Files in this Data Supplement:
Fig. S1. All alleles of sdp exhibit complete DRG loss. (A) sdpw13/w13 exhibits normal morphology and pigmentation. Scale bar: 500 µm. (B) A high-magnification image of 3 dpf Tg(neurog1:egfp)/sdpw13/w13 zebrafish immunostained for Elavl1 reveals no DRG neurons. Scale bar: 50 µm. (C) Approximately 25% of 3 dpf embryos from a cross between sdpw13/+ parents fail to form neurog1+ DRG, indicating that sdpw13 is a fully penetrant recessive mutation. (D) sdpw15/w15 exhibits normal pigmentation but acquires a progressively worsening edema around the eyes and heart beginning at 3 dpf. Scale bar: 500 µm. (E) A high-magnification image of 3 dpf Tg(neurog1:egfp)/sdpw15/w15 zebrafish immunostained for Elavl1 reveals no DRG neurons. Scale bar: 50 µm. (F) Approximately 25% of 3 dpf embryos from a cross between sdpw15/+ parents fail to form neurog1+ DRG, indicating that sdpw15 is a fully penetrant recessive mutation. (G) sdpw14/w14 exhibits normal morphology and pigmentation. Scale bar: 500 µm. (H) A high-magnification image of 3 dpf Tg(neurog1:egfp)/sdpw14/w14 zebrafish immunostained for Elavl1 reveals no DRG neurons. Scale bar: 50 µm. (I) Approximately 25% of 3 dpf embryos from a cross between sdpw14/+ parents fail to form neurog1+ DRG, indicating that sdpw14 is a fully penetrant recessive mutation.
Fig. S2. Derivatives of the neural crest other than DRG are largely unperturbed in sdp embryos. (A) Brightfield micrograph of a sibling 4 dpf zebrafish embryo. Scale bar: 500 µm. (B) Brightfield micrograph of a sdpw12/w124 dpf embryo. The sdpw12/w12 jaw is retracted (arrowhead). (C) Cranial ganglia of a sibling 4 dpf embryo as identified by Elavl1 immunostaining. ad, anterodorsal lateral line ganglion; av, anteroventral lateral line ganglion; f, facial ganglion; g, glossopharyngeal ganglion; m, middle lateral line ganglion; o, octaval/statoacoustic ganglion; p, posterior lateral line ganglion; t, trigeminal ganglion; v, vagal ganglia. Scale bar: 100 µm. (F) All cranial ganglia are present in sdpw12/w12 embryos. (D) Enteric nervous system of a sibling 5 dpf embryo as shown by Elavl1 immunostaining. Scale bar: 100 µm. (G) Enteric nervous system of a sdpw12/w125 dpf embryo. The enteric nervous system migrates the full extent of the gut. (E) Lateral line nerve of a sibling 4 dpf embryo stains positive for acetylated tubulin; myelinating glia are marked by the sox10:eos transgene. Scale bar: 50 µm. (H) Lateral line nerve and associated myelinating glia are present in sdpw12/w12 embryos. (I) Sympathetic neurons (arrowheads) in a sibling 5 dpf embryo immunostained for Elavl1. Scale bar: 150 µm. (J) Sympathetic neurons are also present in 5 dpf sdpw12/w12 embryos. Scale bar: 150 µm. (K) Head cartilages of a sibling 4 dpf embryo. Note the position of the ceratohyal cartilage (arrowhead). ch, ceratohyal; hs, hyosymplectic; pq, palatoquadrate. Scale bar: 100 µm. (K′) Branchial arches of a sibling 4 dpf embryo. bh, basihyal; cb, ceratobranchial; ih, interhyal. Scale bar: 100 µm. (L) Head cartilages of a sdpw12/w124 dpf embryo. The ceratohyal cartilage is retracted (arrowhead). Scale bar: 100 µm. (L′) All branchial arches of the sdpw12/w124 dpf embryo are present. Scale bar: 100 µm.
Fig. S3. Cranial ganglia form normally in sdp embryos. (A) Tg(sox10:eos)/sdpw12/+ embryo at 24 hpf immunostained for Elavl1. ad, anterodorsal lateral line ganglion; ag, anterodorsal lateral line ganglion (Andermann et al., 2002); f, facial ganglion; o, octaval/statoacousic ganglion; t, trigeminal ganglia. Scale bar: 50 µm. (B) Tg(sox10:eos)/sdpw12/+ embryo at 48 hpf immunostained for Elavl1. Scale bar: 50 µm. (C) Tg(sox10:eos)/sdpw12/w12 embryo at 24 hpf immunostained for Elavl1. Scale bar: 50 µm. (D) Tg(sox10:eos)/sdpw12/w12 embryo at 48 hpf immunostained for Elavl1. Scale bar: 50 µm. (E) Quantification of Elavl1+ cell counts per ganglion taken from Tg(sox10:eos)/sdpw12/+ and Tg(sox10:eos)/sdpw12/w12 embryos at 24 hpf. There is no significant difference in Elavl1+ cell count between genotypes in any ganglion counted. (F) Quantification of Elavl1+ cell counts in the trigeminal/facial ganglion taken from Tg(sox10:eos)/sdpw12/+ and Tg(sox10:eos)/sdpw12/w12 embryos at 48 hpf. There is no significant difference in Elavl1+ cell count between genotypes. Error bars represent s.d. n.s., not significant.
Fig. S4. sdpw12/w12 exhibits upregulation of metalloproteases. Whole protein isolate from both sdpw12/w12 (black circles) and sibling embryos (white circles) was assayed for protease activity after a 4 hour incubation. Mean activity is indicated by circles; error bars are represent s.e.m. A two-way analysis of variance showed that the genotype factor was significant, F(1, 37)=14.0447, p=6E-4. The Scheffé post-hoc criterion for significance (**α=0.01, ***α=0.001) revealed that protease activity was significantly elevated in sdpw12/w12 protein isolate compared with siblings (100 ng sdpw12/w12: mean=9,114.8, s.d.=4,084.2; 100 ng sib: mean=4,214.7, s.d.=2,793.7; 250 ng sdpw12/w12: mean=14,427.2, s.d.=3,331.8; 250 ng sib: mean=6,463.0, s.d.=4,579.0; 500 ng sdpw12/w12: mean=18,886.0, s.d.=3,587.3; 500 ng sib: mean=11,019.5, s.d.=6,800.7).
Fig. S5. reck is expressed in vasculature and other ventral tissues throughout early development. (A) crestin expression can be detected at shield stage. Scale bar: 200 µm. (B) crestin expression is faint and diffuse at bud stage. Scale bar: 200 µm. (C) crestin is strongly expressed in a dorsal domain corresponding to the premigratory neural crest (bracket) (9 somites). op, optic placode; ot, otic placode. Scale bar: 200 µm. (D) crestin expression is maintained in the neural crest as it begins migrating in dorsoventral streams (bracket) (18 somites) Scale bar: 200 µm. (E) reck is not yet expressed at shield stage. Scale bar: 200 µm. (F) reck expression is initiated at bud stage in a bilateral patch flanking the midline (arrowhead). Scale bar: 200 µm. (G) reck expression expands to the somitic mesoderm (bracket) (9 somites) Scale bar: 200 µm. (H) reck expression remains restricted to the ventral somitic mesoderm (bracket) (18 somites) Scale bar: 200 µm. (I) At 24 hpf, crestin expression is restricted to streams of neural crest migrating ventrally (arrowheads). Scale bar: 250 µm. (I′) High magnification image of the embryo in I showing streams of migrating neural crest (arrowheads). Scale bar: 100 µm. (I′′) Section through the trunk of an embryo subjected to crestin in situ hybridization; anatomical references are overlaid. da, dorsal aorta; n, notochord; nt, neural tube; pcv, posterior cardinal vein; pn, pronephros; s, somite; ye, yolk extension. (I′′′) In this cross-section, streams of neural crest migrating between the somite and the neural tube are defined by crestin expression (arrowheads); these ventromedially migrating cells give rise to DRG. Scale bar: 30 µm. (J) At 24 hpf, reck expression is strongest in the vasculature (white arrowheads), but a faint dorsal expression domain is also apparent (black arrowhead). Scale bar: 250 µm. (J′) High magnification image of the embryo in J showing reck expression in the ventrally situated vasculature (white arrowheads) as well as the fainter dorsal expression domain (black arrowhead). Scale bar: 100 µm. (J′′) Section through the trunk of an embryo subjected to reck in situ hybridization; anatomical references are overlaid. (J′′′) In cross-section, reck expression is evident in both the developing vasculature (white arrowheads) and in a dorsolateral domain consistent with neural crest (black arrowheads). Scale bar: 30 µm.
Fig. S6. The neural crest of sdp embryos migrates to its normal extent. (A-B) Migrating streams of neural crest as seen in a 24 hpf sox10:eos transgenic background. Somites are immunostained for MF20 (A). Scale bar: 250 µm. The neural crest signal (green) was thresholded (A′) and dorsoventral migration distance was measured across the rostrocaudal axis using a MatLab algorithm (B) (blue line). The raw migration data were smoothed by averaging (black line) and local maxima were detected by iterative point-to-point comparison (red circles). (C-D) sdpw12/w12 embryos exhibit neural crest that appears to migrate the appropriate distance. (E) Neural crest migration distance at 24 hpf is indistinguishable across all three sdpw12 genotypes. White circles, sdp+/+; gray circles, sdpw12/+; black circles, sdpw12/w12. Error bars represent s.d.
Fig. S7. Increased apoptosis is not observed in sdp neural crest. Sdpw12/+ fish were incrossed and the resulting embryos were subjected to Sox10 and TUNEL staining at 30 hpf. Four streams of neural crest cells (open arrowheads) are visible in each image. (A) Embryos heterozygous for sdpw12 exhibit numerous TUNEL+ cells, many of which are in close proximity to Sox10+ cells (filled arrowhead). Scale bar: 50 µm. (A′-A′′′) Orthogonal projections of confocal stacks were generated to ascertain whether TUNEL and Sox10 staining is colocalized, as evident in the coronal (A′), sagittal (A′′) and transverse (A′′′) projections; although TUNEL+ and Sox10+ cells are adjacent, the markers do not colocalize. Scale bar: 10 µm. (B) Embryos homozygous for sdpw12 exhibit a similar distribution of TUNEL+ and Sox10+ cells. Scale bar: 50 µm. (B′-B′′′) Again, in coronal (B′), sagittal (B′′) and transverse (B′′′) projections, no colocalization of markers is observed. Scale bar: 10 µm.
Fig. S8. Abnormal Notch signaling does not appear to be the mechanism by which DRG fail to form in the sdp mutant. (A) Rohon-Béard cells in a 24 hpf sdpw12/+ embryo carrying the neurog1:egfp transgene (green) and immunostained for Elavl1 (red). Scale bar: 100 µm. (B) Rohon-Béard cells are present in comparable density in sdpw12/w12 embryos. Scale bar: 100 µm. (C) Counts of Rohon-Béard cells in all three sdp genotypes; Rohon-Béard cell count does not vary significantly across genotypes, F(2, 17)=0.1940, P=0.8255.
Fig. S9. Treatment of sdp embryos with MMP inhibitors fails to rescue DRG formation. Tg(neurog1:egfp);sdpw12/+ fish were incrossed and the resulting embryos were treated from 16 SS to 3 dpf in the following broad-spectrum MMP inhibitors. neurog1+ DRG neurons were subsequently counted. (A) Chemical structure of GM6001. (B) GM6001 (100 µM; black bars) fails to rescue the sdp DRG phenotype observed in vehicle-treated embryos (white bars). (C) Chemical structure of marimastat. (D) Marimastat (100 µM; black bars) fails to rescue the sdp DRG phenotype observed in vehicle-treated embryos (white bars). (E) Chemical structure of batimastat. (F) Batimastat (500 µM; black bars) fails to rescue the sdp DRG phenotype observed in vehicle-treated embryos (white bars).
Movie 1. Wild-type neural crest migrates in coherent ventral streams. Uninjected Tg(sox10:nls-eos) embryo imaged every 10 minutes beginning at 18 SS. Right panel shows cell traces used to derive migratory data. Stills from this movie are shown in Fig. 7.
Movie 2. Morphant neural crest migrates in coherent ventral streams. Tg(sox10:nls-eos) embryo injected with 1.5 ng reck MO and imaged every 10 minutes beginning at 18 SS. The gross appearance of the neural crest is normal following reck depletion. Right panel shows cell traces used to derive migratory data. Stills from this movie are shown in Fig. 7.
Movie 3. Wild-type neural crest migrates to the ventral edge of the spinal cord and coalesces into a cluster prior to overt neuronal differentiation. Uninjected Tg(sox10:nls-eos)/Tg(neuroD:tagRFP) embryo imaged every 10 minutes beginning at 30 hpf. Right panel shows cell traces used to derive migratory data and calculate the proportion of cells inhabiting the prospective DRG niche. Stills from this movie are shown in Fig. 7.
Movie 4. Morphant neural crest migrates to the ventrally but fails to coalesce into a cluster prior to overt neuronal differentiation. Tg(sox10:nls-eos)/Tg(neuroD:tagRFP) embryo injected with 1.5 ng reck MO and imaged every 10 minutes beginning at 30 hpf. Right panel shows cell traces used to derive migratory data and calculate the proportion of cells inhabiting the prospective DRG niche. Stills from this movie are shown in Fig. 7.