Fig. 3

The reduced fluidity of tbx16 mutant MPZ is due to a decrease in extracellular spaces and junctional fluctuations.

A Measured nuclear density from DAPI stains (insets) (N = 5 embryos for both conditions; center line: mean; box: SE; whiskers: SD). B Segmented location (white) of cell membranes (green). Junction length (l_t) is tracked over time (magenta). C Distribution of normalized junctional fluctuations in tbx16 mutant (red, n = 4058; N = 4 embryos) and sibling (black, n = 4121; N = 5 embryos) embryos. Dashed box highlights tail of distribution. D Representative confocal section showing extracellular spaces (black, inverted LUT). Closeups show zoomed lateral MPZ (L-PMZ) and medial MPZ (M-MPZ). E Volume fraction in the L-MPZ and M-MPZ of tbx16 mutants compared to sibling embryos (p = 0.00689; one-way ANOVA with Tukey test for mean comparison) (N = 20 for sib and N = 22 for tbx16^−/−; center line: mean; box: SE; whiskers: SD). F Representative confocal images of dorsal and ventral slices through posterior tissues of transgenic Cdh2 reporter embryos (zf516Tg) and Cdh2 immunofluorescence stained (sibling and tbx16^−/−) embryos (Methods). Slices are defined as dashed lines in orthogonal yz views (parasagittal sections; top left panel). ROIs (white dash boxes; aDorsal. DMZ, M-MPZ, and L-MPZ) were manually defined on these slices for quantification. Magenta: Cdh2 stained with AlexaFluor 594; Yellow: superfolder GFP labeled Cdh2; Cyan: DAPI stained nuclei. G Normalized Cdh2 levels in aDorsal and DMZ relative to M-MPZ levels. Quantification for both the transgenic reporter line zf516Tg and immunofluorescence staining of wild-type siblings are shown (N = 5 embryos in each group; whiskers: SE; black dot: mean). H Normalized Cdh2 levels in the DMZ and L-MPZ relative to values in the M-MPZ for both tbx16 mutants and siblings (p = 0.03671; Mann-Whitney test). Inset: normalized Cdh2 levels in the M-MPZ of tbx16 homozygous mutants and their wild-type siblings (N = 5 embryos in each group; whiskers: SE; black dot: mean). Scale bars: 20 µm (A); 10 µm (B); 40 µm (D); 10 µm (D) inset; 50 µm (F). Source data are provided as a Source Data file.