Fig. 7

Fig. 7

Loss of Pk1 function causes dorsal NCC clusters to form and be maintained in early stages of neural crest development. Confocal time-lapse imaging of dorsally-mounted Tg(sox10:EGFP) embryos was started at 12 hpf when the neural keel is still developing, with both EGFP and DIC (brightfield) images collected every 5 min. (A-C) Reorganization of EGFP+ NCCs (pseudocolored) in clusters of NCCs at t = 0 to t = 40 mins; dotted line indicates border of the neuroepithelium. The neuroepithelium (NE) is located at top right, and the periphery (P) is located at bottom left, in all panels. Scale bar= 10 µm. (A) In WT embryos, many of the NCCs in contact at t = 0 break contact by 20 min, with almost all contacts breaking by 30 mins and 40 mins. (B, C) In pk1b ^{fh122/ fh122} and pk1a^ch105/ch105 specimens NCC clusters largely remained in contact for 40 mins. (D) To quantify breakage of contacts between NCCs over the time intervals 0–10 min, 10–20 min, 20–30 min and 30–40 min, a ratiometric measure of ‘pair breakage’ within a cluster was used (Methods). In WT embryos, ~ 61% of pairs of cells broke contacts between 0 and 20 min (n = 43 pairs, 3 embryos), with a large majority of pairs losing contact by 20–30 min. In contrast, pairs of NCCs in both pk1b ^{fh122/ fh122} (n = 61 pairs, 3 embryos) and pk1a^ch105/ch105 embryos (n = 58 pairs, 3 embryos) did not break over extended periods of time, with only ~ 27% of pairs in pk1b ^{fh122/ fh122} clusters breaking contact and ~ 21% of pairs in pk1a^ch105/ch105 clusters breaking contact by 30–40 min. (E) To measure the relative proportions of individual cells and cell clusters of varying sizes, the organization of cells at 12 hpf in Tg(sox10:EGFP) embryos was quantified. In WT embryos (n = 37 cells, 3 embryos), 43.9% of NCCs were found as individuals, 27.6% were in pairs, 15.3% in clusters of 3 cells, 10.2% in clusters of 4 cells, and 3.1% in clusters of 5 cells. In pk1b ^{fh122/ fh122} specimens (n = 53 cells, 3 embryos), 5.9% of NCCs were found as individuals, and 17.0% as pairs. Most NCCs were found in cluster sizes of 3 (24.2%) or 4 (22.9%), with clusters consisting of as many as 8 cells. In pk1a^ch105/ch105 specimens (n = 46 cells, 3 embryos), 8.6% of NCCs were found as individuals, and 21.1% as pairs. Most NCCs were found as pairs or in cluster sizes of 3 cells (26.6%), with appreciable percentages of cluster sizes of 4 (11.7%) and 5 (10.9%) and with clusters consisting of as many as 8 cells. (F) As another measure of the relative proportions of individual cells and clusters of varying sizes, the number of cells that persisted in a given configuration (from an individual cell to cells in increasing sizes of clusters) was measured over non-overlapping 20-min time windows. In WT embryos (n = 117 cells, 3 embryos), 53.8% of NCCs remain individual over 20 min, whereas 26.5% were in pairs, 11.1% in clusters of 3 cells, and 8.5% in clusters of 4 cells. In pk1b ^{fh122/ fh122} embryos (n = 99 cells, 3 embryos), 5.05% of NCCs were found as individual cells over the 20-min time window. Most NCCs were found in cluster sizes of 3 (21.2%) or 4 (28.3%), with NCCs found in clusters consisting of as many as 8 cells. In pk1a^ch105/ch105 embryos (n = 148 cells, 3 embryos), 6.1% of NCCs were individual, with most NCCs found in cluster sizes of 3 (27.7%) or 4 (23.6%) and clusters consisting of as many as 8 cells. (G) To assay how persistence—a measure of the total length of a trajectory of a cell as a ratio of the displacement of the cell with a value of 1.0 indicating a targeted route from starting point to end point—varied as a function of cluster size, the persistence of individual cells and clusters of sizes 2 or more was measured for each condition. Persistence is agnostic to the ‘correct’ direction of the cells, and is a measure of the targeted directionality of NCCs, independent of their trajectories. In WT embryos (n = 42 cells, 3 embryos), the persistence of individual NCCs or clusters of 2, 3 or 4 was similar. In both pk1b ^{fh122/ fh122} (n = 49 cells, 3 embryos) and pk1a^ch105/ch105 embryos (n = 64 cells, 3 embryos), the persistence of individual cells and cells in pairs was lower than the persistence of clusters consisting of 3 or more NCCs, or of WT NCCs. Clusters of 3 or more NCCs in both pk1-mutants showed high levels of persistence, with statistically insignificant (p > 0.5) differences in persistence as compared to WT cells. Both pk1-mutants showed small increases in persistence as the size of the cluster increased, with no statistically significant difference between clusters of 5 or more cells.