Figure 1. Ectopic expression of Wnt1 and Wnt3 induces ectopic body axes.

(a) CRISPR-Cas9 knockout of APC results in the formation of ectopic tentacles and pharynges in F0. Left: wild type polyp; right: mosaic mutant polyp. Red arrows—pharynges. (b) Five ectodermal Wnt genes are expressed at mid-gastrula, however, Wnt2 is expressed at a distance to the bend of the blastopore lip (red double-headed arrows). Gene names are colour-coded as on the scheme showing a lateral view on a mid-gastrula with ectodermal Wnt expression domains depicted as coloured lines. en—invaginating endoderm, ec—ectoderm. Black arrowhead points at the bend of the blastopore lip. (c) Results of transplantation of four sequential blastopore lip fragments from donor gastrulae (N=31) to four different recipient gastrulae (N=31 × 4). Possible developmental outcomes: no axis duplication (green bars on graph), incomplete axis duplication (an outgrowth with tentacles and, sometimes, pharynx but without mesenteries; orange bars on graph), complete axis duplication (two contractile axes with head structures; red bars on graph). Red arrows—pharynges. Fragment 1 and fragment 2, closest to the bend of the blastopore lip, are inductive. (d) Co-injection of a plasmid with EF1α promoter driving the expression of a gene of interest and Dextran-Alexa488 into single cells in 8–16 cell stage embryos results in formation of a coherent patch of fluorescent cells. (e) Some embryos injected into single blastomeres at 8–16 cell stage with Wnt1 or Wnt3 or with both these Wnt's develop ectopic axes. WntA, Wnt4 and Chd never induce a second axis. Fluorescent aboral ectoderm from Wnt1 and Wnt3 injected embryos also acquires inductive capacity. The colour code of the bars is the same as on (c). (f) If EF1a::mOrange2 is co-injected with the Wnt plasmids, mOrange2 fluorescence is observed in the induced secondary head (red arrow). Scale bars: 100 μm.