Fig. 3

Expression of proteases in N. vectensis. a–c In situ hybridization of proteases in a the M family—metallopeptidases, astacins, and carboxypeptidases and b the S1 family—trypsins, during early development. The oral pole is to the left in all images; black arrows indicate expression in the ectoderm of the pharynx. c Double fluorescent in situ hybridization showing the co-expression (yellow arrow) of two metalloprotease genes (Nv_88668 and Nv_2109) in the aboral ectoderm of the pharynx and independent expression of both genes in individual cells of the ectodermal mesentery (red and green arrows). Nuclei are labeled with DAPI. d Single-cell expression of genes from panels a and b in metacells identified previously [16]. Metacells C9-C12 are part of the “gland” cell cluster and C26 is part of the “neuron” cell cluster. Two genes (NVJ_200868 and NVJ_2109) are clearly expressed in the ectodermal mesenteries but were not reported in the single-cell study (indicated by dotted lines). e Histogram of the number of expressed trypsins per metacell [16]; red font highlights a small second mode in the distribution. f Scatterplot of the number of cells exhibiting co-expression of multiple trypsins as a function of the number of total cells in which a particular trypsin is expressed. Scale bars = 50 µm