Department of Biochemistry, Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel
*Author for correspondence (e-mail: dale{at}tx.technion.ac.il)
Accepted June 20, 2001
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SUMMARY |
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Key words: Xenopus laevis, XMeis3, Antimorph, Antisense morpholino oligonucleotides, Caudalization, Hindbrain
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INTRODUCTION |
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Several molecules have been identified which participate in the activation and transformation processes. Non-neural ectoderm is induced to anterior-neural tissue by inhibition of bone morphogenetic protein (BMP) activity (Harland and Gerhart, 1997). Secreted BMP antagonist molecules bind the BMP molecule and inhibit its receptor binding activity (Zimmerman et al., 1996; Piccolo et al., 1996; Fainsod et al., 1997; Hsu et al., 1998). BMP antagonists are expressed in Spemanns organizer during gastrulation and induce anterior neural tissue in adjacent ectoderm (Lamb et al., 1994; Hemmati-Brivanlou and Melton, 1994; Sasai et al., 1994).
Three secreted transformation factors have been shown to caudalize neural tissue in whole embryos or explants: retinoic acid (Durston et al., 1989; Sive et al., 1990; Ruiz i Altaba and Jessell, 1991; Sharpe, 1991; Kolm and Sive, 1995; Papalopulu, and Kintner, 1996; Godsave et al., 1998), basic fibroblast growth factor (bFGF; Kenkgaku and Okamato, 1995; Lamb and Harland, 1995; Cox and Hemmati-Brivanlou, 1995) and Xwnt3a (McGrew et al., 1995; McGrew et al., 1997). These factors and/or their receptors are expressed in the neural plate in a temporal and regional manner, supporting their roles as caudalizers of the nervous system. All three of these molecules caudalize in non-equivalent manners and it is still not clear how they interact to specify proper AP pattern in the CNS (Kolm et al., 1997; Gamse and Sive, 2000).
In Xenopus embryos and explants, Meis homeobox proteins have been shown to caudalize and dorsalize the CNS (Salzberg et al., 1999; Maeda et al., 2001). The caudalizing Xenopus Meis3 gene (Salzberg et al., 1999) was originally identified as a Drosophila homothorax (hth) gene homolog (Rieckhof et al., 1997; Kurant et al., 1998). In neurula embryos, XMeis3 is expressed in the hindbrain from rhombomere 2 (r2) to rhombomere 4 (r4), and in the anterior spinal cord (Salzberg et al., 1999). Ectopic XMeis3 expression in embryos causes anterior truncations, with a loss of anterior neural tissues from the cement gland/forebrain until the midbrain-hindbrain junction. In parallel, hindbrain and spinal cord cell types are expanded in embryos that overexpress XMeis3. Expression of pan-neural markers is unaltered by ectopic XMeis3 expression.
In neuralized animal cap explants, ectopic XMeis3 expression inhibits anterior neural induction by BMP antagonists such as noggin or the BMP2/4 dominant-negative (DN) receptor; however, XMeis3 does not inhibit the ability of these BMP antagonists to induce pan-neural markers (Salzberg et al., 1999). Strikingly, in naïve animal cap ectoderm, ectopic XMeis3 expression induces transcriptional activation of hindbrain and spinal cord neural markers, albeit in the absence of pan-neural marker expression (Salzberg et al., 1999). This effect is ectoderm-specific, as XMeis3 does not activate transcription of mesodermal markers in injected animal cap explants (Salzberg et al., 1999). Thus, the XMeis3 protein uncouples neural caudalization from neural induction.
To further examine the role of XMeis3 protein in Xenopus neural development, two distinct strategies have been used to inhibit endogenous XMeis3 protein activity. In the first strategy, fusions of a XMeis3 open reading frame to either the Engrailed transcriptional repressor domain or the VP16 transcriptional activation domain were compared in embryos and explants. We found that the Eng-XMeis3 fusion protein acted as an antimorph, blocking the effects of wild-type XMeis3-encoding RNA in Xenopus embryos and explants, while the VP16-XMeis3 fusion protein acted as a transcriptional activator to caudalize embryos and explants. In embryos, ectopic XMeis3-antimorph (XMeis3-AM) protein expression caused a loss of hindbrain marker expression, with a concomitant posterior expansion of anterior neural markers into the hindbrain region. Spinal cord and pan-neural marker expression was unaltered by the XMeis3-AM protein. In a second experimental approach, inhibition of XMeis3 mRNA translation by injection of XMeis3 antisense morpholino oligonucleotides (AMOs) also disrupted Xenopus hindbrain formation.
In animal cap explants caudalized by bFGF or Wnt3a, antagonism of XMeis3 protein activity did not specifically inhibit caudalizer activity, but it did rostralize the AP extent of posterior neural marker expression. XMeis3 activity is probably required for cells to overcome anterior neural signaling, thus enabling proper hindbrain cell fate identity in the developing Xenopus CNS.
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MATERIALS AND METHODS |
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Xenopus embryos, explants and inducing factors
Ovulation, in vitro fertilization, embryo culture and dissections were carried out as described by Reem-Kalma et al. (Reem-Kalma et al., 1995). Embryos were staged according to Nieuwkoop and Faber (Nieuwkoop and Faber, 1967). Xenopus bFGF (XbFGF) treated (50 ng/ml) animal cap explants were cultured as described by Lamb and Harland (Lamb and Harland, 1995).
RNA injections
Different concentrations of capped sense in vitro transcribed full-length XMeis3 (Salzberg et al., 1999), Eng-XMeis3 and VP16-XMeis3 (0.1-1.8 ng in a volume of 5-10 nl) were injected into the animal hemisphere of embryos at the one or two-cell stages. Capped in vitro transcribed Xenopus noggin RNA (200 pg) and mouse Wnt3a RNA (100 pg) were injected into the animal hemisphere of embryos at the one-cell stage (Smith and Harland, 1992; Baker et al., 1999).
Injection of antisense morpholino oligonucleotides (AMOs)
Antisense morpholino oligonucleotides (AMOs) complementing the 5' region of the XMeis3 mRNA were designed by and purchased from Gene Tools, LLC; Corvallis, OR (www{at}gene-tools.com; Heasman et al., 2000; Nasevicius and Ekker, 2000). The XMeis3 AMO sequence is 5'-ATACCTTTGTGCCATTCCGAGTTGG-3'. A standard control morpholino oligonucleotide (CMO) was also used in each experiment (Gene Tools). AMOs and CMOs were dissolved at 2 mg/ml in sterile water. One-cell embryos were routinely injected in the 10-20 ng range in 5-10 nl volumes. In two-cell stage embryos, one blastomere was injected with 7.5 ng in a 5 nl volume. The AMO was toxic at levels above 30 ng/embryo and experiments were performed at significantly lower concentrations.
In situ hybridization
Whole-mount in situ hybridization was carried out with digoxigenin-labeled probes, as described previously (Hemmati-Brivanlou et al., 1990; Harland, 1991; Knecht et al., 1995). Double in situ hybridization experiments were performed with probes generated from fluorescein and digoxigenin RNA-labeling mixes (Roche). Embryos were stained with BM purple and Fast Red substrates (Roche; Hollemann et al., 1998). In some cases, both probes were stained with BM purple. Two-cell stage albino embryos were injected unilaterally into the animal hemisphere of one-cell with 50-100 pg of RNA encoding the XMeis3-AM protein or 6-7.5 ng of the AMO. Embryos were cultured until late neurula stages and subsequently fixed for in situ hybridization. The uninjected side served as an internal control in all experiments. For lineage tracing analysis, 50 pg of RNA encoding the ß-galactosidase protein (ß-gal) (Smith and Harland, 1991) and RNA encoding the XMeis3-AM protein were co-injected unilaterally at the two-cell stage. Embryos were stained in red for ß-gal activity and fixed for whole-mount in situ hybridization as described previously (Bonstein et al., 1998). The perturbations seen in the embryos were always seen on the red stained ß-gal/XMeis3-AM or AMO injected side (data not shown).
RT-PCR analysis
RT-PCR was performed as described previously (Wilson and Melton, 1994), except that random hexamers (100 ng/reaction) were used for reverse transcription. Primers for EF1, En2, Krox20 and HoxB9 have been described elsewhere (Hemmati-Brivanlou and Melton, 1994). The primers for HoxD1 and RAR
2.2 have been described by Kolm et al. (Kolm et al., 1997). The otx2 and XAG1 primers are described elsewhere (Knecht et al., 1995). The XE10 primers are described elsewhere (Weinstein et al., 1996). The HoxB3 primers have been described by Hooiveld et al. (Hooiveld et al., 1999).
Western blot analysis
Western blot analysis was performed as described (Henig et al., 1998). For constructing the XMeis3-Myc vector, a full-length XMeis3 PFU generated fragment was subcloned 5' to the Myc fusion site in the pCS2+MT vector. This plasmid was linearized with NotI and transcribed with Sp6 to generate RNA encoding XMeis3-Myc fusion protein. XMeis3-Myc RNA (1.6 ng) was co-injected with 16 ng of XMeis3-AMO or CMO into one-cell stage embryos. Protein was isolated from a pool of ten embryos per group at stage 12.5. A total of 50 µg protein was loaded per sample for electrophoresis. Western blot analysis was performed using the 9E10 Myc antibody. As a control for protein loading, total Erk protein was detected by the p44/p42 antibody (New England Biolabs). As a positive control, in vitro transcribed/translated Meis3-Myc protein (TNT system; Promega) was loaded for electrophoresis.
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RESULTS |
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Ectopic XMeis3-antimorph (XMeis3-AM) expression eliminates the hindbrain region and expands anterior neural tissues
To address the role of endogenous XMeis3 protein in early development, we injected 400-800 pg of in vitro synthesized XMeis3 antimorph (XMeis3-AM) -encoding RNA into the animal hemisphere of one-cell Xenopus embryos. Embryos were scored at tailbud to tadpole stages for phenotypes (Fig. 2D). Overexpression of XMeis3-AM RNA caused anterior expansions in over 80% (n=40/48) of the injected embryos in the shown experiment. In comparison with control embryos, these embryos had enlarged cement glands and a shortened body axis (Fig. 2D).
To further examine the effects of ectopic XMeis3-AM expression on spatial expression of neural markers, whole-mount in situ hybridization was performed on XMeis3-AM-injected late neurula stage embryos. Embryos were unilaterally injected (50-100 pg of XMeis3-AM RNA) into the animal hemisphere of one blastomere at the two-cell stage. Complementing the observation of cement gland expansion (Fig. 2D), in nearly 60% of XMeis3-AM injected embryos (n=33/56), expression of the forebrain/midbrain-specific otx2 (Blitz and Cho, 1995) and forebrain-specific cpl-1 (Knecht et al., 1995) markers was posteriorly expanded (Fig. 3A-D). As seen in the double in situs with Krox20 or En2, otx2 expression was dramatically expanded into the hindbrain region (Fig. 3B-C). In the most extreme phenotypes, otx2 expression extended posteriorly into the r4/r5 boundary (Fig. 3B). cpl-1 expression was also shifted into the hindbrain region, as shown by the double in situ with En2 (Fig. 3D). In the cement gland, XAG1 and XA1 (not shown) expression (Sive et al., 1989) spread posteriorly in over 60% of the embryos (n=25/40). XAG1 expression appeared to extend into both spinal cord and lateral epidermal regions (Fig. 3E).
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Expression of the pan-neural nrp1 marker (Fig. 3F; Table 1) (Richter et al., 1988) and the spinal cord-specific HoxB9 marker (Fig. 3G; Table 1) (Wright et al., 1990) was unaltered by ectopic XMeis3-AM expression, despite overlapping XMeis3 mRNA expression in the hindbrain and anterior spinal cord (Salzberg et al., 1999). Interestingly, expression of the neuron specific n-tubulin marker (Hollemann et al., 1998) was highly inhibited (Fig. 3N; Table 1) by ectopic XMeis3-AM activity. In strong phenotypes, both the r2-derived trigeminal neuron as well as the more posterior neural expression was eliminated. In more moderate phenotypes, the trigeminal was still missing but posterior expression was less inhibited (not shown). Thus, XMeis3 may have a role in early neuron specification.
These results demonstrate that ectopic XMeis3-AM expression can cause an anterior transformation of the hindbrain by inhibiting caudalization. Thus, functional XMeis3 protein appears to be required for correct specification of the hindbrain in early Xenopus development.
Antisense morpholino oligonucleotides also inhibit XMeis3 caudalizing activity and hindbrain pattern
An additional molecular tool for antagonism of in vivo XMeis3 protein activity is antisense morpholino oligonucleotides (AMOs). AMOs have recently been shown to inhibit mRNA translation in Xenopus and zebrafish embryos (Heasman et al., 2000; Nasevicius and Ekker, 2000). AMOs, complementary to the 5' UTR and spanning the initial translated codons of the XMeis3 mRNA (see Materials and Methods) were injected at the one-cell stage into the animal hemisphere of embryos. In every experiment, a control morpholino oligonucleotide (CMO) was injected at an identical concentration to the AMO (see Materials and Methods). As described previously for XMeis3-AM, the specific inhibitory effect of AMOs on XMeis3 activity was screened by co-injection with wild-type XMeis3 RNA. Injection of XMeis3 AMOs together with XMeis3 wild-type RNA inhibited caudalizing activity in both animal cap explants and whole embryos (Fig. 4A). In animal cap explants, XMeis3 ectopically activated expression of the Krox20, HoxB3 and HoxB9 genes (Fig. 4A, lane 7), but in explants co-expressing XMeis3 and the AMO, posterior neural marker expression was eliminated (Fig. 4A, lane 9). In whole embryos, ectopic XMeis3 significantly increased Krox20 and HoxB3 gene expression (Fig. 4A, lane 2); this increase was inhibited by co-expression with the AMO (Fig. 4A, lane 5). Strengthening this observation, expression of the AMO in embryos significantly reduced normal Krox20 and HoxB3 expression levels, in comparison with CMO-injected control embryos (Fig. 4A, compare lanes 2 and 4). HoxB9 expression was not disrupted in AMO-injected embryos (Fig. 4A, lanes 2-5).
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In XMeis3-Myc/CMO-injected embryos, XMeis3-Myc protein acts as a caudalizer, increasing Krox20 expression in these embryos (Fig. 4C, compare lanes 2 and 3). However, when XMeis3-Myc/AMO was co-injected, levels of Krox20 RNA were highly reduced, significantly below levels in control embryos (Fig. 4C, lanes 2-4).
To further determine the role of endogenous XMeis3 protein in the embryo, we used the AMO to inhibit endogenous XMeis3 mRNA translation during early development. We injected 12.5-20 ng of the AMO into the animal hemisphere of one-cell stage embryos; these embryos were scored at tailbud to tadpole stages for phenotypes (Fig. 5). Like ectopic XMeis3-AM expression, the AMO (17.5-20 ng) caused anterior expansions and cement gland enlargement in over 80% (n=41/49) of the injected embryos (Fig. 5A, lower panel), in comparison with control CMO-injected embryos (Fig. 5A, upper panel). As in the case of the XMeis3-AM phenotypes, these embryos also had a much shorter body axis; body length was reduced by approximately 25-33% in AMO phenotypic embryos. At lower AMO concentrations (12.5-15 ng), body length was still altered in over 75% of the embryos (n=37/54), and anterior expansion phenotypes were weaker (Fig. 5A, middle panel). Thus, like the XMeis3-AM protein, injection of the AMO caused a prominent dose-dependent anteriorized phenotype in embryos.
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To demonstrate AMO specificity, XMeis3 and Drosophila hth-encoding RNAs were separately co-injected into embryos together with the AMO. We have previously shown that ectopic hth expression can caudalize Xenopus embryos and animal cap explants, in the same way as XMeis3 (Salzberg et al., 1999). As the hth gene lacks the XMeis3 5' region encoded by the AMO, we expect that its caudalizing activity should not be affected by the AMO. Indeed, both RNAs caudalized embryos in a similar manner: 85% of the XMeis3/CMO injected embryos (n=8) and 75% of the hth/CMO (n=16) -injected embryos had small cement glands (Fig. 5C, upper panel). XMeis3/AMO-injected embryos had rescued caudalized phenotypes: nearly 80% of the embryos (n=18) had normal or expanded cement glands (Fig. 5C, lower panel). In sharp contrast, 70% (n=20) of the hth/AMO-injected embryos had small cement glands (Fig. 5C, lower panel), like the XMeis3/CMO-injected group (Fig. 5C, upper panel). In the AMO-injected control group, 75% (n=16) of the embryos had expanded cement glands (lower left panel), in comparison with the CMO-injected (upper left panel) group (n=10). Similar results were also seen in animal cap explants; expression of Krox20 was reduced in XMeis3/AMO versus XMeis3/CMO-injected explants, but levels of Krox20 expression were identical in hth/AMO- and hth/CMO-expressing explants (not shown). These results show that the AMO cannot inhibit hth caudalizing activity, thus, the AMO is indeed specific to the XMeis3 gene.
To further examine the role of the AMO in neural patterning, whole embryos at the two-cell stage were injected unilaterally into one blastomere with 6-7.5 ng of AMO, and whole-mount in situ hybridization was performed. We saw a dramatic reduction in hindbrain marker expression on the injected side: Krox20 (Fig. 6A), XE10 (Fig. 6B) and HoxB3 (Fig. 6C). HoxB9 expression in the spinal cord was not decreased in AMO-injected embryos (Fig. 6A,C). In AMO-injected embryos, XE10 expression was exclusively inhibited in the hindbrain (where XMeis3 expression overlaps), but not in ectoderm regions found lateral to the neural tube, where XMeis3 was not expressed (Fig. 6B). We also examined how AMO injection altered endogenous XMeis3 expression in r2-r4 and the anterior spinal cord. In moderate phenotypes (Fig. 6D), we detected a shift of the XMeis3 rhombomeric expression from r2-r4 to r5-r7 with a fusion of the expression domain to the spinal cord. In the same embryo, En2 expression is pushed to the approximate r2/r3 boarder (Fig. 6D). In more extreme phenotypes (Fig. 6E), the XMeis3 expression pattern is again shifted posteriorly, but XMeis3 mRNA levels are also highly reduced. These data strongly corroborate the results obtained with the XMeis3-AM protein (Fig. 3), providing substantial proof that XMeis3 protein activity is obligatory for proper cell fate determination in the hindbrain.
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In noggin-neuralized animal cap explants, XMeis3-AM protein modified caudalization by XbFGF and mouse Wnt3a. In these caps, the perturbation of XMeis3 activity did not inhibit caudalization per se, but did bias neural marker expression in a more anterior manner. In both noggin/XbFGF- and XbFGF-treated animal caps explants, XMeis3-AM protein decreased expression of spinal cord-specific HoxB9 marker, yet increased expression of the more anterior En2 marker (Fig. 7A). This effect was dependent on the initial AP coordinates of the explant. When caps are treated with XbFGF, only HoxB9 is induced (Fig. 7A, lane 3), yet in the presence of the XMeis3-AM protein, both HoxB9 (reduced levels) and En2 are expressed (Fig. 7A, lane 4). In XbFGF/noggin-treated caps, both En2 and HoxB9 are expressed (Fig. 7A, lane 5); however, in the presence of XMeis3-AM protein, En2 is exclusively expressed and at increased levels (Fig. 7A, lane 6). Thus, the final extent of anteriorization in the XMeis3-AM injected explants appears determined by the initial AP patterning coordinates in the explant that were pre-determined by FGF±noggin. The presence of the XMeis3-AM protein shifted neural marker expression in favor of the more anterior mid-hindbrain junction, while inhibiting expression of more posterior spinal cord and hindbrain markers.
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In caps that solely expressed mouse Wnt3a, XMeis3-AM activated En2 expression and inhibited hindbrain marker expression, but it did not inhibit HoxB9 expression (Fig. 7B, compare lanes 5 and 7). In some instances, it even stimulated HoxB9 expression (not shown).
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DISCUSSION |
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Further supporting this observation, studies in transgenic flies that express either Xenopus Eng-XMeis3 or Drosophila Eng-HTH chimera proteins demonstrated hth loss-of-function like phenotypes (Inbal et al., 2001). In a complementary manner, transgenic flies expressing VP16-HTH chimera protein displayed hth gain-of-function like phenotypes; VP16-HTH also rescued phenotypes in hth mutant embryos (Inbal et al., 2001). Our previous studies demonstrated that ectopic expression of either wild-type HTH or XMeis3 proteins caudalized Xenopus embryos and animal cap explants (Salzberg et al., 1999). These results suggest that the Meis family transcriptional activator function has been conserved for nervous system development in such diverse organisms as flies and frogs.
Like the XMeis3-AM protein, injection of AMOs also inhibited wild-type XMeis3 caudalizing activity in embryos and animal cap explants. XMeis3-Myc protein levels were eliminated by the AMO. These results show that the AMO also acts as a potent inhibitor of XMeis3 activity, by preventing mRNA translation.
To address the role of XMeis3 during Xenopus CNS development, one-cell embryos were injected with the ENG-XMeis3 (XMeis3-AM) RNA, VP16-XMeis3 RNA or AMOs. Ectopic expression of VP16-XMeis3 RNA caudalized Xenopus embryos in a manner similar to the wild-type XMeis3-encoding RNA. By contrast, both XMeis3-AM and AMOs had distinct posterior-truncation/anterior-expansion phenotypes. In these embryos, the cement gland was expanded and the body axis was shortened.
To further address this point, albino embryos were unilaterally injected with XMeis3-AM or AMOs into one blastomere at the two-cell stage. At neurula stages, a wide array of neural markers were examined by whole-mount in situ hybridization. Confirming the phenotypic observations, in XMeis3-AM-injected embryos, we saw an expansion of expression of anterior markers such as XAG1, cpl-1, otx2 and En2 into more posterior regions of the brain. In the most extreme cases, we saw otx2 expression shifted as far back as r4/r5 and En2 expression was eliminated. In more moderate phenotypes, otx2 expression was shifted to r1/r2 and En2 expression was shifted to r3/r4. The posterior spread and loss of En2 and endogenous XMeis3 expression, and the concomitant loss of the r2-derived trigeminal neuron demonstrate that patterning in the most anterior hindbrain r1/r2 regions is greatly disrupted by the loss of endogenous XMeis3 activity. Rhombomeric expression of the Krox20, XE10, HoxB1 and HoxB3 markers was severely reduced by the XMeis3-AM protein or the AMO. In the most moderate hindbrain phenotypes, we could detect a two rhombomeric-shift of Krox20 expression from r3/r5 to r5/r7. By contrast, the spinal cord and pan-neural markers, HoxB9 and nrp1 were unaffected by XMeis3-AM or AMO activity.
It also appears that neurogenesis may be affected by the loss of XMeis3 activity. In XMeis3-AM injected embryos, n-tubulin expression is always lost in the r2-derived trigeminal neuron, and this could be a reflection of rhombomeric identity loss in this region. In most cases, injection of the XMeis3-AM also inhibited posterior n-tubulin expression. Neither XMeis3 nor noggin strongly induces n-tubulin expression in animal cap explants; however, in the presence of both molecules, we detected high levels of n-tubulin expression in animal caps (S. E. and D. F., unpublished). Thus, further experiments need to be performed to determine the exact role for XMeis3 protein in specifying neuron cell fates along the AP axis.
These results show that functional XMeis3 protein maintains a proper AP balance required for hindbrain formation. The spread of expression of anterior neural markers posteriorly into the hindbrain suggests that XMeis3 is essential for actively maintaining a caudalized state in the hindbrain. While XMeis3 does not seem required for neural induction, it seems to fine tune the AP pattern in the forebrain-hindbrain region. The conversion of hindbrain regions to more anterior fates, with concomitant posterior expansion of XAG1, cpl-1, otx2 and En2 expression emphasizes the role of XMeis3 in this AP fine-tuning process. XMeis3 is expressed in the anterior spinal cord; however, it may not be required for proper spinal cord formation.
Animal cap assays shed an interesting light on the interactive role of XMeis3 with caudalizing signaling molecules such as XbFGF and Wnt3a, confirming a role for XMeis3 as a neural patterning gene. Ectopic XMeis3-AM expression did not specifically inhibit caudalizing activity by these signaling molecules. However, the lack of XMeis3 activity did lead to a rostral shift in the AP levels of these explants that was dependent on the initial AP coordinates in the explants. In the case of animal cap explants treated solely with XbFGF, these caps expressed HoxB9 but not En2; however, these explants expressed both HoxB9 (reduced) and En2, when XMeis3 activity was inhibited. XbFGF/noggin-treated animal caps expressed both HoxB9 and En2, yet in the presence of the XMeis3-AM, these caps ceased to express HoxB9 and had increased levels of En2. Thus, in XMeis3-AM-expressing animal caps, the final AP output was determined by the initial AP status of the explant. XMeis3-AM protein shifted posterior neural marker expression to the anterior mid-hindbrain junction, while inhibiting expression of spinal cord and hindbrain markers. Our previous studies have shown that XMeis3 caudalization activity requires functional FGF/mitogen-activated protein kinase signaling (Ribisi et al., 2000); however, this relationship is not reciprocal, as bFGF caudalizing activity per se is not dependent on XMeis3 activity. This result strongly supports a role for XMeis3 as a cell patterning protein that interprets and maintains a given AP status in the CNS.
The interpretation of experiments in which animal cap explants are caudalized by mouse Wnt3a is more complicated, but supportive of the results with bFGF. In noggin/Wnt3a-expressing caps, a similar rostralization was observed in the presence of XMeis3-AM, a gain of En2 expression, with a concomitant loss of HoxB9, HoxB3 and Krox20 expression. However, a somewhat contrasting result was seen with the co-injection of mouse Wnt3a and XMeis3-AM in the absence of noggin. In these experiments, there was indeed an increase in En2 expression and a decrease in hindbrain marker expression, but we did not observe a reduction in HoxB9 expression. In some experiments, we even saw an increase in HoxB9 expression (not shown). It appears that in the absence of a strong neural inducer, mouse Wnt3a is a relatively weak inducer of hindbrain in comparison with XMeis3; however, mouse Wnt3a may actually induce spinal cord better than XMeis3. We have found that when XMeis3 induces maximal levels of hindbrain markers, HoxB9 expression is reduced in animal cap explants (not shown). Apparently, when mouse Wnt3a caudalizes alone, its induction of the HoxB9 spinal cord marker is optimal when some XMeis3 target genes are inhibited by the XMeis3-AM. Perhaps, antagonism of specific XMeis3 target genes by the antimorph protein may enable mouse Wnt3a to more efficiently activate spinal cord markers instead of hindbrain markers in the absence of neural induction. However, in the presence of noggin, XMeis3 target genes appear to be required for high HoxB9 and hindbrain marker expression by mouse Wnt3a. In whole embryos, HoxB9 expression is unchanged or even slightly increased in the presence of XMeis3-AM or the AMO. Inhibition of Xwnt-3a activity reduces HoxB9 expression in embryos and explants (McGrew et al., 1997), so a delicate balance between Wnt and XMeis3 activities may maintain optimal HoxB9 expression levels in the spinal cord. The implications of these wnt/XMeis3 interactions are still unclear, and further experiments are being carried out to understand how Wnt and XMeis3 pattern posterior neural tissue.
Using two distinct molecular approaches, we have inhibited XMeis3 protein activity in Xenopus embryos. In these embryos, a clear perturbation of the posterior CNS is observed, most specifically in the hindbrain region. XMeis3 appears to give distinct spatial identity to hindbrain cells. Without proper XMeis3 activity, anterior neural tissue spreads posteriorly and hindbrain identity is lost. The hindbrain is trapped in a more rostral cell fate. XMeis3 caudalizes the CNS to hindbrain, without inducing neural tissue. When viewing the activation and transformation model of neural induction, functional XMeis3 activity may be prerequisite for the transformation step. XMeis3 probably interprets spatial information along AP axis in hindbrain cells, thus enabling them to differentiate in a proper manner. Future studies will focus on how XMeis3 functions as a transcriptional activator to caudalize the brain. By identifying genes directly targeted by XMeis3, we intend to determine the genetic hierarchy regulating hindbrain formation in the developing CNS.
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ACKNOWLEDGMENTS |
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