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XMeis3 protein activity is required for proper hindbrain patterning in Xenopus laevis embryos

Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel

View larger version (25K): [in a new window]   Fig. 1. Fusion constructs used for expression in Xenopus tissue (see Materials and Methods). (Top) Wild-type full-length XMeis3 protein. (Middle) Eng-XMeis3/XMeis3-antimorph protein. (Bottom) VP-XMeis3 activator protein.

View larger version (53K): [in a new window]   Fig. 2. Eng-XMeis3 RNA encodes an antimorph protein; VP16-XMeis3 RNA encodes a caudalizing protein. (A) One-cell stage embryos were injected in the animal hemisphere with 1.0 ng of XMeis3 RNA (lane 4), 1.6 ng of Eng-XMeis3 RNA (XMeis3-AM; lane 5) or both (lane 6). Eighteen animal cap explants were removed from uninjected (lane 3) and injected groups (lanes 4-6) 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, HoxD1 and HoxB9. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from normal embryos. (B) One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of VP16-XMeis3 RNA (lane 4). Eighteen animal cap explants were removed from uninjected (lane 3) and injected groups (lane 4) 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, HoxB3, HoxD1 and RAR2.2. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from normal embryos. (C) Embryos at the one-cell stage were injected with 1.3 ng of in vitro transcribed VP16-XMeis3. The upper embryo serves as an uninjected control. Injected embryos had anterior truncations and highly reduced cement gland formation (lower panel). The dorsal anterior index (DA) (Kao and Elinson, 1988) was 2.5 (n=39); over 20% of the embryos completely lacked cement glands and another 80% had extreme posterior truncations, with partial cement gland formation (lower panel). Embryos are oriented posterior to anterior: left to right. Embryos were fixed for photography at stages 35/36. (D) Embryos at the one-cell stage were injected with 1.0 ng of XMeis3-AM antimorph RNA. The top embryo serves as an uninjected control. In this representative experiment, over 80% of the XMeis3-AM injected displayed phenotypes. Embryos are oriented posterior to anterior: left to right. Embryos were fixed for photography at stages 30/31.

View larger version (56K): [in a new window]   Fig. 3. Expression pattern of neural markers in embryos injected with XMeis3-AM RNA. Two-cell albino embryos were injected unilaterally into the animal hemisphere of one blastomere with 50-100 pg of XMeis3-AM RNA. The red arrow delineates the dorsal midline. In all embryos, XMeis3-AM injection is on the left side. In all cases (except Fig. 3M), embryos are viewed dorsally; embryos are oriented anterior (top) to posterior (bottom). (A) In situ hybridization with otx2. Expression is expanded posteriorly on the XMeis3-AM injected side. The red lines delineate the AP extent of otx2 expression on the uninjected versus injected side. (B) In situ hybridization with otx2 and Krox20. otx2 expression is expanded posteriorly and Krox20 expression (blue arrows) is lost on the XMeis3-AM-injected side. The red lines delineate the AP extent of otx2 expression on the uninjected versus injected side. (C) In situ hybridization with otx2 and En2 (red). Expression of otx2 and En2 (blue arrows) is expanded posteriorly. (D) In situ hybridization with cpl-1 and En2 (red). cpl-1 expression is expanded posteriorly and En2 expression (blue arrow/uninjected side) is lost on the XMeis3-AM-injected side. The red lines delineate the AP extent of cpl-1 expression on the uninjected versus injected side. (E) In situ hybridization with XAG1; expression is expanded posteriorly and laterally on the XMeis3-AM injected side. (F) In situ hybridization with nrp1; expression is unchanged on the XMeis3-AM injected side. (G) In situ hybridization with Krox20 and HoxB9; Krox20 expression (blue arrows/uninjected side) is eliminated on the XMeis3-AM-injected side. HoxB9 expression is unchanged on the XMeis3-AM injected side. (H) In situ hybridization with XE10. XE10 expression is eliminated on the XMeis3-AM-injected side. (I) In situ hybridization with HoxB1. HoxB1 expression is eliminated on the XMeis3-AM injected side. (J) In situ hybridization with HoxB3. HoxB3 expression is eliminated on the XMeis3-AM-injected side. (K) In situ hybridization with En2 (red) and Krox20. En2 expression is pushed posteriorly to the r3/r4 boarder and Krox20 expression is pushed posteriorly to the r5/r7 boarder on the XMeis3-AM-injected side. (L) In situ hybridization with En2 and Krox20. En2 expression is pushed posteriorly to r3 on the XMeis3-AM-injected side. The blue arrow delineates the reduced Krox20 expression (fused stripes) on the injected side. (M) In situ hybridization with En2 (red) and Krox20. Expression of En2 and Krox20 (blue arrows/uninjected side) is eliminated on the XMeis3-AM-injected side. An anterior view of the embryo: dorsal (top) to ventral (bottom). (N) In situ hybridization with n-tubulin. n-tubulin expression is eliminated on the XMeis3-AM-injected side. The trigeminal neuron is marked by blue arrows on both sides.

View larger version (19K): [in a new window]   Fig. 4. XMeis3 antisense morpholino oligonucleotide (AMO) inhibits posterior neural marker expression by blocking translation of XMeis3 RNA. (A) One-cell stage embryos were injected in the animal hemisphere with 15 ng of AMO (lanes 4-5 and 8-9), 15 ng of control morpholino oligonucleotide (CMO; lanes 2-3 and 6-7) and 1.0 ng of XMeis3 RNA (lanes 3, 5, 7 and 9). Eighteen animal cap explants were removed from all injected groups (lanes 6-9) of blastula embryos (stage 8-9). Explants from each group were grown to stage 20 and total RNA was isolated. In parallel, total RNA was also isolated from pools of seven embryos from each injected group (lanes 1-5). RT-PCR analysis was performed with the markers: Krox20, HoxB3 and HoxB9. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from normal CMO-injected embryos (lane 2). (B) Western analysis of XMeis3-Myc protein. One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of RNA encoding the XMeis3-Myc fusion protein (lanes 2-3) and 16 ng of AMO (lane 3) or 16 ng of CMO (lane 1-2). Protein was isolated from pools of seven embryos per group at stage 12.5: control (lane 1), XMeis3-Myc/CMO (lane 2), XMeis3-Myc/AMO (lane 3). As a positive control, in vitro synthesized XMeis3-Myc protein was also examined on the filter (lane 4). Analysis was performed using the 9E10 Myc antibody. As a positive control, total Erk protein was detected with the p44/p42 antibody. (C) Embryos from the above experiment (Fig. 4B) were grown to late neurula stages. Total RNA was also isolated from pools of seven embryos from the control (lanes 1-2) and injected groups (lanes 3-4). RT-PCR analysis was performed with the markers XMeis3 and Krox20. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from uninjected control embryos (lane 2).

View larger version (57K): [in a new window]   Fig. 5. XMeis3 antisense morpholino oligonucleotide causes posterior truncations and anterior expansions in embryos. (A, top) Embryos at the one-cell stage were injected with 20 ng of the CMO. These embryos resembled uninjected controls. All embryos were fixed for photography at stage 38. (A, middle) Embryos at the one-cell stage were injected with 12.5 – 15 ng of the AMO. (A, bottom) Embryos at the one-cell stage were injected with 17.5-20 ng of the AMO. (B) Embryos from the experiment in A were grown to late neurula stages. Total RNA was also isolated from pools of seven embryos from each of the injected groups (lanes 1-6). RT-PCR analysis was performed with the markers: En2, Krox20 and HoxB9. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from control CMO injected embryos (lane 2). (C) Embryos at the one-cell stage were injected with 15 ng of the CMO (top) or the AMO (bottom). CMO- and AMO-injected embryos were co-injected with either 1.6 ng of XMeis3 RNA (middle) or 1.6 ng of hth RNA (right). White arrows mark the cement glands. All embryos were fixed for photography at stage 27/28.

View larger version (57K): [in a new window]   Fig. 6. XMeis3 antisense morpholino oligonucleotide eliminates hindbrain marker expression. Two-cell albino embryos were injected unilaterally into the animal hemisphere of one blastomere with 6-7.5 ng of the XMeis3 AMO. In situ hybridization was performed in late neurula stage embryos. In all cases, embryos are viewed dorsally; embryos are oriented anterior (top) to posterior (bottom). The red arrow delineates the dorsal midline. Embryos were injected on the right side. (A) In situ hybridization with Krox20 and HoxB9; Krox20 expression is eliminated on the AMO-injected side. HoxB9 expression is unchanged on the AM- injected side. (B) In situ hybridization with XE10; expression is eliminated on the AMO-injected side. (C) In situ hybridization with HoxB3 and HoxB9; HoxB3 expression is eliminated on the AMO-injected side. HoxB9 expression is unchanged on the AMO-injected side. (D) In situ hybridization with XMeis3 and En2 (red); expression of both markers is posteriorized on the AMO-injected side. (E) In situ hybridization with XMeis3; expression is inhibited on the AMO-injected side. The XMeis3 expression in r2 is indicated by arrows on both sides.

View larger version (54K): [in a new window]   Fig. 7. XMeis3-antimorph protein anteriorizes neural marker expression in animal cap explants caudalized by bFGF or Wnt3a. (A) One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of XMeis3-AM RNA (lane 4) or 0.2 ng of noggin RNA (lane 5) or both (lane 6). Eighteen animal cap explants were removed from uninjected (lanes 2-3) and injected groups of blastula embryos (stage 8-9). Explants from each group were aged until stage 10.25 and XbFGF was added at 50 ng/ml. Explants from each group were grown to late neurula stage and total RNA was isolated. RT-PCR analysis was performed with the markers: En2 and HoxB9. EF1 served as a control for quantifying RNA levels in the different samples. -RT-PCR (lane 1) was performed on total RNA isolated from uninjected embryos. (B) One-cell stage embryos were injected in the animal hemisphere with 1.6 ng of XMeis3-AM RNA (lane 6-7 and 9), 0.2 ng of noggin RNA (lane 4 and 8-9) and/or 0.1 ng of mouse Wnt3a RNA (lanes 5, 7-9). Eighteen animal cap explants were removed from uninjected (lane 3) and injected groups of blastula embryos (stage 8-9). Explants from each group were grown to late neurula stage and total RNA was isolated. In parallel, total RNA was also isolated from uninjected control embryos (lanes 1-2). RT-PCR analysis was performed with the markers En2, Krox20, HoxB3 and HoxB9. EF1 served as a control for quantifying RNA levels in the different samples. For controls, RT-PCR (lane 2) and -RT-PCR (lane 1) were performed on total RNA isolated from uninjected control embryos.