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FIG. 7. (A) BMP4 misexpression by bead implantation into the anterior neural plate of stages 13-14 embryos results in the suppression of most neural markers. Left column shows embryos implanted with control, BSA beads in the anterior neural plate. Embryos were fixed at stage 17 and stained by in situ hybridization to the range of neural markers on the left. Right column shows embryos implanted with beads soaked in BMP4 in the anterior neural plate. Suppression of the neural markers listed on the left can be seen in the vicinity of the bead, with the exception of Pax-6, which remained unchanged. Pax-6 was analyzed by double in situ hybridization with XSox3, where XSox3 expression is shown in light blue and Pax-6 in magenta. Note that, while the XSox3 gene was downregulated (i.e., absence of blue stain), Pax-6 expression remains (i.e., presence of magenta stain) in the anterior right neural plate, where the bead was implanted. (B) Suppression of XSox3 (light blue) is also observed when BMP4 beads are implanted at stages 16 and 20 and analyzed at stages 19 and 25, respectively. Bead implantation at stage 13 is shown for comparison. These embryos were analyzed by double in situ hybridization to XSox3 (light blue) and nrp1 (light magenta). Expression of nrp1 is not affected by BMP4 bead implantation throughout the neurula stages (right panel). Note retention of magenta stain (i.e., nrp1 expression), but a significant reduction in light blue stain (i.e., XSox3 expression) in embryos on the right. Embryos implanted with control BSA beads are shown on the left and have no affect on either nrp1 or XSox3. |
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FIG. 6. (A) The expression of BMP4 behind the Pax-6 promoter inhibits most neural markers at early neurula stages of development. Left-hand column: embryos transgenic with Pax-6GFP (controls); right-hand column: embryos transgenic with Pax-6BMP4. All the embryos shown are also transgenic for CSGFP3 (as observed at stage 15 by fluorescence microscopy). Embryos were fixed between stages 15 and 19 and stained by whole-mount in situ hybridization to the neural plate markers listed on the left. A reduction in Xotx2, Xrx1, XSox3, and XBF-1 expression and, to some extent, a reduction in XBF-2 and Pax-6 expression was seen in Pax-6BMP4 transgenic embryos. In contrast, the expression of the pan-neural marker nrp1 was not significantly affected by the Pax-6BMP4 transgene, whereas X-dll3 was significantly expanded into the anterior neural plate by the Pax-6BMP4 transgene. Bottom panels show cross sections of the anterior neural region of Pax-6GFP control (left) and Pax-6BMP4 (right) transgenic embryos stained for X-dll3. (B) Bar graph comparing transgenic embryos containing Pax-6GFP with those containing Pax-6BMP4. Percentage of embryos showing no change (blue), decrease in expression (red), or increase in expression (yellow) of the markers listed underneath. Total numbers of embryos (n ﰆ x) are noted beneath each set of columns. (C) Image of microcephalic phenotype obtained by stage 40 in transgenic embryos containing Pax-6BMP4 (lower embryo) compared with transgenic embryos containing Pax-6GFP (upper embryo). |
