Fig. 4. Overexpression of zebrafish charon leads to a lack of mesendoderm. Zebrafish charon RNA (25 or 100 pg) was injected into one-to-four cell stage embryos. The RNA injection led to variable levels of defects in the formation of mesendoderm, which is observed in one-eyed pinhead (oep) mutant and antivin/lefty1-injected embryos. Non-injected control embryos at 10 hpf [at the time of yolk plug closure (YPC), which is equivalent to bud stage (A) and at the pharyngula stage (24 hpf, B,C,D)]. The phenotypes of the injected embryos were classified into three categories (Class I-III) with increasing severity. Class I embryos at YPC (E) and at the pharyngula stage (F-I). Class II embryos at YPC (J) and at the pharyngula stage (K,L,M). Class III embryos at YPC (N) and at the pharyngula stage (O,P,Q). (A,B,E,F,J,K,N,O) Lateral views. (C,G,L,P) Ventral views of the head. (D,H,M,Q) Dorsal views of the head. (I) Lateral view of the trunk. Arrowheads indicate the anterior border of the dorsal axial mesendoderm and notochord. The numbers of each class of the embryos are shown in Table 1. (R-Y) Misexpression of charon caused a defect in the axial mesendoderm formation. The embryos receiving 25 pg of charon RNA lacked no tail (ntl) expression in the dorsal midline and dorsal forerunner cells at YPC (S,U) and goosecoid (gsc) expression at the 90% epiboly stage (W). In the charon RNA-injected embryos, the expression of six3.2 (a marker for forebrain, arrowhead) was slightly expanded and the expression domain of pax2.1 (a marker for the mid-hindbrain boundary, arrow) was slightly shifted posteriorly at YPC (Y). (R,T,W,X) Uninjected control embryos. (R,S) Dorsal views. (T-W) Lateral views. (X,Y) Animal pole views, with ventral to the top.