DEV038448 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Early components of the hi1520 mutant phenotype include proliferation and cell death. (A,B,D,E) Wild type (A,D) and clint1 mutants (B,E) immunolabeled for BrdU (A,B) or stained with AO (D,E) at 36 hpf. Arrows label proliferation (B) and cell death (E). (C,F) Quantification of proliferation (C) and cell death (F) in wild type (circles) and clint1 mutants (triangles). Scale bars: 200 µm

• Supplemental Figure S2 -

  Fig. S2. Functional neutrophil behaviors in clint1 mutants. (A-C) Quantification of migratory characteristics of neutrophils in clint1;mpo:GFP mutants before (A-C; clint1 mutants) and after (B,C; clint1 post-wound) wounding. (A) Plot of average velocity (µm/minute) versus percentage of time stopped. (B) Plot of average velocity (µm/minute) versus directionality (D/T). Each data point indicates parameters for an individual neutrophil from three clint1;mpo:GFP embryos before (blue) and after (orange) wounding. Data were averaged per embryo and are represented by dashed lines. (C) Table summarizing neutrophil tracking data from time-lapse movies of clint1;mpo:GFP embryos before (clint1 mutants, D, see Movie 2) and after (clint1 post-wound, E, see Movie 3) wounding. Standard deviations are shown. (D,E) Total migration paths taken by neutrophils overlaid onto the first frame of representative movies. (D) Arrowheads identify bidirectional tracks. Yellow squares identify starts of tracks. (E) Asterisk identifies wound. (F) Quantification of leukocyte displacement in embryos raised in sterile E3 (circles) or sterile E3 with significant amounts of E. coli added (triangles). NS, not significant. Scale bars: 50 µm.

• Supplemental Figure S3 -

  Fig. S3. clint1-specific MOs phenocopy hi1520 mutant phenotypes. Standard control MO (con MO) (A,B), clint1-ex1 MO (C,D) or clint1-atg MO (E,F) injected wild-type embryos immunolabeled at 48 hpf for Mpo (green) and BrdU incorporation (red). Arrowheads identify epidermal aggregates (C,E) and proliferative cells (D,F), and arrows identify neutrophils (D,F). Scale bar: 200 µm.

• Supplemental Figure S4 -

  Fig. S4. The zebrafish proteins Clint1 and Vti1b co-localize but have non-overlapping roles in epidermal development. (A-D) Expression of mCherry-clint1 in zebrafish epidermal cell (A) or mCherry-clint1 (B,D) and vti1b-GFP (C,D) in HEK293 cells. (E) Schematic of vti1b exon structure and primers used to investigate effects of vti1b-ex1 MO on splicing. (F-K) Control MO (F,G), vti1b-atg MO (H,I) or vti1b-ex1 MO (J,K) injected embryos immunolabeled for MPO (green) and p63 (red). (L) RT-PCR amplification of vti1b and ef1a from control and clint1-ex1 MO-injected wild-type embryos. Arrow and asterisks indicate full-length and alternatively spliced vti1b transcripts, respectively. Scale bars: 10 µm in A,D; 200 µm in K.

• Supplemental Figure S5 -

  Fig. S5. Onset of clint1 mutant phenotype is unaffected by inhibition of myeloid development. (A-D′) Caudal fin fold views of wild-type (A-D) and clint1 mutant (A′-D′) embryos injected with control MO (A,A′,B,B′) or pu.1 MO (C,C′,D,D′) and immunolabeled for MPO (green) and BrdU (red, arrows). Note proliferation (arrows) in both the presence (B′) and absence (D′) of neutrophils in clint1 mutants at 48 hpf. Scale bar: 200 µm.

• Supplemental Figure S6 -

  Fig. S6. Normal proliferation of clint1 morphant cells in a wild-type environment and unaffected Pkcζ localization in clint1 mutants. (A-B′) Cells from uninjected (A,B) or clint1-ex1 MO-injected (A′,B′) Tg(β-actin:hras-eGFP) embryos were transplanted to the ventral ectoderm of non-transgenic wild-type embryos. (C) Quantification of GFP-positive cells in unlabeled recipients at 2 and 3 dpf. (D,E) Transverse sections of wild-type (D) and clint1 mutant (E) embryos immunolabeled for Pkcζ (green) and p63 (red) and with DAPI (blue) at 48 hpf.

• Movie 1 -

  Movie 1. Confocal in vivo time-lapse recording of a GFP^lo cell displaying phagocytic behavior by engulfing debris in lateral epidermis of clint1;mpo:GFP embryo. Movie starts at 3 dpf and lasts for 120 minutes. Note the pseudopod extension that attaches to debris. Images from this movie were used for Fig. 3C,D.

• Movie 2 -

  Movie 2. In vivo time-lapse recording of neutrophil migration in clint1;mpo:GFP embryo before wound. Movie starts at 3 dpf and lasts for 180 minutes. Neutrophils display bidirectional trafficking between vasculature and epidermis. Images from this movie were used for Fig. S2D, and data were included in quantifications shown in Fig. S2A-C.

• Movie 3 -

  Movie 3. In vivo time-lapse recording of neutrophil migration in clint1;mpo:GFP embryo post-wound. Movie starts after wounding of embryo shown in Movie 2 and lasts for 180 minutes. Neutrophils are recruited to the wound and continue to display bidirectional trafficking between vasculature and epidermis. Images from this movie were used for Fig. S2E, and data were included in quantifications shown in Fig. S2B,C.

• Movie 4 -

  Movie 4. In vivo time-lapse recording of mCherry-clint1 expression in zebrafish epidermal cell. Movie starts at 3 dpf and lasts for 17 minutes. clint1 expression appears to be in intracellular vesicles. Images from this movie were used for Fig. S4A.

• Movie 5 -

  Movie 5. Confocal in vivo time-lapse recording of a cluster of mGFP-labeled wild-type epidermal cells in a wild-type environment. Movie starts at 48 hpf and lasts for 90 minutes. Cells are maintained in a rigid epithelium. Images from this movie were used for Fig. 8A and were quantified in Fig. 8C.

• Movie 6 -

  Movie 6. Confocal in vivo time-lapse recording of a cluster of mGFP-labeled clint1 morphant epidermal cells in a clint1-deficient environment. Movie starts at 48 hpf and lasts for 90 minutes. Cells display a fibroblastoid shape and migratory behavior. Cells 1-7 change positions within the cluster. Images from this movie were used for Fig. 8B and were quantified in Fig. 8D.