Bali, N. | Search bioRxiv

bioRxiv2025

Next-generation hybridization chain reaction tools with enhanced sensitivities to detect challenging targets

Compared to traditional enzyme-based in situ amplification methods, Hybridization Chain Reaction v3.0 (HCR v3.0) offers high specificity for spatial RNA visualization but lacks the sensitivity required to robustly detect short or low-abundance targets, particularly in thick tissue with high autofluorescence. Here, we describe three HCR variants that combine the specificity of HCR v3.0 with additional signal amplification through catalytic reporter deposition (HCR-Cat), immunostaining (HCR-Immuno), or iterative HCR and immunodetection (HCR-Multi). These methods substantially enhance detection sensitivity, enabling robust spatial visualization of low-abundance transcripts, improved performance in challenging tissue environments, and compatibility with fluorescence- and alkaline phosphatase-based chromogenic detection. These methods enable transcript-resolution imaging, even when using a limited number of probes, thereby expanding the range of biological applications accessible to HCR.

neuroscience↗

bioRxiv2020

Sticks and Stones, a conserved cell surface ligand for the Type IIa RPTP Lar, regulates neural circuit wiring in Drosophila

Control of tyrosine phosphorylation is an essential element of many cellular processes, including proliferation, differentiation neurite outgrowth, and synaptogenesis. Receptor-like protein-tyrosine phosphatases (RPTPs) have cytoplasmic phosphatase domains and cell adhesion molecule (CAM)-like extracellular domains that interact with cell-surface ligands and/or co-receptors. We identified a new ligand for the Drosophila Lar RPTP, the immunoglobulin superfamily CAM Sticks and Stones (Sns). Lar is orthologous to the three Type IIa mammalian RPTPs, PTPRF (LAR), PTPRD (PTP{delta}), and PTPRS (PTP{sigma}). Lar and Sns bind to each other in embryos and in vitro. The human Sns ortholog, Nephrin, binds to PTPRD and PTPRF. Genetic interaction studies show that Sns is essential to Lars functions in several developmental contexts in the larval and adult nervous systems. In the larval neuromuscular system, Lar and sns transheterozygotes (Lar/sns transhets) have synaptic defects like those seen in Lar mutants and Sns knockdown animals. Lar and Sns reporters are both expressed in motor neurons and not in muscles, so Lar and Sns likely act in cis (in the same neurons). Lar mutants and Lar/sns transhets have identical axon guidance defects in the larval mushroom body in which Kenyon cell axons fail to stop at the midline and do not branch. Pupal Kenyon cell axon guidance is similarly affected, resulting in adult mushroom body defects. Lar is expressed in larval and pupal Kenyon cells, but Sns is not, so Lar-Sns interactions in this system must be in trans (between neurons). Lastly, R7 photoreceptor axons in Lar mutants and Lar/sns transhets fail to innervate the correct M6 layer of the medulla in the optic lobe. Lar acts cell-autonomously in R7s, while Sns is only in lamina and medulla neurons that arborize near the R7 target layer. Therefore, the Lar-Sns interactions that control R7 targeting also occur in trans.

neuroscience↗