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Fig. 4. Knock-down of endogenous Pa2G4 disrupts the formation of the neural border zone and its derivatives. (A) Xenopus embryos showing “typical” cleavage patterns at the 8-cell stage (8CS) and 16-cell stage (16CS). Diagram on right shows the 16CS fate map of which blastomeres are the major progenitors of the neural plate (NP), neural crest (NC), pre-placodal ectoderm (PPE) or epidermis (EPI) (based on Moody, 1987). Green asterisks indicate the sites of microinjection that target knock-down to the precursors of the neural crest and cranial placodes. (B)In situ hybridization assays of gene expression at neural plate or neural tube stages monitored after unilateral knock-down of endogenous Pa2G4. Right side of each image (except zic2-b) is the MO-injected side, and the left is the uninjected, control side that demonstrates the normal gene expression pattern. Genes expressed in the neural border zone are either undetected (arrow, msx1) or much smaller in size (bar, pax3). Genes expressed in the neural crest are either undetected (arrow, foxd3) or much more faintly stained (red arrow, sox9-a). Genes expressed in the PPE and placodes are greatly reduced in size/staining intensity (green arrows in sox9-a, sox9-b, six1, sox11, irx1-a, irx1-b). Concomitantly, the domain of neural plate/neural tube gene expression is expanded on the knock-down side of the embryo (red bars in sox11, irx1-a, irx1-b, sox2, zic2-a). Knock-down of endogenous Pa2G4 also results in ectopic expression of zic2 in the lateral epidermis, as well as maintained expression of foxd4l1 in the neural plate at stages long after it should be extinguished (blue arrows). (C) Knock-down of endogenous Pa2G4 disrupts gene expression in the otocyst and branchial arches. Otic genes are either reduced in intensity or undetected in the otocyst on the knock-down side (right image in each panel, red arrows). Left image in each panel shows gene expression on the control side of the same embryo. Green arrows denote reduction in gene expression in the neural crest-derived branchial arches. Two different embryos show that expression of otx2 in the ventral otocyst was either undetectable (right image) or reduced in size (black bar, middle image) in comparison to the control side (black bar, left image). (D) Measurement of the size of the otx2 expression domain shows that for those morphant embryos in which there was some otx2 expression, it is significantly smaller compared to the control side (* indicates p<0.001, paired t-test; bars indicate SEM). For all phenotypes in this figure, please see Table 1 for frequencies and the number of embryos analyzed.
