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Figure S2: ISH of rescued embryos.
Lateral views of tailbud stage embryos stained with Sox10 and Twist probes. The right
panels correspond to the side injected with the mRNA (A’-E’). Embryos were injected at
the one cell stage with MO13 and 19 and at the 8-cell stage in one dorsal animal
blastomere with the various ADAM13 constructs and a fluorescent lineage tracer.
Embryos were sorted using the lineage tracer (left/right/dorsal anterior expression), and
processed for in situ hybridization using a combination of Sox10 and Twist probes. The
lengths of CNC segments were measured on each side or each embryo. The percent
rescue (green value) corresponds to the percentage of embryos with a 20% or more increase in segment length on the side injected with each mRNA compared to the contralateral side (2MO alone). The percentage of inhibition (red value) corresponds to a 20% decrease in the length of CNC segment on the side injected with each mRNA compared to the contralateral side. The total number of embryos scored from 3 independent experiments is: A13 N=59 (A’), A13-Plk/A N=51 (B’), A13-Plk/D N=64 (C’), A13-Gsk/A N=52 (D’), A13-Gsk/D N=69 (E’). These data confirm that the phospho-mimetic mutants A13-Plk/D and A13-Gsk/D can rescue CNC positioning, while A13-Gsk/A does not. In this assay the A13-Plk/A provides a similar rescue as the wild type ADAM13, but also worsened CNC positioning more frequently (22%). |
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FIGURE 4: ADAM13 requires Plk activity in the CNC. (A) Histogram showing the percentage of embryos with inhibited CNC migration in a targeted injection assay. Values are normalized to injection of GFP alone and are from at least three independent experiments. Error bars are SD. n, number of embryos scored. *p < 0.05, **p < 0.01. (B) In situ hybridization detecting both of the CNC markers Sox10 and Twist in early tailbud embryos (st. 21), showing that migration, but not induction, is decreased by Plk-DN. Injected side of each embryo is on the left. (C) Analysis of CNC migration from the in situ hybridizations in B, showing the percentage of embryos for each case with severe, weak, or no defect in CNC migration on the injected side compared with the noninjected side of each embryo. |
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Figure S3: ISH of grafted embryos.
Embryos were injected at the 2-cell stage either with RFP alone or MO13, MO19 and A13-Plk/A and RFP mRNA. CNC were grafted at stage 15 into sibling non-injected host embryos and grown until tailbud stage as previously described (Alfandari et al., 2001). Fluorescent photographs were taken prior to fixation and ISH were performed with a combination of Sox10 and Twist probes. |
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Figure S4: ISH versus lineage tracer.
Variability of phenotypes in fluorescence-based methods to track CNC migration (grafts or targeted injection) and in situ hybridization methods to track CNC positioning (ISH). In the first method, cell position can be visualized in the same
embryo over time to test cell migration. In the second method, embryos are fixed either before or after migration and stained for CNC markers giving the position of
CNC. When reagents that inhibit migration of all CNC without affecting gene expression are used, the results from both techniques are similar. On the other hand, when reagents inhibit a subset of CNC and the cells that are inhibited turn off CNC markers, the inhibition is obvious in lineage tracer experiments but not with ISH
techniques. One example of ADAM13 and 19 inhibition is given on the right. The majority of grafted CNC (red) did not migrate (yellow oval), but a small percentage of cells can be seen to migrate in spite of the absence of ADAM13 and 19 (white arrowhead). The ISH of the same embryo reveals a weaker but normal positioning of
CNC. Note that the majority of the cells that did not migrate are not obviously stained by ISH. |
