| |
Figure 3. Local perturbation of Vmem signal disrupts endogenous forebrain marker expression during neural development. A, Control (Ctrl; uninjected) embryos at the indicated stage (Ai, Aiii,
Av, Avii, Aix) and embryosmicroinjected(Inj)with Kv1.5mRNA(Aii, Aiv, Avi, Aviii, Ax) intheright dorsal cell atthefour-cellstage. Insitu hybridizationforemx(Ai, Aii[n 7 of 10], Avii, Aviii[n
10 of 14]),bf1(Aiii, Aiv[n 6 of 9], Aix, Ax[n 6 of 9]), andotx2(Av, Avi [n 9 of 13])show asignificantly decreased or missing expression (red arrowheads) ofemx,bf1, andotx2only on the
injectedside of the embryos, while expression on the uninjected (Uninj)side is intact (green arrowheads). Quantification of thein situsignal (Axi–xiii) as ratio of thesignal intensity for uninjected
verses its contralateral injectedside for emx (Axi), bf1 (Axii), and otx2 (Axiii)show asignificant change in theinsitusignal for these probes upon Kv1.5mRNA injection. The data are represented as
mean SEM(n
8 for each). The data are analyzed viattest. ***p 0.001. B, Control(untreated) embryos(Bi, Bv, Bix), embryostreated with LiCl(0.2 M for 10min at 32-cellstage; Bii, Bvi, Bx),
UV light (75 s; Biii, Bvii, Bxi) and microinjected with Kv1.5 mRNA in the two dorsal cells at four-cell stage (Biv, Bviii, Bxii). Phase contrast images (Bi–iv) of embryos at stage 30 show that LiCl
treatment (Bii) dorsalizes the embryos with a majority of dorsal tissue and lack of ventral tissue development, while UV treatment (Biii) ventralizes the embryos with increased ventral tissue
specification and lack of dorsal tissue development as compared with control embryos (Bi), whichshow correctly balanced development of dorsal and ventral tissues.Kv1.5-injected embryos (Biv)
appear normal similar to control embryos with balanced development of dorsal and ventral tissues. In situ hybridization for dorsalization markers chordin(Bv–viii) andcerberus (Bix–xii) show
normal expression (green arrows) in control embryos (Bv, Bix), a significantly increased expression (yellow arrows) in LiCl-treated embryos (Bvi [n 9 of 10], Bx [n 9 of 9]), a significantly
decreased expression (red arrow) in UV-treated embryos (Bvii [n 14 of 15], Bxi [n 11 of 11]), and relatively unchanged expression (green arrows) in Kv1.5-injected embryos (Bviii [n 20 of
20], Bxii [n 18 of 18]) compared with control embryos. |
