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First published online 3 December 2003
doi: 10.1242/dev.00905

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Xenopus Meis3 protein forms a hindbrain-inducing center by activating FGF/MAP kinase and PCP pathways

Department of Biochemistry, The Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

View larger version (52K): [in a new window]   Fig. 1. XMeis3-expressing cells juxtaposed to neuralized tissue induce elongation in a non-autonomous manner. (A) Schematic drawing of recombinant explant strategy. In this system, a pigmented animal cap (AC) explant taken from an embryo ectopically expressing XMeis3-encoding RNA is recombined with an albino AC taken from an embryo ectopically expressing BMP-DNR-encoding RNA. Uninjected pigmented AC explants were recombined with uninjected albino AC explants (B) or with albino explants from embryos injected with BMP-DNR-encoding RNA (0.2-0.4 ng per embryo) (C). Pigmented AC explants expressing XMeis3-encoding RNA (0.8-1.0 ng per embryo) were recombined with uninjected AC explants (D) or with explants expressing BMP-DNR-encoding RNA (E). AC explants were removed from blastula embryos (stage 8-9), recombined and cultured until late neurula (stages 18-19).

View larger version (78K): [in a new window]   Fig. 2. XMeis3-expressing cells caudalize adjacent anterior neural fated cells. XMeis3-injected (1.0 ng RNA) AC explants were taken from pigmented embryos and recombined with AC explants removed from albino embryos neuralized with the BMP DNR (0.2 ng RNA). In situ hybridization was performed to the otx2 (forebrain) and Krox20 (hindbrain) markers. (A) AC/BMP DNR explants. Expression of otx2 is high (n=7/12) in the neuralized albino side of these recombinant explants. (B) XMeis3/BMP DNR explants. Expression of otx2 is strongly inhibited (80%, n=11/14) in the albino side, and these cells undergo elongation. Notice that residual otx2 expression is detected at the distal end of the elongated explants and high expression is detected in non-elongated explants (14%, 2/14, asterisks). (C) AC/BMP DNR explants. No elongation or Krox20 expression is detected (n=0/6) on the albino side. (D) XMeis3/BMP DNR explants. Krox20 expression (67%, n=8/12) is induced in the elongating albino side. (E) In some elongating albino explants, two Krox20 stripes are detected, similar to the endogenous r3/r5 pattern in the embryo. (F,G) XMeis3/BMP DNR explants in which N17Ras- (1.0 ng) and BMP-DNR-encoding RNAs were co-injected on the albino side. In these explants, elongation is inhibited. (F) Expression of otx2 is rescued (100%; n=9/9). (G) Krox20 expression is inhibited in all of the explants (n=12/12).

View larger version (89K): [in a new window]   Fig. 3. XMeis3 induces hindbrain and neurogenic, but not mesodermal, marker expression. (A) In situ hybridization to the r5/r6 HoxB3 gene. HoxB3 is expressed in >70% of the elongating BMPDNR-expressing explants (n=17/24). XMeis3-expressing AC explants recombined with a control AC explants did not express HoxB3 (not shown; n=0/13). (B) In situ hybridization to the neurogenic n-tubulin gene, which is expressed in >50% of the elongating BMP-DNR-expressing explants (n=18/34). XMeis3-expressing explants recombined with neither a control AC explant nor an uninjected control AC explant recombined with a BMP-DNR-expressing explant expressed n-tubulin (n=0/53; not shown). (C) Immunohistochemistry to the notochord-specific Tor70 marker. The elongated DMZ explants stains strongly (n=8/8) in comparison to (D) the elongated XMeis3/BMP DNR explants (n=0/18). Some non-specific background is seen in the unbleached pigmented animal cap cells.

View larger version (80K): [in a new window]   Fig. 4. XMeis3 induces convergent extension and membrane localization of dsh protein in recombinant explants. (A) AC/AC recombinant explant in which the albino side is injected with RNA (50 pg) encoding membrane-localized GFP. (B) DLMZ albino explant injected with membrane-localized GFP. The explant has undergone convergent extension and cells are intercalated. (C,D) XMeis3/BMP DNR recombinant explants in which the elongated BMP DNR albino side is co-injected with membrane-localized GFP. Cells in the elongated side have intercalated and undergone convergent extension in comparison to control explants (A). (E) AC/BMP DNR recombinant explant (late neurula) injected with RNA encoding dsh-GFP (50 pg) on the albino side (rhodamine detection). The dsh-GFP is detected diffusely throughout the cells in the non-elongating explant. (F) AC/BMP DNR recombinant explant (early neurula, pre-elongation) injected with dsh-GFP on the albino side (HRP detection). The dsh-GFP is detected diffusely throughout the cells in the explant. (G) XMeis3/BMP DNR recombinant explant (late neurula) injected with dsh-GFP on the albino side (rhodamine detection). The dsh-GFP is localized to the intercalated elongating cells. (H) XMeis3/BMP DNR recombinant explant (early neurula, pre-elongation) injected with dsh-GFP on the albino side (HRP detection). The dsh-GFP is membrane localized. (I) XMeis3/BMP + FGFI DNR recombinant explant (late neurula) injected with dsh-GFP on the albino side (rhodamine detection). The dsh-GFP is detected diffusely throughout the cells in the non-elongating explant. (J) XMeis3/BMP + FGFI DNR recombinant explant (early neurula, pre-elongation) injected with dsh-GFP on the albino side (HRP detection). The dsh-GFP is detected diffusely throughout the cells in the explant.

View larger version (37K): [in a new window]   Fig. 5. XMeis3 activates FGF/MAPK caudalizing activities. (A) Embryos at the one-cell stage were injected with 1.0 ng of XMeis3-encoding RNA, 1.0 ng of FGFI-DNR-encoding RNA or both. Animal cap explants removed at blastula stages were grown to early and late gastrula stages. Protein was isolated and western blot analysis of phosphorylated-ERK and total ERK protein was performed. One representative experiment is shown. (B) The bar graphs for (A) describe chemiluminescent quantitation of samples performed using an Image Maker VPS-CL monitor (Amersham-Pharmacia). To quantify the samples, the relative amounts of phosphorylated ERK and total ERK were calculated and plotted on the bar graphs. (C) One-cell-stage embryos were injected in the animal hemisphere with 1.0 ng of XMeis3 RNA, 1.0 ng of FGF1 DNR RNA or both. 18 animal cap explants were removed from uninjected and injected groups of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. RT-PCR analysis was performed with the markers Krox20, HoxB9 and HoxD1. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR and –RT-PCR was performed on total RNA isolated from normal embryos. (D) One-cell-stage embryos were injected in the animal hemisphere with 1.0 ng XMeis3 RNA. 36 animal cap explants were removed from uninjected and injected groups of blastula embryos (stage 8-9). 18 explants from each group were grown to stages 11.5 and 12.5, and total RNA was isolated. RT-PCR analysis was performed with the markers Krox20 and FGF3. EF1 served as a control to quantify RNA levels in the different samples. For controls, RT-PCR and –RT-PCR (not shown) were performed on total RNA isolated from normal embryos.

View larger version (66K): [in a new window]   Fig. 6. Inhibition of Wnt PCP activity inhibits XMeis3-induced convergent extension but not neural marker expression. (A) XMeis3/BMP DNR recombinant explants in which the neuralized albino cells undergo elongation (60%, n=14/24). (B) XMeis3/BMP DNR explants co-injected in the albino explant with 0.5 ng of wnt11 dominant-negative ligand (14% weak elongation, n=2/24), (C) Xdsh-(d2) (22% weak elongation, n=4/18) or (D) Xdd1 (no elongations, n=0/18) encoding RNAs. Elongation is inhibited in the neuralized albino side in all of the co-injected groups (B-D). (E) One-cell-stage embryos were injected in the animal hemisphere with 1.0 ng of XMeis3 RNA, 0.5 ng of wnt11 dominant-negative ligand RNA or both. 18 animal cap explants were removed from uninjected and injected groups of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. RT-PCR analysis was performed with the markers Krox20 and HoxD1. EF1 served as a control to quantify RNA levels in the different samples. For controls, RT-PCR and –RT-PCR were performed on total RNA isolated from normal embryos.

View larger version (57K): [in a new window]   Fig. 7. XMeis3 caudalizing activity functions in the absence of canonical Wnt signaling. All embryos were injected in the animal hemisphere with RNA or DNA at the one-cell stage and animal caps were removed at blastula stages 8-9. (A) XMeis3/AC control recombinant explants (no elongations, n=0/28). (B) XMeis3/BMP DNR recombinant explants, in which the neuralized albino cells undergo elongation (83% elongations, n=25/30). (C) XMeis3/BMP DNR explants co-injected in the albino neuralized explant with ß-catenin MO. These explants elongate (85% elongation, n=17/20). (D) Albino explants were also co-injected with the ß-catenin-promoter-driven luciferase reporter construct 3X TCF-luc on the albino side as described in (A,B), except that 0.5 ng GSK-3-encoding RNA was injected instead of ß-catenin MO. At early neurula stages, 8-15 explants were lysed per group and luciferase activity assayed. The graph shows relative luciferase activity in each sample, with the control (A) taken as 100% luciferase activity. (E) One-cell-stage embryos were injected in the animal hemisphere with 1.0 ng XMeis3 RNA, 0.5 ng GSK-3 RNA or both. 18 animal cap explants were removed from uninjected and injected groups of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. RT-PCR analysis was performed with the markers Krox20 and HoxB9. EF1 served as a control to quantify RNA levels in the different samples. For controls, RT-PCR and –RT-PCR was performed on total RNA isolated from normal embryos.