Journal: bioRxiv

Article Title: Stepwise developmental mimicry generates proximal-biased kidney organoids

doi: 10.1101/2024.06.28.601028

Figure Lengend Snippet: a . Whole-mount immunofluorescent stain of day 12 control and LY294002-treated kidney organoids. Insets highlight JAG1 and HNF1B protein expression. Scale bars: 500 microns. b . Measurements from (a) for JAG1 + and HNF1B + nephron size (µm ) and HNF1B + intensity (relative fluorescent units) for n = 3 day 12 organoids. SEM error bars are shown. Statistical significance is determined using Student’s t test. c . Bulk RNA-sequencing (TPM) of select genes on days 10, 12, 14, and 18. SEM error bars are shown from n = 2 whole organoids. d . Whole-mount immunofluorescent stain of control and proximal-biased day 14 organoids with insets showing HNF4A expression. Scale bars: 200 microns. e . Immunofluorescent antibody stains of week 16 human kidneys during HNF4A + , HNF4G + proximal tubule elongation. White arrowheads indicate autofluorescence from endothelial cells. Scale bars: 10 microns. f . Whole-mount immunofluorescent stain of control and proximal-biased day 18 organoids with insets showing HNF4A expression. Scale bars: 200 microns. g . Quantification of total number of HNF4A + organoid nephron segments from n = 4 whole day 18 organoids. SEM error bars are shown. Statistical significance is determined using Student’s t test. h . Quantification of the average area (µm ) of HNF4A + organoid nephron segments from n = 3 whole day 18 organoids. SEM error bars are shown. Statistical significance is determined using Student’s t test. i . Area sum (µm ) quantification of HNF4A + organoid nephron segments from n = 3 whole day 18 organoids.

Article Snippet: Primary antibodies used in this study were: WT1 (abcam, ab89901, 1:1000), JAG1 (R&D Systems, AF599, 1:300), HNF1B (Thermo Fisher Scientific, MA5-24605, 1:500), HNF4A (R&D Systems, MAB4605, 1:200), CDH1 (BD Biosciences, 610181, 1:300), ZO-1 (Thermo Fisher Scientific, 33-9100, 1:200), PAX2 (R&D Systems, AF3364, 1:50), SIX1 (Cell Signaling Technology, 12891S, 1:300), HES1 (Cell Signaling Technology, 11988, 1:300), POU3F3 (Novus Biologicals, NBP1-49872, 1:500), HNF4G (Thermo Fisher Scientific, PA5-82189, 1:200), LRP2 (My Bio Source, MBS690201, 1:500), HAVCR1 (R&D Systems, AF1750, 1:200), γH2AX (Cell Signaling Technology, 2577), LAMB1 (Santa Cruz Biotechnology, sc-33709, 1:250), ATP1A1 (Abcam, ab7671, 1:200), and SOX9 (Abcam, ab185230, 1:300).

Techniques: Staining, Control, Expressing, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Measurement of adhesion and traction of cells at high yield (MATCHY) reveals an energetic ratchet driving nephron condensation

doi: 10.1101/2024.02.07.579368

Figure Lengend Snippet: A biophysical atlas of primary mouse nephrogenic niche cells reveals an energetic ratchet accompanying nephron progenitor differentiation. (A) Left , Whole-mount immunofluorescence micrograph of E17 kidney cortical surface showing CITED1+ SIX2+ nephron progenitor niche compartments and LHX1, JAG1 differentiation markers. Right , schematic of niche anatomy and stages of nephron progenitor differentiation. NPC, nephron progenitor cell; PN, primed nephron progenitor; PTA, pre-tubular aggregate; RV, renal vesicle; BRV, beyond renal vesicle (comma-shaped body, S–shaped body, etc.). (B) Top , tSNE plot of cell clusters and feature plots of cadherin expression over the nephron differentiation trajectory from scRNA-seq data published in Combes et al. 2019. Arrows indicate clusters having appreciable marker expression. Bottom , gene set enrichment analysis results for the listed sets, comparing NPC/PN stages to PTA/RV stages. (C) Montage of phase and immunofluorescence micrographs and traction force microscopy output (displacement, disp., and stress fields) for primary mouse E17 embryonic kidney nephrogenic zone cells representative of each cell type along the differentiation trajectory. (D) Top , t-SNE dimensionality reduction plots based on expression of the markers in (C) showing annotation of clusters by cell type (left), and by traction force (right). Bottom , Violin plots of traction force by cell type showing means and quartiles (gray lines, n = 8 replicate wells; red points are means of replicates). (E) Plot of cell-cell contact angle between the indicated homotypic and heterotypic pairs (mean ± S.D., n > 100 pairs per comparison), and heat-map matrix of median contact angles. Arrows highlight relationship between homotypic contact angle for a given cell type and that for its heterotypic contact with the most closely related cell type along the differentiation trajectory. (F) Biophysical atlas showing median traction and homotypic adhesion data mapped as colors onto the niche schematic. Statistics in (D) and (E) are one-way ANOVA, Tukey’s test, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The wells were then incubated with fluorophore-conjugated primary antibodies: rabbit anti-Six2 (11562-1-AP, Proteintech, RRID: AB_2189084) labeled with Alexa Fluorphore 555 (Novus, 333-0010), mouse anti-Lhx1 (CF504527, OriGene, RRID: AB_2724601) labeled with Alexa Fluorphore 647 (Novus, 336-0010), and rabbit Cited1-488 (Proteintech, CL48826999100UL, RRID: AB_2919211) as the first set and rabbit anti-SIX2-555, mouse anti-LHX1-647, and goat anti-JAG1 (AF599, R&D Systems, RRID: AB_2128257) labeled with Alexa Fluophore 488 (Novus, 332-0005).

Techniques: Immunofluorescence, Expressing, Marker, Microscopy, Comparison