Fig 2: Immunofluorescence staining reveals that low FSS enhances the accumulation of IFT88 at the basal body, and this protein may participate in cilia assembly. (Aa-Ac) Stationary cultured cells. (Ba-Dc) Cells were loaded with low FSS. (Ea-Gc) Cells were loaded with laminar FSS. IFT88 was labeled with red fluorescence and Dvl2 with green. (Aa-Da) Loading of low FSS increased the expression of IFT88 (arrows). (Ea-Ga) Loading of laminar FSS led to only a few cells expressing the protein (arrows). (Ac-Gc) Yellow fluorescence indicated the colocalization of these two proteins, which could be observed in nearly all cells at 12 h (arrows), and after 18 h in response to low FSS, these two proteins were separated, rendering it difficult to capture them in the same focal plane (arrow); however, the proteins were dispersed in some cells under the action of laminar FSS at 18 h (arrows). Scale bars=20 µm. Dvl2, dishevelled segment polarity protein 2; FSS, fluid shear stress; IFT88, intraflagellar transport 88.

Fig 3: A knockdown of Dvl1/2/3 in the testis in vivo perturbs spermatogenesis.a Regimen used to treat adult rats with n = 6 rats per treatment or control group. Testes received the Dvl1, Dvl2, and Dvl3-specific siRNA duplexes (triple knockdown) vs. non-targeting negative control siRNA duplexes on day 0, 2, and 4 (thrice) and rats were killed on day 6 for either IB, histological analysis, or immunofluorescence (IF) analysis. This regimen was established based on results of pilot experiments as a knockdown of Dvl3 alone failed to induce considerable phenotype in the testis, thus a triple knockdown by RNAi was used instead. b Representative IB data from n = 3 experiments, illustrating successful knockdown of Dvl1, Dvl2, and Dvl3, without affecting the expression of other PCP proteins such as Fzd3 and Vangl2, nor other BTB-associated except for a considerable downregulation of actin barbed end capping and bundling protein Eps8 (known to maintain actin filament bundles at the ES), and upregulation of MARK2 regulatory protein (known to promote MT catastrophe, i.e., destabilizing MTs), consistent with findings in vitro (see Fig. 4c). Uncropped IBs were shown in Figure S5. Histograms in the lower panel are the composite IB data such as those shown in the upper panel from n = 3 independent experiments. Each bar is a mean ± SD of three experiments. *, P < 0.01 when compared with the corresponding control by Student’s t test. c Histological analysis was performed using paraffin sections (~ 5 µm thick) of testes (fixed in modified Davidson fixative) and stained with hematoxylin and eosin. Control testes transfected with non-targeting negative control siRNA duplexes had normal spermatogenesis in the seminiferous epithelium (left panel). However, considerable defects in spermatogenesis were detected in the epithelium from testes following Dvl1/2/3 knockdown by RNAi as noted in the second and third columns; selected areas in these two columns were boxed in either red or blue and magnified in the corresponding insets to illustrate defects. These include: (i) step 19 spermatids (white arrowheads) found in stage I–IV tubules, co-existing with steps 15–17 spermatids, and also some multinucleated round spermatids (green arrowheads); (ii) step 19 spermatids remained trapped deep inside the epithelium in stage VII or VIII tubules (blue arrowheads) when they should have been transported near the tubule lumen to prepare for their release at spermiation, also defects in spermatid polarity were noted with these spermatids no longer pointed toward the basement membrane but deviated by 90°–180° from the intended orientation (yellow arrowheads); (iii) step 19 spermatids were detected in the epithelium, co-existing with step 9,10, 11, or 12 spermatids (white arrowheads) and mult-nucleated round spermatids (green arrowheads) in stages IX–X and XI–XII tubules, and some step 19 spermatids had defects in polarity (yellow arrowhead); and (iv) considerable thinning of the epithelium was also detected, possibly due to unwanted release of elongated spermatids as a result of defects in cytoskeletal organization. Scale bar, 80 µm in first panel, which applies to all other images in first, second and third panels; 40 µm in red and blue insets, which apply to all corresponding insets. d Percentage (%) of defective tubules from at least 100 randomly scored tubules from cross-sections of a rat testis with n = 4 rats. *, P < 0.01 compared with the control group by Student’s t test

Fig 4: Disrupted Wnt5a signaling of Prickle1 mutant alleles.(A) Prickle1 proteasomal degradation on Wnt5a signaling. HEK293 cells were transfected with combinations of GFP-Prickle1, Wnt5a and Ryk. Anti-GFP antibody is used to immunoprecipitate Prickle1. Anti-ubiquitin antibody was used to detect ubiquitinated Prickle1 on an immunoblot. Increase in ubiquitinated Prickle1 is detected upon Wnt5a and Ryk transfection and treatment with MG-132 (lane2 and lane3 with lane1). The same blot was then probed with anti-GFP antibody to detect unmodified GFP-tagged Prickle1 (lower blot). The probable intact GFP-tagged Prickle1 protein roughly inversely correlates to the ubiquitination level (upper panel). (B) Upregulation of Prickle1 protein in Wnt5a-/- limb tissues of distal paws at E17.5. (C) Wnt5a stimulates phosphorylation of Dvl2 in MEFs. (D) Reduced unphosphorylated form (native form) of Dvl2 in the mutant kidney and limb tissues at P0, mimicking application of Wnt5a to cultured MEF cells. (E) Decreased dephosphorylated Dvl2 in Wnt5a-/- limb tissues of the distal paws at E17.5. Same Western blot was reprobed by anti-RNA polymerase II (PoII) for loading control. ps-Dvl2: Phosphorylation-shifted Dvl2. (F) A proposed role of Prickle1 in regulation of Wnt5a signaling- controlled cellular events. Prickle1 inhibits Dvl2 phosphorylation, which is critical for localized actin assembly and asymmetrical cellular localization of Dvl2. Aberrant Dvl2 phosphorylation would lead to defects in cell shape, cell migration and arrangement during tissue morphogenesis. Wnt5a signaling adjust status of Dvl2 phosphorylation partially through degradation of Prickle1.

Fig 5: Loss of Nedd4 and Nedd4l enhances ISC expansion upon RSPO stimulation AExperimental design of RSPO3 stimulation model.B, CRepresentative images of EdU (B) and Olfm4 (C) staining in intestinal tissues obtained from the indicated genotypes. Scale bar, 50 µm.DModel of proposed mechanism of NEDD4/NEDD4L-mediated regulation of Wnt pathway. NEDD4 and NEDD4L target two Wnt pathway components: (i) LGR4/5 receptor for lysosomal and proteasomal degradation, and (ii) DVL2 for proteasomal degradation.