anti gfrα1  (R&D Systems)

Journal: bioRxiv

Article Title: Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

doi: 10.64898/2026.03.31.715459

Figure Lengend Snippet: GDNF-RET signaling tunes branching in mouse and human ureteric bud tissues. A. GDNF-RET signaling interactions involved in kidney branching. B. Immunofluorescence of cleared E14 mouse kidney. Top left , ECAD and SIX2. Bottom left, RET and GFRA1. Top right , isolated RET. Bottom right , isolated GFRA1 signal. C. Air-liquid interface (ALI) culture of mouse embryonic kidney explants. D. Design of GDNF-RET signaling perturbation experiments in ALI culture. E. Immunofluorescence of E13 kidneys grown in ALI culture for 4 days in 100 nM Selpercatinib (+RETi), 100 ng ml -1 GDNF (+GDNF), or control media. Inset , close-up of tip domains. Explants are immunostained for EpCAM (epithelium), RET (tip cells), and JAG1 (early nephrons). F. Tip number on days 1-4 across all conditions, N = 4 kidneys per condition. P -values by one-way Kruskal-Wallis test with Dunn’s post hoc test. G. Differentiation of iUB organoids from hiPSCs H. Immunofluorescence of epithelial (ECAD) and tip (RET) markers in a day 12 iUB organoid. I. Design of GDNF-RET signaling perturbations for iUB organoids. J. Merged brightfield and GATA3:mCherry images of iUB organoids at days 9 and 12. Organoids were cultured in control (-GDNF) or complete branching medium (+GDNF, 50 ng ml -1 ), excess GDNF (++GDNF, 250 ng ml -1 ), or complete branching medium with 100 nM Selpercatinib (+RETi). See: Fig. S4F . K. Projected area (x10 4 µm 2 ) on days 9 and 12 for all conditions, n = 47, 56, 40, 46 iUB organoids (-GDNF, +RETi, +GDNF, ++GDNF) from 2 independent biological replicates. L. Circularity (a.u.) on days 9 and 12. M. Bud number on days 9 and 12. P -values in panels K, L, M by one-way ANOVA with Dunnett’s post hoc test using +GDNF as reference group.

Article Snippet: MDCK cells were not starved and were treated with 50 ng ml -1 GDNF (#212-GD-050, R&D Systems) and 100 ng ml -1 recombinant human Gfrɑ1 (#714-GR-100, R&D Systems) at defined time points.

Techniques: Immunofluorescence, Isolation, Control, Cell Culture

Journal: bioRxiv

Article Title: Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

doi: 10.64898/2026.03.31.715459

Figure Lengend Snippet: Blue light stimulation of optoRET drives ERK signaling and ligand-independent budding in iUB organoids. C. Schematic of hiPSC-optoRET generation using piggyBac transposase and differentiation into iUB-optoRET organoids. D. Immunofluorescence of iUB-optoRET monolayers stimulated for 2 hr under the indicated conditions (±light). Blue light stimulation was provided by an optoPlate-96 (470 nm, 50 mW cm -2 , 1 s every 10 s) for 2 hrs. Top , optoRET (detected by anti-GFP) and nuclei (DAPI). Bottom , intensity-coded ppERK (a.u.). E. Violin plot of ppERK (a.u.) for iUB-optoRET cells under -light and +light conditions, n = 2458, 2030 cells (control, +light) pooled from 2 independent biological replicates. P -value by Welch’s t-test. F. Immunofluorescence density plot of ppERK (a.u.) as a function of optoRET(eGFP) intensity (a.u.) for cells shown in B. n = 2458, 2030 cells (control, +light) pooled from two independent biological replicates. G. Stimulation conditions for iUB-optoRET organoids: control (−light/−GDNF), +light, +GDNF, and combined (+light/+GDNF) treatments. H. Live fluorescence images of GATA3 mCherry iUB-optoRET organoids under optogenetic and ligand-based stimulation conditions. Blue light stimulation was provided by an optoPlate-96 (320 mW cm -2 , 0.5 s every 4 min) between days 7-11 and the +GDNF and +light/+GDNF groups received 50 ng ml -1 GDNF. I. Bud number on day 11 across all conditions, n = 50, 50, 51, 50 organoids (control, +light, +GDNF, +light/+GDNF) from 2 independent biological replicates. P -values by one-way Kruskal-Wallis test with Dunn’s post hoc test. J. Differential expression analysis from bulk RNA-seq of iUB-optoRET organoids grown in ±light stimulation conditions (see also: Fig. S14 ). Top , relative density of tip and trunk marker genes. Bottom , volcano plot of all genes organized by significance (-log 10 ( p )) and log 2 (fold-change) with tip and trunk-specific markers highlighted. Data are derived from 3 biological replicates. K. Gene Set Enrichment Analysis (GSEA) as a function of normalized enrichment score (NES). Hallmark gene sets are color coded by false discovery rate (FDR), size coded by the size of the gene set.

Article Snippet: MDCK cells were not starved and were treated with 50 ng ml -1 GDNF (#212-GD-050, R&D Systems) and 100 ng ml -1 recombinant human Gfrɑ1 (#714-GR-100, R&D Systems) at defined time points.

Techniques: Immunofluorescence, Control, Fluorescence, Quantitative Proteomics, RNA Sequencing, Marker, Derivative Assay

Journal: bioRxiv

Article Title: Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling

doi: 10.64898/2026.03.31.715459

Figure Lengend Snippet: Spatially patterned optoRET stimulation drives asymmetric budding in iUB organoids. A. Strategy for targeted optogenetic stimulation of iUB-optoRET organoids. B. Average projections of GATA3:mCherry for small ( top panels ) and large ( bottom panels ) iUB-optoRET organoids under control, +GDNF, +light whole, and +light half conditions. Light stimulation was provided by 488 nm DMD-based light projection (0.5 s every 4 mins) for 4 days. Organoids in the +GDNF group received 50 ng ml -1 GDNF. Top row , average projections across individual organoids at day 7. Middle row , average projections across individual organoids at day 11. Bottom row , normalized average projections for individual organoids at day 11. C. Representative average projections of individual small ( top ) and large ( bottom ) iUB-optoRET organoids on day 11. The cyan dashed line divides the non-illuminated side ( left , -light half) from the illuminated side ( right , +light half). Magenta dots indicate bud locations. D. Confocal immunofluorescence images of optoRET+ and RET+ cells within iUB-optoRET bud tips in +light whole condition. Endogenous RET was visualized with an antibody against the extracellular domain (ECD). Left , ECAD, optoRET, and RET(ECD). Middle , optoRET and tip outline. Right , RET(ECD) and tip outline. E. Radial histograms of bud orientation angle (°) for small organoids across all conditions, n = 19, 20, 19, 20 organoids (control, +GDNF, +light whole, and +light half) pooled from 2 biological replicates. P -values by Kolmogorov-Smirnov test against a uniform reference distribution.

Article Snippet: MDCK cells were not starved and were treated with 50 ng ml -1 GDNF (#212-GD-050, R&D Systems) and 100 ng ml -1 recombinant human Gfrɑ1 (#714-GR-100, R&D Systems) at defined time points.

Techniques: Control, Immunofluorescence