Fig. 1.

Amphiura filiformis development and identification of skeletogenic cells. a Live imaging of embryos. Early cleavage stage shows tetrahedral arrangement of cells at 3 hpf, similar to Ophiopholis aculeata [28]. Mid-cleavage stage shows equally sized cells at 6 hpf. Early blastula stage shows spherical embryo with blastocoel at 10 hpf. Hatched blastula embryos have distinct blastocoel at 16 hpf and animal–vegetal orientation is visible through thickening at vegetal side of the embryo. At 23 hpf, mesenchyme blastula stage shows first ingressing cells from the vegetal side of the embryo that fill up the blastocoelar space by 27 hpf. At 30 hpf, calcein (green) stained gastrula embryo shows two newly formed spicules that extent to a tri-radiate structure as visible on a bright field by 36 hpf. b High-resolution time-courses for genes analyzed in this study shown as heatplot were obtained by QPCR. Expression values are relative to Afi-16S (see Additional file 1 for explanation of calculation and QPCR controls; exact numbers are shown in Table S1). c WMISH of skeletogenic marker genes identifies the vegetal plate and the primary ingressing cells as the SM cell lineage in A. filiformis. The two regulatory genes Afi-alx, Afi-jun and the skeleton matrix gene, Afi-p19, are expressed in the vegetal plate of the blastula embryos, then in the first ingressing mesenchymal cells and at later stage in a location congruent to where spicules are formed. Other two orthologs of sea urchin skeletogenic matrix genes, Afi-p58a and Afi-p58b, are also detected in the first ingressing cells of the mesenchyme blastula stage and at later stage in the same location where the spicules are formed. ECl early cleavage, Cl cleavage, EBl early blastula, Bl-VV blastula vegetal view, Bl blastula, MBI mesenchyme blastula, LMBl late mesenchyme blastula, G gastrula, LG late gastrula. Scale bars are 50 μm