Files in this Data Supplement:
Fig. S1. lef1nl2 mutants lack terminal NMs and have a truncated pLL nerve. (A,B) Wild-type sibling and lef1nl2 mutant larvae at 4 dpf. The pLL nerve is visualized by expression of the TgBAC(neurod:EGFP) transgene. NMs are labeled with DASPEI. (A) Wild-type embryo contains NMs L1-L5, a secondary (2°) NM and the terminal cluster (tc). (B) lef1nl2 mutant contains NMs L1-L5 and a 2° NM, but lacks a terminal cluster (tc). In addition, the lef1nl2 mutant has a truncated pLL nerve (white arrow). (C,D) Functional hair cells in the final deposited NM labeled with FM 1-43 (red) in wild-type (C) and lef1nl2 larvae (D) at 4 dpf. Nuclei are labeled with DAPI. (E) Quantification of FM 1-43-labeled hair cells showing no significant difference between wild-type and mutant larvae (F) Average number of DAPI-labeled nuclei per NM in wild-type and lef1nl2 (n=6, mean ± s.e.m.). Scale bar: 20 µm.
Fig. S2. Adult lef1nl2 mutants show defects in fin and tooth formation. (A,B) Bright-field images of adult wild-type sibling (A) and lef1nl2 mutant (B) fish. Note that the lef1nl2 mutant lacks a caudal fin. (C-D′′) Alizarin Red staining in calcified bone in wild-type (C) and lef1nl2 mutant (D) fish. Lepidotrichia in both paired pectoral fins (arrows) and the caudal fin (arrowheads) are formed in the wild-type fish (C-C′′). By contrast, these structures are stunted in the lef1nl2 mutant (D-D′′). (E,F) Alizarin Red staining of the pharyngeal teeth and bone of the jaw and Alcian Blue staining of cartilage in the gill rakers. (E) Wild-type jaw with several pharyngeal teeth (black brackets). (F) By contrast, the lef1nl2 mutant jaw contains only one tooth (black arrow). Gill rakers appear normal in both wild-type and lef1nl2 mutant fish.
Fig. S3. Adult pLL is truncated in lef1nl2 mutants. DASPEI labeling of the pLL in adult wild-type sibling and lef1nl2 mutant fish carrying the TgBAC(neurod:EGFP)nl1 transgene. (A) The wild-type fish shows NM stitches in the lateral (white arrowheads) and ventral (red arrowhead) stripes. (B) The lef1nl2 mutant has a truncated lateral stripe and fully formed ventral stripe (blue arrow). Adults were between 4 months post-fertilization and 22-23 mm in length.
Fig. S4. Primordium patterning is disrupted following induction of Tg(hsp70l:dkk1-GFP). RNA in situ hybridization at 32 hpf to visualize expression of patterning markers in wild-type embryos (left panels) and Tg(hsp70l:dkk1-GFP) embryos heat-shocked at 28 hpf reveals that these factors are downregulated following Dkk1 transgene induction; lef1 (A,B), fgf10a (C,D) and pea3 (E,F). Expression of the chemokines cxcr4b (G,H) and cxcr7b (I,J) are indistinguishable between wild-type and heat-shocked Tg(hsp70l:dkk1-GFP) embryos. Scale bar: 20 µm.
Fig. S5. Expression of tcf genes in the primordium. At 30 hpf, tcf7 is expressed in the leading zone of the primordium (A), tcf7l1a is expressed throughout the primordium, but not in leading edge cells (B) and tcf7l1b is expressed in a deposited NM, but not in the primordium (C) in a wild-type embryo. Scale bar: 20 µm. (D) Pectoral fin development in embryos injected with the tcf7-MO in a heterozygous (E) or lef1nl2 mutant (F) background at 6 dpf. Pectoral fins are stunted or absent in the lef1nl2;tcf7-MO embryo when compared with the lef1nl wild-type sibling injected with tcf7-MO (red arrows). Scale bar: 50 µm.
Fig. S6. NM number is decreased in lef1nl2 mutant/tcf7 morphant and Tg(hsp70l:dkk1-GFP) embryos. (A) Quantification of NM number at 2 dpf in wild-type sibling and lef1nl2 mutant embryos injected with the tcf7 MO showing a significant decrease in the number NMs in lef1nl2/tcf7-MO embryos (n=11, **P<0.0001, Student�s t-test). (B) Quantification of NMs (L2-L5) at 2 dpf in control and Tg(hsp70l:dkk1-GFP) embryos heat shocked at 28 hpf. There is a significant decrease in NM number in Tg(hsp70l:dkk1-GFP) embryos following heat shock. NM L1 was excluded from the data as it was specified before heat shock (n=15, ***P<<0.0001, Student�s t-test). Data are presented as mean±s.d.
Fig. S7. Loss of Lef1 function does not lead to cell death in the primordium. (A-B′′′) Confocal projections of 40 hpf wild-type (A) and lef1nl2 mutant (B) primordium that have been labeled by TUNEL for cell death (A′,B′), with Tg(-8.0cldnb:lynGFP) to visualize primordium (A′′,B′′) and by DAPI to visualize nuclei (A′′′,B′′′). There are no detectable TUNEL-positive cells in lef1nl2 mutants when compared with wild-type controls. (C-D′′′) Tunnel labeling of wild-type (C) and lef1-MO-injected (D) embryos at 35 hpf. Little TUNEL labeling was detected in lef1-MO embryos. Scale bar: 20 µm.
Fig. S8. Activation of the Tg(hsp70l:dkk1-GFP) transgene leads to cell death in the primordium. (A-B′′′) Confocal projections of 34 hpf primordia of control embryos (A) or Tg(hsp70l:dkk1-GFP) embryos heat shocked at 28 hpf (B). Cell death was detected by TUNEL (A′′,B′′); primordia were visualized by expression of Tg(-8.0cldnb:lynGFP) transgene (A′,B′) and nuclei were labeled by DAPI (A′′′,B′′′). Although no TUNEL-labeled cells were present in the control primordium (A-A′′′), a number of cells are TUNEL-positive in Tg(hsp70l:dkk1-GFP) embryos heat shocked at 28 hpf (B-B′′′), Scale bar: 20 µm.
Fig. S9. Converted Kaede cells divide outside of the leading zone in lef1nl2 mutants. Schematic event map showing the location of converted Kaede cells at 24 hpf in wild-type (blue) and lef1nl2 mutant (green) embryos and the location of individual cell division events during live imaging of primordium migration between 24 hpf and 48 hpf. (n=4 wild-type and 4 leflnl2 embryos).
Movie 1. Wild-type primordium migration, rosette renewal and NM deposition. Time-lapse of confocal projections of a wild-type embryo expressing Tg(-8.0cldnb:lynGFP) transgene. The embryo was imaged every 6 minutes for 13 hours beginning at 34 hpf. During this time, three NMs were deposited from three rosettes present at the start of imaging (red, blue and yellow arrows) and two new rosettes were formed in the primordium (green and pink arrows). Scale bar: 20 µm.
Movie 2. Rosette renewal and primordium organization is disrupted in a lef1nl2 mutant. Time-lapse of confocal projections of a lef1nl2 mutant embryo expressing the Tg(-8.0cldnb:lynGFP) transgene. The embryo was imaged every 6 minutes for 14 hours beginning at 34 hpf. At the start of imaging, three rosettes were visible in the primordium, which were deposited over the course of migration. One additional proto-NM was formed and was deposited (green arrow). The leading region of the primordium fails to form a new rosette and migration stalls (pink arrow). Scale bar: 20 µm.
Movie 3. Converted Kaede cells proliferate and remain in the leading zone of the wild-type primordium. Time-lapse of confocal projections of a wild-type primordium that contains four brightly labeled and one faintly labeled red Kaede-positive cells in a Tg(-8.0cldnb:lynGFP) transgenic background. The embryo was repositioned between 328.5 and 335.8 minutes to capture complete migration of the primordium. Cells divide in the leading zone (blue arrows) and in rosettes (yellow arrows). All red Kaede cells remain in the primordium. Frames were collected every 7.3 minutes for 1204.5 minutes between 26 and 45 hpf. Scale bar: 20 µm.
Movie 4. In a lef1nl2 mutant lineage, labeled cells proliferate in the primordium and NMs. Time-lapse confocal projection of a lef1nl2 mutant primordium containing two converted Kaede cells. These cells divide in the leading zone (blue arrows) and are then sorted out of the leading zone and incorporated into NMs. Cells continue to divide in NMs (yellow arrows). The embryo was repositioned between minutes 693.5 and 700.8. One red Kaede cell is lost during the repositioning. Frames were collected every 7.3 minutes for 1146.1 minutes between 26 and 45 hpf. Scale bar: 20 µm.
Movie 5. Wild-type cells remain in the leading region of the primordium during migration. Time-lapse of confocal projections of a wild-type, Tg(-8.0cldnb:lynGFP)-positive primordium that contains rhodamine-dextran labeled wild-type donor cells (yellow arrowhead) in the leading region (white arrow). Note that donor cells remain in the leading region of the primordium during the course of migration. Frames were collected every 8 minutes for 818 minutes beginning at 32 hpf. Scale bar: 20 µm.
Movie 6. lef1nl2 mutant cells are sorted out of the leading region of a wild-type primordium. Time-lapse of confocal projections of a wild-type, Tg(-8.0cldnb:lynGFP)-positive primordium that received rhodamine-dextran labeled cells from the lef1nl2 mutant. At the onset of migration, a subset of donor cells (yellow arrowhead) was localized to the leading region of the primordium (white arrow). By the end of the movie (800 minutes), the donor cells (yellow arrowhead) have been sorted out of the leading region (white arrow). Frames were collected every 8 minutes for 800 minutes beginning at 32 hpf. Scale bar: 20 µm.
Movie 7. Loss of Wnt signaling in leading edge cells leads to a failure of primordium migration. Time-lapse of confocal projections of a wild-type Tg(-8.0cldnb:lynGFP)-positive host primordium that contains rhodamine-dextran-labeled, Tg(hsp70l:dkk1-GFP) donor cells (yellow arrows) in the leading edge. Embryos were heat-shocked at 28 hpf. Over the course of the movie, the primordium lost its organization, elongated and eventually stalled. Note that a subset of host cells (white arrow) migrated beyond the donor cells (yellow arrowhead). Frames were collected every 6 minutes for 714 minutes beginning at 37 hpf. Scale bar: 20 µm.