.

evolutionary biology↗

bioRxiv2023

Butterfly wing iridescence is regulated by araucan, a direct target of optix and spalt

Butterfly wings exhibit a remarkable diversity of structural iridescent colors, yet the genetic regulation of iridescence remains poorly understood. Here, we show that the Iroquois-complex transcription factor gene araucan plays a role in modulating wing scale iridescence in the common buckeye butterfly, Junonia coenia. Using CRISPR-Cas9 knockouts, we demonstrate that loss of araucan function causes dorsal wing scales to shift from golden-brown to blue iridescence, and eyespot center scales to shift from saturated purple-violet to dull grey-brown. These phenotypes are associated with changes in thickness of the lower lamina in scale cells, a structural feature known to influence thin-film interference, along with reduction of pigmentation in ground scales. Together, these results identify araucan as a single transcription factor that determines coloration by simultaneously regulating both photonic architecture and light-absorbing pigmentation, and show that these effects can be regulated in pattern-specific manner. Our findings provide a framework for linking gene regulation to the spatially coordinated evolution of structural and pigmentary components of color.

genetics↗

bioRxiv2023

Frizzled2 receives the WntA morphogen during butterfly wing pattern formation

Butterfly color patterns provide visible and biodiverse phenotypic readouts of the patterning processes that occur in a developing epithelium. While the secreted ligand WntA was shown to instruct the color pattern formation in butterflies, its modes of reception and signal transduction remain elusive. Butterfly genomes encode four homologues of the Frizzled-family of Wnt receptors. Here we show that CRISPR mosaic knock-outs of frizzled2 (fz2) phenocopy the color pattern effects of WntA loss-of-function in multiple nymphalids. While WntA mosaic clones result in intermediate patterns of reduced size, consistently with a morphogen function, fz2 clones are cell-autonomous. Shifts in pupal expression in WntA crispants show that WntA and fz2 are under positive and negative feedback, respectively. Fz1 is required for Wnt-independent planar cell polarity (PCP) 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.

developmental biology↗

bioRxiv2023

Genetics of continuous colour variation in a pair of sympatric sulphur butterflies

Continuous colour polymorphisms can serve as a tractable model for the genetic and developmental architecture of traits, but identification of the causative genetic loci is complex due to the number of individuals needed, and the challenges of scoring continuously varying traits. Here we investigated continuous colour variation in Colias eurytheme and C. philodice, two sister species of sulphur butterflies that hybridise in sympatry. Using Quantitative Trait Locus (QTL) analysis of 483 individuals from interspecific crosses and an high-throughput method of colour quantification, we found that two interacting large effect loci explain around 70% of the heritable variation in orange-to-yellow chromaticity. Knockouts of red Malphighian tubules (red), a candidate gene at the primary QTL likely involved in endosomal maturation, resulted in depigmented wing scales showing disorganised pterin granules. The Z sex chromosome contains a large secondary colour QTL that includes the transcription factor bric-a-brac (bab), which we show can act as a modulator of orange pigmentation in addition to its previously-described role in specifying UV-iridescence. We also describe the QTL architecture of other continuously varying traits, and that wing size maps to the Z chromosome, supporting a Large-X effect model where the genetic control of species-defining traits is enriched on sex chromosomes. This study sheds light on the genetic architecture of a continuously varying trait, and illustrates the power of using automated measurement to score phenotypes that are not always conspicuous to the human eye. ForewordThe colour phenotypes in this article involve nuanced gradations of yellow and orange that may be difficult to perceive for people who are colour vision deficient. Hue-shifted versions of all main figures are accessible online for dichromat readers (BioRxiv preprint: Supplementary Material).

genetics↗

bioRxiv2021

A genetic switch for male UV-iridescence in an incipient species pair of sulphur butterflies

Mating cues evolve rapidly and can contribute to species formation and maintenance. However, little is known about how sexual signals diverge and how this variation integrates with other barrier loci to shape the genomic landscape of reproductive isolation. Here, we elucidate the genetic basis of UV iridescence, a courtship signal that differentiates the males of Colias eurytheme butterflies from a sister species, allowing females to avoid costly heterospecific matings. Anthropogenic range expansion of the two incipient species established a large zone of secondary contact across the eastern US with strong signatures of genomic admixtures spanning all autosomes. In contrast, Z chromosomes are highly differentiated between the two species, supporting a disproportionate role of sex chromosomes in speciation known as the large-X (or large-Z) effect. Within this chromosome-wide reproductive barrier, linkage mapping indicates that cis-regulatory variation of bric a brac (bab) underlies the male UV-iridescence polymorphism between the two species. Bab is expressed in all non-UV scales, and butterflies of either species or sex acquire widespread ectopic iridescence following its CRISPR knock-out, demonstrating that Bab functions as a suppressor of UV-scale differentiation that potentiates mating cue divergence. These results highlight how a genetic switch can regulate a premating signal and integrate with other reproductive barriers during intermediate phases of speciation. Significance statementIncipient species are at an intermediate stage of speciation where reproductive isolation is counteracted by the homogenizing effects of gene flow. Human activity sometimes leads such species to reunite, as seen in the Orange Sulphur butterfly, which forms large hybridizing populations with the Clouded Sulphur in alfalfa fields. Here we show that the sex chromosome maintains these species as distinct, while the rest of their genome is admixed. Sex chromosomes notably determine which males display to females a bright, iridescent ultraviolet signal on their wings. Genetic mapping, antibody stainings, and CRISPR knock-outs collectively indicate that the gene bric a brac controls whether UV-iridescent nanostructures develop in each species, elucidating how a master switch gene modulates a male courtship signal.

evolutionary biology↗

bioRxiv2020

The gene cortex controls scale colour identity in Heliconius

In Heliconius butterflies, wing pattern diversity is controlled by a few genes of large effect that regulate colour pattern switches between morphs and species across a large mimetic radiation. One of these genes, cortex, has been repeatedly associated with colour pattern evolution in butterflies. Here we carried out CRISPR knock-outs in multiple Heliconius species and show that cortex is a major determinant of scale cell identity. Chromatin accessibility profiling and introgression scans identified cis-regulatory regions associated with discrete phenotypic switches. CRISPR perturbation of these regions in black hindwing genotypes recreated a yellow bar, revealing their spatially limited activity. In the H. melpomene/timareta lineage, the candidate CRE from yellow-barred phenotype morphs is interrupted by a transposable element, suggesting that cis-regulatory structural variation underlies these mimetic adaptations. Our work shows that cortex functionally controls scale colour fate and that its cis-regulatory regions control a phenotypic switch in a modular and pattern-specific fashion.

evolutionary biology↗