Figure 1.
Whole mount in situ hybridization for rbc3a mRNA, anterior to the left. (A) Dorsal expression at gastrulation onset (6 hpf) includes the embryonic shield (arrowhead). Animal pole view, dorsal to the right. (B–E) Expression in NC cells starts at premigratory stages,11.5 hpf (B, C) and 13 hpf (D, E), with lower levels in the presumptive notochord (not) and in the tailbud (C, E). Dorsal views (B, D); lateral views (C, E). (F) Expression at 15 hpf in the pineal gland (pg), otic vesicle (ot), somites, tailbud, and premigratory NC (arrowheads) in the trunk. (G, H) Expression at 24 hpf in the pineal gland, cranial sensory ganglia (black arrows), somites, and tail NC (arrowhead). Scale bars, 100 µm.
Figure 2.
Rbc3a loss of function disrupts NC migration.
(A–C) Live 24 hpf controls (A), rbc3a-MO1–injected (B), and rbc3aQ850X mutant (C) embryos. Morphant/mutant morphological defects include the mhb and cell aggregates in the dorsal midline (black arrows in B and C). (D–E′) NC defects in live sox10:gfp transgenics injected with rbc3a-MO1. (D, E) Merged bright-field and fluorescent images of the cranial region at 24 hpf, lateral views, show GFP+ NC cells accumulated dorsally (white arrowheads). (D′, E′) Dorsal views showing midline position of aggregates over the mhb and further posteriorly (white arrowheads). (F–Q) Whole mount in situ hybridization for markers of different NC lineages in controls (row 1), rbc3a-MO1–injected (row 2), and rbc3aQ850X mutants (row 3) at 28 hpf, dorsal views, anterior to the left. dlx2 (F–H) expression in skeletogenic NC and foxd3 (I–K) expression in gliogenic NC appear unaffected, while mitfa in presumptive melanocytes (L–N) and gch in xanthophores (O–Q) are expressed in dorsal midline aggregates in rbc3a morphants/mutants (white arrows). Abbreviations: 1–4, pharyngeal arches; ot, otic vesicle; mhb, midbrain-hindbrain boundary. Scale bars, 100 µm.
Figure 3.
Rbc3a knockdown disrupts endosomal maturation but not acidification in NC.
(A–D) Confocal images of whole mount immunohistochemical staining for endocytic markers in NC cells in sox10:lyn-gfp transgenics, which labels NC cell membranes (green). (A′–D′) 4× insets of (A–D). (A–B′) Anti-EEA1 marks early endosomes (red). rbc3a-MO1–injected embryos show large EEA1+ aggregates in NC cells (white arrowheads in B′). (C–D′) Anti-LAMP1 marks late endosomes/lysosomes (red). No increase in number or size of LAMP1+ vesicles was observed with rbc3a-MO1 injection. (E, F) Automated quantification of average % area (E) and average particle size (F) per NC cell stained positive for EEA1, LAMP1, or Lysotracker Red (Lyso) using ImageJ Particle Analyzer. rbc3a-MO1 injection produced significantly higher EEA1+ and Lysotracker+ relative area and particle size but significantly smaller LAMP1+ relative area and particle size per NC cell. Error bars represent ± SEM. (G, H) Live whole-mount images of sox10:gfp+ NC cells labeled with Lysotracker (red). rbc3a-MO1–injected embryos show many large, acidic Lyso+ vesicles, which colocalize with EEA1 (I′,J′, white arrows) but not LAMP1 (K′, L′). ** p<0.001, *** p<0.0001. Scale bar, 10 µm.
Figure 4.
V0a1 knockdown disrupts NC migration and early endosome maturation.
(A, B) Fluorescent images of live sox10:gfp embryos at 24 hpf, dorsal views, showing aggregates of GFP+ cells in the dorsal midline. Ot, otic vesicle. Scale bar, 100 µm. (C) Percentages of embryos with GFP+ aggregates in embryos injected with increasing amounts of v0a1-MO. (D–E′) EEA1 staining (red) in sox10:gfp+ NC cells (green), in controls (D, D′), and v0a1-deficient embryos (E, E′) showing EEA1+ vesicle enlargement at 24 hpf. Scale bar, 10 µm. (F–G′) Lysotracker staining (red) in sox10:gfp+ NC cells in controls (F, F′) and V0a1-deficient embryos (G, G′) showing enlarged acidic intracellular compartments in NC cells. (H, I) Automated quantification of average % area (H) and average particle size (I) per NC cell stained positive for EEA1, LAMP1, or Lysotracker Red (Lyso) using ImageJ Particle Analyzer. V0a1 knockdown produced significantly higher EEA1+ and Lysotracker+ relative area and average particle size but significantly less LAMP1+ relative area and particle size per NC cell. Error bars represent ± SEM. * p<0.05, ** p<0.001, *** p<0.0001.
Figure 5.
Rbc3a knockdown disrupts subcellular localization of Bcat and Fz7 in NC cells.
(A) Quantitative RT-PCR analysis of early Wnt target genes with important roles in EMT reveals reduced expression of snai2, gbx2, and twist1a at 11–12 hpf in rbc3a-MO1–injected embryos. Error bars represent triplicate experiments ± SEM. * p<0.05. (B–E″) Immunostaining with an anti-Bcat antibody (red) in whole-mounted, sox10:lyn-gfp transgenic embryos to label NC cell membranes (green) and DAPI to label nuclei (blue). Bcat levels in the nucleus are (B–C″) reduced in rbc3a-MO1–injected embryos at 11 hpf and (D–E″) elevated in the nucleus of MO-injected embryos at 24 hpf compared with wild-type (WT) controls. Scale bar, 10 µm. (F, G) Dorsal images of tcf:gfp Wnt reporter transgenic fish at 24 hpf identifies distinct aggregates of GFP+ cells (white arrowheads) in the dorsal midline of rbc3a-MO1–injected embryos (G) but not wild-type (F) embryos. Mhb, midbrain-hindbrain boundary; ot, otic vesicle. Scale bar, 100 µm. (H–K′) Immunohistochemical staining for GFP (green) after microinjection of fz7-yfp mRNA in sox10:lyn-tdtomato (red) transgenic embryos. At both 11 hpf (H–I′) and 24 hpf (J–K′), the number of YFP+ puncta per cell increased in NC cells in rbc3a-MO1–injected embryos. YFP colocalizes with tdTomato at the membranes of NC cells in rbc3a-MO1–injected embryos at 24 hpf (K′). Scale bar, 10 µm.
Figure 6.
Rbc3a knockdown reduces expression of cadherins in NC cells.
(A) Quantitative real-time PCR for ecad, ncad, and cdh11 at four timepoints during the onset of NC migration in rbc3a-MO1–injected embryos. ecad expression is significantly down-regulated at 13 hpf and remains low. ncad expression is also significantly reduced by 15 hpf. In contrast, cdh11 expression increases at 12 hpf. Error bars represent triplicate experiments ± SEM. (B–G′) Confocal images of immunohistochemical staining for Ncad (red) in sox10:lyn-gfp transgenics (green) at 11 hpf (B–C′), 14 hpf (D–E′), and 24 hpf (F–G′). Ncad levels are reduced at the membranes of GFP+ cells by 14 hpf in rbc3a-MO1–injected embryos and almost completely absent in the NC of rbc3a-MO1–injected embryos by 24 hpf, while still present at the membrane in surrounding cells. * p<0.05, ** p<0.01. Scale bar, 10 µm.
Figure 7.
Model for Rbc3a and V0a1 functions in NC cells.
(A) Rbc3a promotes association of a specific v0a1 isoform to early endosomes. In the absence of Rbc3a, endosomes acidify, but do not mature. (B) Rbc3a controls maturation of early endosomes (EE) containing Fz-Wnt complexes to contribute to multivesicular bodies (MVBs), which promote canonical Wnt signaling and EMT. In the absence of Rbc3a (or v0a1), canonical Wnt signaling decreases early (∼11 hpf) and increases later (∼20 hpf). Defects in EE maturation disrupt Fz-Wnt recycling and degradation, producing high levels of Fz-Wnt complexes at the cell membrane and aberrantly high levels of canonical Wnt signaling, which drives NC cells to a pigment progenitor fate.