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In vertebrates, BMP signaling before gastrulation suppresses neural development. Later in development, BMP signaling specifies a dorsal and ventral fate in the forebrain and dorsal fate in the spinal cord. It is therefore possible that a change in the competence of the ectoderm to respond to BMP signaling occurs at some point in development. We report that exposure of the anterior neural plate to BMP4 before gastrulation causes suppression of all neural markers tested. To determine the effects of BMP4 after gastrulation, we misexpressed BMP4 using a Pax-6 promoter fragment in transgenic frog embryos and implanted beads soaked in BMP4 in the anterior neural plate. Suppression of most anterior neural markers was observed. We conclude that most neural genes continue to require suppression of BMP signaling into the neurula stages. Additionally, we report that BMP4 and BMP7 are abundantly expressed in the prechordal mesoderm of the neurula stage embryo. This poses the paradox of how the expression of most neural genes is maintained if they can be inhibited by BMP signaling. We show that at least one gene in the anterior neural plate suppresses the response of the ectoderm to BMP signaling. We propose that the suppressive effect of BMP signaling on the expression of neural genes coupled with localized suppressors of BMP signaling result in the fine-tuning of gene expression in the anterior neural plate.
FIG. 1. The expression of genes in the neural plate are reduced by BMP4 mRNA injection into one dorsal cell of 4- to 8-cell- stage embryos. Embryos were coinjected with 200 pg of BMP4 and lacZ mRNA (light blue; right) or were uninjected (left) and were analyzed by whole-mount in situ hybridization (magenta) to the neural markers indicated. Embryos shown are at stages 19 0.
FIG. 3. The Pax-6 promoter drives the expression of GFP in a similar domain to that of endogenous Pax-6. Panels A, D, G, and J show images of embryos stained for endogenous Pax-6 RNA by whole-mount in situ hybridization at stages 14, 25, 30, and 32, respectively. Panels B, E, H, and K show images of Pax-6GFP transgenic embryos stained for GFP RNA by whole-mount in situ hybridization at stages 14, 25, 30, and 32, respectively. At stage 14, both endogenous Pax-6 (A) and GFP transgene (B) are expressed in a broad arc at the anterior neural plate, which gives rise to the forebrain and eyes. Expression is also apparent in two lateral stripes in the posterior neural plate that will give rise to the hindbrain and neural tube. (C) A stage 14 transgenic embryo stained for endogenous Pax-6 RNA (magenta) and GFP RNA (light blue). (I) A stage 32 half transgenic embryo stained by whole-mount in situ hybridization to Pax-6 (light blue) and GFP (magenta). Note strong expression of GFP transgene in the telencephalon (arrow). (F), (L) Fluorescence images in Pax-6GFP transgenic embryos at stages 25 and 32, respectively.
FIG. 4. Expression of endogenous Pax-6 compared with that of GFP driven by the Pax-6 promoter as seen in sagittal sections of stage 32 embryos. Top row shows in situ hybridization to GFP driven off the Pax-6 promoter and bottom row shows in situ hybridization to endogenous Pax-6 mRNA. Both the transgene and the endogenous gene are expressed in the retina (A and D) but the transgene is absent from the lens (A). The transgene also shows higher expression in the telencephalon (arrow, A) but lower expression in the diencephalon (B versus E). GFP is expressed in the hindbrain (B), in a similar domain to Pax-6 (compare B to E). Both GFP and Pax-6 are expressed in the neural tube (C and F, respectively). Abbreviations: HB, hindbrain; L, lens; Di, diencephalon; NR, neural retina.
FIG. 5. The cointegration of two constructs into one transgenic embryo occurs at high frequency when the same restriction enzyme is used to linearize both constructs and to cut the sperm DNA. Ninety-four percent of resulting transgenic embryos (stage 35) contained both the XCarGFP (expressing in the somites) and the CryGFP3 (expressing in the lens) transgenes (C), whereas 3% contained only the CryGFP3 transgene (A) and 3% only the XCarGFP transgene (B). Similarly, 91% of embryos in a separate experiment contained both the CMVRFP and Pax-6GFP constructs when added together in the same transgenic reaction (D).
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 ï°† 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).
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 anteriorright 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.
FIG. 8. BMP4 is expressed in the non-neural ectoderm and both BMP4 and 7 are expressed in the prechordal mesoderm, underlying the anteriormost neural plate. (A) Anterior view of a stage 17 embryo stained by whole-mount in situ hybridization to BMP4 (purple stain). BMP4 is expressed around and bordering the keyhole-shaped neural plate with stronger expression anteriorly. The dotted white line represents the angle of sectioning shown in B, C, and D. (B and C) Sagittal section through an embryo stained for BMP4 mRNA, anterior is to the left. Low power (B) and high power (C) reveal the expression of BMP4 in the prechordal meso- derm (arrow). (D) Sagittal section through a stage 17 embryo stained by whole-mount in situ hybridization to BMP7 mRNA. The anterior-most portion of the section is shown. BMP7 is expressed in the prechordal mesoderm (arrow), underlying the prechordal plate.
FIG. 9. (A) Injection of XBF-1 RNA suppresses BMP4 expression in the ectoderm. XBF-1 was coinjected with lacZ RNA (blue). (B) Animal caps combined with BSA beads express a basal level of msx1 (left panel). Animal caps combined with BMP4-soaked beads express a higher level of msx1 (middle panel). Animal caps injected with XBF-1 mRNA and combined with BMP4 beads show reduced msx1 expression (right panel).
