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Fig. 3 Upregulation of ncam1a in ncam1b mutant embryos. (A) ddPCR measurements normalized to the housekeeping gene β-actin show significant upregulation of ncam1a and in ncam1b mutants, but not in ncam1b morphants, at 36 hpf. Mean gene expression levels were determined using biological triplicates or quadruplicates, each representing the mean of technical duplicates. Wild type (WT), n1b -/- PMSC mutants, and n1b -/- Del mutants were analyzed, with pools of 100 embryos per condition. (B–D) Ncam1a expression (orange) in the migrating primordium (primI). (B) Tg(ClaudinB::lynGFP), serving as wild type, and (C) ncam1b -/- Del; Tg(ClaudinB::lynGFP). (B’ + C’) Utilizing ClaudinB::lynGFP expression, a threshold was set to delineate the outline of primI (white border), Ncam1a expression in this region (B’’ + C’’) was quantified by mean grey value measurement of using Fiji. (D) The mean grey value of the n1b -/- Del mutant normalized to the WT shows a significant upregulation of Ncam1a expression. Statistic was made using one sample t-test. Scale bar 20 µm. Abbreviations: WT: Wild type; n1b -/- PMSC: ncam1b -/- Premature Stop Codon mutant; n1b -/- Del: ncam1b deletion mutant; IHC: Immunohistochemistry.