Biology subjects

Negishi, T.

Publications and source records attributed to Negishi, T..

4 recordsLinked to original sources

bioRxiv2025

Differential functions of multiple Wnts and receptors in cell polarity regulation in C. elegans

Metazoan species possess multiple Wnt ligands and receptor genes that regulate diverse developmental processes. Because these genes often act redundantly, analysis of single-gene mutants does not necessarily reveal the full roles of Wnt signaling. In C. elegans, three Wnt genes (cwn-1, egl-20, and cwn-2) and three receptor genes (lin-17/Fzd, mom-5/Fzd, and cam-1/Ror) redundantly regulate the polarity of asymmetrically dividing seam cells. Here, we comprehensively analyzed genetic interactions among these Wnt and receptor genes. In mom-5 mutant backgrounds, additional mutations in Wnt genes disrupted cell polarization. In contrast, in cam-1 mutant backgrounds, Wnt mutations frequently caused abnormal polarity orientation. These findings indicate that MOM-5 and CAM-1 play distinct roles in establishing cell polarization and determining its orientation, respectively. lin-17 mutations suppressed polarity reversal in multiple Wnt compound mutants, suggesting that LIN-17 may function as a molecular switch for polarity orientation. Although all three Wnt genes regulate polarity orientation in a gradient-independent manner in the absence of receptor mutations, in lin-17 mutant backgrounds, reversing the expression gradients of cwn-1 and egl-20, but not cwn-2, enhanced polarity reversal. This suggests that cwn-1 and egl-20 act not only permissively but also instructively to regulate polarity orientation. Together, our results reveal distinct and cooperative functions of multiple Wnt ligands and receptors that ensure robust control of cell polarity.

developmental biology↗

bioRxiv2025

A single-chain antibody-based AID2 system for conditional degradation of GFP-tagged and untagged proteins

Protein knockdown using an improved auxin-inducible degron (AID2) technology has proven to be a powerful tool for studying protein function. The current approach requires the fusion of target proteins with a degron tag, a process typically achieved through CRISPR knock-in. However, knock-in remains challenging in non-model organisms and humans, limiting the broader applicability of AID2. To overcome this limitation, we developed a single-chain antibody AID2 (scAb-AID2) system. This approach employs an adaptor composed of a single-chain antibody fused with a degron, which recognises a target protein and induces rapid degradation in the presence of the inducer 5-Ph-IAA. We demonstrated that scAb-AID2, in combination with an anti-GFP nanobody, degraded GFP-fused proteins in human cells and C. elegans. Furthermore, we showed that endogenous p53 and H/K-RAS were conditionally degraded in cells expressing an adaptor encoding an anti-p53 nanobody and -RAS monobody, respectively, and led to aphidicolin sensitivity in cell culture and growth inhibition in mouse xenografts. This study paves the way for broader application of AID2-based target depletion in model and non-model organisms and for advancing therapeutic strategies.

bioengineering↗

bioRxiv2024

The spatiotemporal distribution of LIN-5/NuMA regulates spindle orientation and tissue organization in the C. elegans germ line

Mitotic spindle orientation sets the cell division plane and is thus critical for maintaining tissue organization. The C. elegans gonad is tube-shaped, with germ cells forming a circumferential monolayer around a shared inner core of cytoplasm called the rachis. Each germ cell is connected to the rachis via a stable cytoplasmic bridge, polarizing germ cells along their rachis-basal axis. How this tissue organization is maintained during development is unclear, as germ cells lack the canonical cell-cell junctions that, in other tissue types, ensure proper spindle orientation. Here we use live-cell imaging of C. elegans germ cells, both in situ and in gonad explants, to show that the microtubule force generator dynein and its conserved regulator LIN-5/NuMA regulate spindle orientation in C. elegans germ cells and are required for germline tissue organization. We uncover a cyclic, polarized pattern of LIN-5/NuMA cortical localization that predicts centriole/centrosome positioning throughout the cell cycle, providing a means to align spindle orientation with the tissue plane. This work reveals a new mechanism by which oriented cell division can be achieved to maintain tissue organization during animal development.

cell biology↗

bioRxiv2018

Nodal and Eph signalling relay drives the transition between apical constriction and apico-basal shortening during ascidian endoderm invagination

Gastrulation is the first major morphogenetic event during animal embryogenesis. Ascidian gastrulation starts with the invagination of 10 endodermal precursor cells between the 64- and late 112-cell stages. This process occurs in the absence of endodermal cell division and in two steps, driven by myosin-dependent contractions of the acto-myosin network. First, endoderm precursors constrict their apex. Second, they shorten apico-basally, while retaining small apical surfaces, thereby causing invagination. The mechanisms controlling the endoderm mitotic delay, the step 1 to step 2 transition, and apico-basal shortening have remained elusive. Here, we demonstrate the conserved role during invagination of Nodal and Eph signalling in two distantly related ascidian species (Phallusia mammillata and Ciona intestinalis). We show that the transition to step 2 is controlled by Nodal relayed by Eph signalling and that Eph signalling has a Nodal-independent role in mitotic delay. Interestingly, both Nodal and Eph signals are dispensable for endodermal germ layer fate specification. Summary statementIdentification of a regulatory developmental signalling sub-network driving endoderm cell shape changes during ascidian endoderm invagination, not involved in cell fate specification.

developmental biology↗