A micro-RNA is the effector gene of a classic evolutionary hotspot locus.

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.

Genome Assembly and Annotation of the Dark-Branded Bushbrown Butterfly Mycalesis mineus (Nymphalidae: Satyrinae).

We report a high-quality genome draft assembly of the dark-branded bushbrown, Mycalesis mineus, a member of the Satyrinae subfamily of nymphalid butterflies. This species is emerging as a promising model organism for investigating the evolution and development of phenotypic plasticity. Using 45.99 Gb of long-read data (N50 = 11.11 kb), we assembled a genome size of 497.4 Mb for M. mineus. The assembly is highly contiguous and nearly complete (96.8% of Benchmarking Universal Single-Copy Orthologs lepidopteran genes were complete and single copy). The genome comprises 38.71% of repetitive elements and includes 20,967 predicted protein-coding genes. The assembled genome was super-scaffolded into 28 pseudo-chromosomes using a closely related species, Bicyclus anynana, with a chromosomal-level genome as a template. This valuable genomic tool will advance both ongoing and future research focused on this model organism.

Lepidopteran prolegs are novel traits, not leg homologs.

Lepidopteran larvae have both thoracic legs and abdominal prolegs, yet it is unclear whether these are serial homologs. A RNA-seq analysis with various appendages of Bicyclus anynana butterfly larvae indicated that the proleg transcriptome resembles the head-horn transcriptome, a novel trait in the lepidoptera, but not a thoracic leg. Under a partial segment abdominal-A (abd-A) knockout, both thoracic leg homologs (pleuropodia) and prolegs developed in the same segment, arguing that both traits are not serial homologs. Further, three of the four coxal marker genes, Sp5, Sp6-9, and araucan, were absent from prolegs, but two endite marker genes, gooseberry and Distal-less, were expressed in prolegs, suggesting that prolegs may be using a modular endite gene-regulatory network (GRN) for their development. We propose that larval prolegs are novel traits derived from the activation of a pre-existing modular endite GRN in the abdomen using abd-A, the same Hox gene that still represses legs in more lateral positions.

Spatial and temporal regulation of Wnt signaling pathway members in the development of butterfly wing patterns.

Wnt signaling members are involved in the differentiation of cells associated with eyespot and band 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/ß-catenin dependent (canonical) and Armadillo/ß-catenin independent (noncanonical) Wnt signaling in eyespot and band development in Bicyclus anynana by localizing Armadillo (Arm), the expression of all eight Wnt ligand and four frizzled receptor transcripts present in the genome of this species and testing the function of some of the ligands and receptors using CRISPR-Cas9. We show that distinct Wnt signaling pathways are essential for eyespot and band patterning in butterflies and are likely interacting to control their active domains.

The genetic basis of wing spots in Pieris canidia butterflies.

Spots in pierid butterflies and eyespots in nymphalid butterflies are likely non-homologous wing colour pattern elements, yet they share a few features in common. Both develop black scales that depend on the function of the gene spalt, and both might have central signalling cells. This suggests that both pattern elements may be sharing common genetic circuitry. Hundreds of genes have already been associated with the development of nymphalid butterfly eyespot patterns, but the genetic basis of the simpler spot patterns on the wings of pierid butterflies has not been investigated. To facilitate studies of pierid wing patterns, we report a high-quality draft genome assembly for Pieris canidia, the Indian cabbage white. We then conducted transcriptomic analyses of pupal wing tissues sampled from the spot and non-spot regions of P. canidia at 3-6 h post-pupation. A total of 1352 genes were differentially regulated between wing tissues with and without the black spot, including spalt, Krüppel-like factor 10, genes from the Toll, Notch, TGF-ß, and FGFR signalling pathways, and several genes involved in the melanin biosynthetic pathway. We identified 14 genes that are up-regulated in both pierid spots and nymphalid eyespots and propose that spots and eyespots share regulatory modules despite their likely independent origins.

Butterfly eyespots exhibit unique patterns of open chromatin.

Background: How the precise spatial regulation of genes is correlated with spatial variation in chromatin accessibilities is not yet clear. Previous studies that analysed chromatin from homogenates of whole-body parts of insects found little variation in chromatin accessibility across those parts, but single-cell studies of Drosophila brains showed extensive spatial variation in chromatin accessibility across that organ. In this work we studied the chromatin accessibility of butterfly wing tissue fated to differentiate distinct colors and patterns in pupal wings of Bicyclus anynana. Methods: We dissected small eyespot and adjacent control tissues from 3h pupae and performed ATAC-Seq to identify the chromatin accessibility differences between different sections of the wings. Results: We observed that three dissected wing regions showed unique chromatin accessibilities. Open chromatin regions specific to eyespot color patterns were highly enriched for binding motifs recognized by Suppressor of Hairless (Su(H)), Krüppel (Kr), Buttonhead (Btd) and Nubbin (Nub) transcription factors. Genes in the vicinity of the eyespot-specific open chromatin regions included those involved in wound healing and SMAD signal transduction pathways, previously proposed to be involved in eyespot development. Conclusions: We conclude that eyespot and non-eyespot tissue samples taken from the same wing have distinct patterns of chromatin accessibility, possibly driven by the eyespot-restricted expression of potential pioneer factors, such as Kr.

Butterfly eyespots evolved via cooption of an ancestral gene-regulatory network that also patterns antennae, legs, and wings.

Butterfly eyespots are beautiful novel traits with an unknown developmental origin. Here we show that eyespots likely originated via cooption of parts of an ancestral appendage gene-regulatory network (GRN) to novel locations on the wing. Using comparative transcriptome analysis, we show that eyespots cluster most closely with antennae, relative to multiple other tissues. Furthermore, three genes essential for eyespot development, Distal-less (Dll), spalt (sal), and Antennapedia (Antp), share similar regulatory connections as those observed in the antennal GRN. CRISPR knockout of cis-regulatory elements (CREs) for Dll and sal led to the loss of eyespots, antennae, legs, and also wings, demonstrating that these CREs are highly pleiotropic. We conclude that eyespots likely reused an ancient GRN for their development, a network also previously implicated in the development of antennae, legs, and wings.

Interaction network analysis of YBX1 for identification of therapeutic targets in adenocarcinomas.

Human Y-box binding protein-1 (YBX1) is a member of highly conserved cold-shock domain protein family, which is involved in transcriptional as well as translational regulation of many genes. Nuclear localization of YBX1 has been observed in various cancer types and it's overexpression has been linked to adverse clinical outcome and poor therapy response, but no diagnostic or therapeutic correlation has been established so far. This study aimed to identify differentially expressed novel genes among the interactors of YBX1 in different cancer types. Analysis of RNA-Seq data for colorectal, lung, prostate and stomach adenocarcinoma identified 39 unique genes, which are differentially expressed in the four adenocarcinoma types. Gene-enrichment analysis for the differentially expressed genes from individual adenocarcinoma with focus on unique genes resulted in a total of 57 gene sets specific to each adenocarcinoma. Gene ontology for commonly expressed genes suggested the pathways and possible mechanisms through which they affect each adenocarcinoma type considered in the study. Gene regulatory network constructed for the common genes and network topology was analyzed for the central nodes. Here 12 genes were found to play important roles in the network formation; among them, two genes FOXM1 and TOP2A were found to be in central network formation, which makes them a common target for therapeutics. Furthermore, five common differentially expressed genes in all adenocarcinomas were also identified.

Expression and network analysis of YBX1 interactors for identification of new drug targets in lung adenocarcinoma.

Y-Box Binding protein 1 (YBX-1) is known to be involved in various types of cancers. It's interactors also play major role in various cellular functions. Present work aimed to study the expression profile of the YBX-1 interactors during lung adenocarcinoma (LUAD). The differential expression analysis involved 57 genes from 95 lung adenocarcinoma samples, construction of gene network and topology analysis. A Total of 43 genes were found to be differentially expressed from which 17 genes were found to be down regulated and 26 genes were up-regulated. We observed that Polyadenylate-binding protein 1 (PABPC1), a protein involved in YBX1 translation, is highly correlated with YBX1. The interaction network analysis for a differentially expressed non-coding RNA Growth Arrest Specific 5 (GAS5) suggests that two proteins namely, Growth Arrest Specific 2 (GAS2) and Peripheral myelin protein 22 (PMP22) are potentially involved in LUAD progression. The network analysis and differential expression suggests that Collagen type 1 alpha 2 (COL1A2) can be potential biomarker and target for LUAD.