Supplementary Figure 1: Additional characterization of pllp expression and pllp loss-of-function

(A) In situ hybridization of pllp probe at 48–72 hpf. Embryos and larvae were collected and fixed at different time points, and incubated with a DIG-labelled PLLP antisense RNA probe and AP-linked anti-DIG antibody. ISH from all different time points were performed at the same time and developed for 2 h before fixation and cleanup. Arrow indicates gut. (B) TgBAC(pllp-GFP) transgenic zebrafish at 48 hpf. Transgenic fish were bred and embryos and larvae were analysed by epifluorescence microscopy. (C) Transverse sections of TgBAC(pllp-GFP) fish at 48–96-hpf. Sections from the posterior midgut (about 1/3 total gut length before the cloaca) were stained to analyse GFP expression using phalloidin (which labels F-actin in apical microvilli) and DAPI (for DNA). Arrows indicate apical localization. Scale bars, 10 μm (magnification, 5 μm). (D) TALEN-generated pllp^pd1116 mutant null allele. TALENs were generated to target the first exon of zebrafish pllp and injected into 1-cell embryos. After raising the founders, we cloned an allele, pd1116 that harbours a null mutation. We confirmed RNA nonsense mediated decay of the pllp mRNA in the homozygous mutant embryos by measuring pllpmRNA levels in pllp^pd1116 mutant larvae at 120 hpf by RT-qPCR. Results are mean ± s.d.% expression relative to control and normalized with rRNA 18S expression (n = 3 RNA extracts; ^∗P < 0.05 (Student’s t test); Statistic source data can be found in Supplementary Table 3). (E) Live whole-mount images of WT and pllp^pd1116 larvae at 144 hpf. (F) Quantification of total number of intestinal cells per section of 6 dpf pllp^pd1116 larvae. 144 hpf larvae were fixed, sectioned and stained with DAPI and F-actin to quantify the total number of intestinal cells per section. Results are represented as mean ± s.d. total number of nuclei in a 2 μm-thick cross-sections (n = 10 sections from 5 WT and 5 pllp^pd1116 fish selected from 3 independent experiments). (G) Toluidin-stained EM sections of pllp^pd1116 mutant larvae (400x magnification). Yellow arrows indicate enlarged immature endocytic compartments. Red bars are shown to compare the difference in cell height. Scale bars, 5 μm. (H) Silencing of TgBAC(pllp-GFP) in morpholino-injected 4 dpf larvae. TgBAC(pllp-GFP) embryos were injected with MO1 and MO2 morpholinos at one-cell stage and allowed to grow until 4 dpf. Whole animal lysates from 20 larvae were analysed by Western blot, using anti-GFP antibodies. (I) Quantification of % of morpholino-injected larvae presenting disrupted epithelial columnar organization. WT embryos were injected with MO1 and MO2 morpholinos at one-cell stage and grown until 4 dpf. Posterior gut sections were stained with Phalloidin (green) and DAPI (red) and analysed by confocal microscopy. Data are presented as % of total larvae with disrupted columnar organization (n = 58 control, 25 MO1 and 20 MO2-injected larvae pooled from three independent morpholino injection experiments; ^∗P < 0.05 (Student’s t test)). (J) Confocal images showing the phenotype of morpholino-injected larvae quantified in (I). L, lumen. Scale bars, 5 μm. (K) Quantification of microvilli length measured in TEM at 6000x magnification. Results are represented as mean ± s.d. length (in μm) (n = 20 microvilli length averages per cell from 5 WT and 5 mutant cells, randomly selected from 2 independent experiments; ^∗P < 0.05 (Student’s t test)). (L) Quantification of apical endosomes in WT and pllp^pd1116 larvae. Diameters of apical endosomes (within 0-3 μm from the apical surface) were measured from TEM images. Results are represented as mean ± s.d. number of endosomes per cell (n = 10 cells from 5 WT and 5 mutant fish, randomly selected from 2 independent experiments; ^∗P < 0.05 (Student’s t test)) (M) Quantification of apical membrane expansion in pllp^pd1116 larvae. Perimeter of the whole luminal apical membrane (red) was divided by the perimeter of the basal membrane (blue). Results are represented as mean ± s.d. apical/basal perimeter ratios per cell (n = 20 cells per condition, from 4 control and 4 mutant fish, randomly selected from 4 independent experiments; ^∗P < 0.05 (Student’s t test)). (N) Quantification of larvae survival in the first 18 dpf. WT and pllp^pd1116 5 dpf larvae (n = 25) were raised in 1L tanks with limited food supply and were assessed by observing heartbeat every day until 18 dpf. Solid lines indicate WT larvae. Dotted lines indicate pllp^pd1116 larvae. (O) Rescue of dextran endocytosis in pllp^pd1116 larvae carrying the TgBAC(pllp-GFP)pd1115 transgene. 144 hpf larvae were gavaged with Dextran-TR, incubated for 2 h, sectioned and stained with DAPI (blue, DNA). Scale bars, 10 μm. (P) Quantification of phenotype rescue. 144 hpf larvae from a cross of TgBAC(pllp-GFP) pllp^+/pd1116 with pllp^{pd1116/pd1116} fish were gavaged with Dextran-TR, incubated for 2 h, sorted by transgenic GFP expression, and then the percentage of larvae with disrupted endocytosis (<50%) and disrupted cell height (<15μm) phenotypes were quantified in each case (GFP+, N = 39; GFP-, N = 85). (Q) Rescue of Rab11 subapical localization in pllp^pd1116 larvae carrying the TgBAC(pllp-GFP) transgene. 144 hpf WT larvae were fixed, sectioned and stained with the anti-Rab11 antibody (red) and DAPI (blue, DNA). Scale bars, 10 μm.