Biology subjects

Yoda, S.

Publications and source records attributed to Yoda, S..

6 recordsLinked to original sources

bioRxiv2025

Morphogen-driven melanin pathway dynamics regulated by Wnt1 and apontic-like underlie larval spot coloration in Bombyx mori

In insects, conspicuous larval pigmentation patterns serve critical ecological roles such as warning signals and mimicry, yet their underlying genetic regulation remains poorly understood. In this study, I investigated the molecular mechanisms underlying black and yellow pigmentation patterns in three distinct larval spot types of the silkworm Bombyx mori: large, diffuse L-spots of the Multilunar (L) mutant; small, sharply defined +p-spots of the Normal strain; oval pM-hybrid spots of an interspecific hybrid with Bombyx mandarina. Each spot type comprises a yellowish center surrounded by a black periphery, forming crescent-shaped pigmentation patterns. Chemical treatments confirmed that both colors are melanin-based. Using quantitative PCR and RNA interference (RNAi), I analyzed six melanin synthesis genes (Tyrosine Hydroxylase, Dopa Decarboxylase, laccase2, yellow, tan, and ebony) and discovered that black pigmentation involves both dopa/dopamine- and NBAD-melanin synthesis, whereas yellow pigmentation primarily reflects only the latter. I further examined Wnt1 and apontic-like (apt-like) using qPCR, RNAi, and TALEN-mediated mosaic analysis. Wnt1 expression localized to presumptive spot areas, functioning dose-dependently to regulate both spot size and pigment composition: high Wnt1 levels induced larger spots with yellow centers, while reduced Wnt1 expression resulted in black pigmentation and smaller spots. Wnt1-activated transcription factor apt-like was required for pigmentation in all spot types without influencing spot size. Taken together, the results of this study reveal a morphogen-driven gene regulatory network in which Wnt1 dosage and downstream transcriptional cascades orchestrate pigment placement and patterning, offering new insights into the modular genetic control of insect pigmentation.

genetics↗

bioRxiv2024

Refined CRISPR/Cas9 genome editing in pea aphids uncovers the essential roles of Laccase2 in overwintering egg adaptation

The production of overwintering eggs is a critical adaptation for winter survival among many insects. Melanization contributes to eggshell pigmentation and hardening, consequently enhancing resistance to environmental stress. The complex life cycle of the pea aphid (Acyrthosiphon pisum), a model hemipteran insect with remarkable reproductive capacity, involves cyclical parthenogenesis. It enables the production of black overwintering eggs that undergo obligate diapause to survive under unfavorable conditions. Laccase2 (Lac2) is essential for cuticle sclerotization and pigmentation in other insects. We hypothesized that Lac2 plays a critical role in aphid eggshell pigmentation and survival during diapause. To test the hypothesis, we used CRISPR/Cas9 ribonucleoprotein microinjections and a novel Direct Parental CRISPR (DIPA-CRISPR) method to knockout Lac2. In Lac2 knockout (KO) crispants (G0), pigment-less eggs correlated with induced indel rates. Additionally, eggshell pigmentation was completely lost in homozygous Lac2 knockouts, leading to embryonic lethality. Observation of late-stage embryos in KO diapause eggs suggested that lethality occurred during late embryogenesis or hatching. Furthermore, eggshell stiffness was significantly reduced in Lac2 KOs, highlighting the role of this gene in eggshell hardening. Moreover, fungal growth was observed in KO eggs. These findings reveal the essential roles of Lac2 in eggshell pigmentation, hardening, late embryonic development, hatching, and fungal protection, which are critical for pea aphid survival during overwintering diapause. This study also advances CRISPR/Cas9-mediated genome editing in pea aphids by addressing the challenges associated with their unique biology, including complex life cycles, obligatory diapause, bacterial endosymbiosis, inbreeding depression, and high nuclease activity. Our optimized protocol achieved efficient targeted mutagenesis and germline transmission, thereby generating stable KO lines. Additionally, we successfully applied DIPA-CRISPR to aphids by inducing mutations via adult oviparous female injections in fertilized eggs. These robust genome-editing protocols will facilitate functional studies in aphids, a key model for research on evolution, ecology, development, and agriculture. Author SummarySurviving harsh winters is a challenge for many insects, and the production of specialized overwintering eggs is a common adaptation strategy. These eggs are protected from cold, desiccation, and fungal infections by their hardened, pigmented shells. The pea aphid, a hemipteran insect with a complex life cycle, relies on these eggs to survive winter. Aphids alternate between asexual reproduction in the warmer months and sexual reproduction in the fall, producing overwintering eggs that remain dormant until spring. In this study, we explored the role of Laccase2 (Lac2), a key gene that contributes to the strengthening and darkening of insect exoskeletons. We disrupted Lac2 in pea aphids using advanced genome-editing techniques, including a new approach called DIPA-CRISPR. This resulted in pigment-free eggs with weakened shells that were more prone to fungal infections and failed to hatch. This demonstrates that Lac2 is essential for the survival of overwintering eggs. In addition, we refined the CRISPR/Cas9 genome editing methods for pea aphids, enabling efficient and precise genetic studies. These findings and the tools we developed can facilitate research on ecology, evolution, and pest control, while shedding light on how insects adapt to challenging environments.

genetics↗

bioRxiv2024

Aspergillus nidulans cell wall integrity kinase, MpkA, impacts cellular phenotypes that alter mycelial-material mechanical properties

Mycelial materials are an emerging, natural material made from filamentous fungi that have the potential to replace unsustainable materials used in numerous commercial applications (e.g., packaging, textiles, construction). Efforts to change the mechanical properties of mycelial-materials have typically involved altering growth medium, processing approaches, or fungal species. Although these efforts have shown varying levels of success, all approaches have shown there is a strong correlation between phenotype (of both fungal mycelia and mycelial materials assembly) and resultant mechanical properties. We hypothesize that genetic means can be used to generate specific fungal phenotypes, leading to mycelial materials with specific mechanical properties. To begin to test this hypothesis, we used a mutant of the model filamentous fungus, Aspergillus nidulans, with a deletion of the last kinase in the cell wall integrity (CWI) signaling pathway, mpkA. We generated one set of mycelial materials from the {Delta}mpkA deletion mutant (A1404), and another from its isogenic parent (A1405; control). When subjected to tensile testing, and compared to material generated from the control, {Delta}mpkA material has similar elastic modulus, but significantly increased ultimate tensile strength, and strain at failure. When subjected to a fragmentation assay (i.e., resistance to shear-stress), the {Delta}mpkA material also had higher relative mechanical strength. To determine possible causes for this behavior, we carried out a comprehensive set of phenotype assessments focused on: three-dimensional structure, hyphal morphology, hyphal growth behaviors, and conidial development. We find, compared to the control, material generated from the {Delta}mpkA mutant manifests significantly less development, a modified cell wall composition, larger diameter hyphae, more total biomass, higher water capacity and more densely packed material, which all appear to impact the altered mechanical properties.

bioengineering↗

bioRxiv2023

DrugMap: A quantitative pan-cancer analysis of cysteine ligandability

Cysteine-focused chemical proteomic platforms have accelerated the clinical development of covalent inhibitors of a wide-range of targets in cancer. However, how different oncogenic contexts influence cysteine targeting remains unknown. To address this question, we have developed DrugMap, an atlas of cysteine ligandability compiled across 416 cancer cell lines. We unexpectedly find that cysteine ligandability varies across cancer cell lines, and we attribute this to differences in cellular redox states, protein conformational changes, and genetic mutations. Leveraging these findings, we identify actionable cysteines in NF{kappa}B1 and SOX10 and develop corresponding covalent ligands that block the activity of these transcription factors. We demonstrate that the NF{kappa}B1 probe blocks DNA binding, whereas the SOX10 ligand increases SOX10-SOX10 interactions and disrupts melanoma transcriptional signaling. Our findings reveal heterogeneity in cysteine ligandability across cancers, pinpoint cell-intrinsic features driving cysteine targeting, and illustrate the use of covalent probes to disrupt oncogenic transcription factor activity.

systems biology↗

bioRxiv2022

Functional involvement of multiple genes as members of the supergene unit in the female-limited Batesian mimicry of Papilio polytes

Supergenes are sets of genes and genetic elements that are inherited like a single gene and control complex adaptive traits, but their functional roles and units are poorly understood. In Papilio polytes, female-limited Batesian mimicry is thought to be regulated by a ~130kb inversion region (highly diversified region: HDR) containing three genes, UXT, U3X and doublesex (dsx) which switches non-mimetic and mimetic types. To determine the functional unit, we here performed electroporation-mediated RNAi analyses (and further Crispr/Cas9 for UXT) of genes within and flanking the HDR in pupal hindwings. We first clarified that non-mimetic dsx-h had a function to switch from male to non-mimetic female and only dsx-H isoform 3 had an important function in the formation of mimetic traits. Next, we found that UXT was involved in making mimetic type pale-yellow spots and adjacent gene sir2 removed excess red spots in hindwings, both of which refine more elaborate mimicry. Furthermore, downstream gene networks of dsx, U3X and UXT screened by RNA sequencing showed that U3X upregulated dsx expression and repressed UXT expression. These findings demonstrate that a set of multiple genes, not only inside but also flanking HDR, can function as supergene members, which extends the definition of supergene unit than we considered before. Also, our results indicate that dsx-H functions as the switching gene and some other genes such as UXT and sir2 within the supergene unit work as the modifier gene. Article summarySupergenes are thought to control complex adaptive traits, but their detailed function are poorly understood. In Papilio polytes, female-limited Batesian mimicry is regulated by an ~130kb inversion region (highly divergent region: HDR) containing three genes. Our functional analysis showed that doublesex switches the mimicry polymorphism, and that an inside gene UXT and an outside gene sir2 to the HDR work to refine more elaborate mimicry. We here succeed in defining the unit of mimicry supergene and some novel modifier genes.

genetics↗

bioRxiv2021

Structural and functional analysis of lorlatinib analogs reveals roadmap for targeting diverse compound resistance mutations in ALK-positive lung cancer

The treatment approach to advanced, ALK-positive non-small cell lung cancer (NSCLC) utilizing sequential ALK tyrosine kinase inhibitors (TKIs) represents a paradigm of precision oncology. Lorlatinib is currently the most advanced, potent and selective ALK tyrosine kinase inhibitor (TKI) in the clinic. However, tumors invariably acquire resistance to lorlatinib, and after sequential ALK TKIs culminating with lorlatinib, diverse refractory compound ALK mutations can emerge. Here, we determine the spectrum of lorlatinib-resistant compound ALK mutations identified in patients after treatment with lorlatinib, the majority of which involve ALK G1202R or I1171N/S/T. By assessing a panel of lorlatinib analogs against compound ALK mutant in vitro and in vivo models, we identify structurally diverse lorlatinib analogs that harbor differential selective profiles against G1202R- versus I1171N/S/T-based compound ALK mutations. Structural analysis revealed that increased potency against compound mutations was achieved primarily through two different mechanisms of improved targeting of either G1202R- or I1171N/S/T-mutant kinases. Based on these results, we propose a classification of heterogenous ALK compound mutations designed to focus the development of distinct therapeutic strategies for precision targeting of compound resistance mutations following sequential TKIs.

cancer biology↗