New roles for Wnt and BMP signaling in neural anteroposterior patterning
2019; Springer Nature; Volume: 20; Issue: 6 Linguagem: Inglês
10.15252/embr.201845842
ISSN1469-3178
AutoresHanna Polevoy, Yoni Evgeni Gutkovich, Ariel Michaelov, Yael Volovik, Yaniv M. Elkouby, Dale Frank,
Tópico(s)Axon Guidance and Neuronal Signaling
ResumoArticle1 April 2019free access Transparent process New roles for Wnt and BMP signaling in neural anteroposterior patterning Hanna Polevoy Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yoni E Gutkovich Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Ariel Michaelov Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yael Volovik Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yaniv M Elkouby Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Dale Frank Corresponding Author [email protected] orcid.org/0000-0002-1857-6820 Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Hanna Polevoy Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yoni E Gutkovich Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Ariel Michaelov Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yael Volovik Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Yaniv M Elkouby Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Dale Frank Corresponding Author [email protected] orcid.org/0000-0002-1857-6820 Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel Search for more papers by this author Author Information Hanna Polevoy1,‡, Yoni E Gutkovich1,‡, Ariel Michaelov1,‡, Yael Volovik1, Yaniv M Elkouby1,† and Dale Frank *,1 1Department of Biochemistry, Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa, Israel †Present address: Department of Developmental Biology and Cancer Research, The Institute for Medical Research Israel-Canada, Hebrew University Hadassah Medical School, Jerusalem, Israel ‡These authors contributed equally to this work *Corresponding author. Tel: +972 4 829 5286; Fax: +972 4 855 3299; E-mail: [email protected] EMBO Rep (2019)20:e45842https://doi.org/10.15252/embr.201845842 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract During amphibian development, neural patterning occurs via a two-step process. Spemann's organizer secretes BMP antagonists that induce anterior neural tissue. A subsequent caudalizing step re-specifies anterior fated cells to posterior fates such as hindbrain and spinal cord. The neural patterning paradigm suggests that a canonical Wnt-signaling gradient acts along the anteroposterior axis to pattern the nervous system. Wnt activity is highest in the posterior, inducing spinal cord, at intermediate levels in the trunk, inducing hindbrain, and is lowest in anterior fated forebrain, while BMP-antagonist levels are constant along the axis. Our results in Xenopus laevis challenge this paradigm. We find that inhibition of canonical Wnt signaling or its downstream transcription factors eliminates hindbrain, but not spinal cord fates, an observation not compatible with a simple high-to-low Wnt gradient specifying all fates along the neural anteroposterior axis. Additionally, we find that BMP activity promotes posterior spinal cord cell fate formation in an FGF-dependent manner, while inhibiting hindbrain fates. These results suggest a need to re-evaluate the paradigms of neural anteroposterior pattern formation during vertebrate development. Synopsis In Xenopus, the caudalizing activities inducing hindbrain (Wnt-Meis3-FGF) are uncoupled in time and space from the spinal cord inducing (BMP-FGF-Cdx) activities. These observations do not support a model in which a Wnt-signaling gradient demarcates all anteroposterior neural fates. Inhibition of canonical Wnt-signaling or its downstream transcription factors eliminates hindbrain, but not spinal cord fates. BMP-activity promotes posterior spinal cord cell fates in an FGF-dependent manner, while inhibiting hindbrain fates. A simple high to low Wnt-gradient cannot explain specification of all cell-fates along the neural anteroposterior axis. Introduction In vertebrates, the nervous system has a distinct anteroposterior (AP) pattern. After initial neural induction, the nervous system is subdividing into the forebrain, midbrain, hindbrain, and spinal cord regions [rev. in 1, 2]. Historically, much of our knowledge of these earliest patterning events has been elucidated in amphibian and chick model systems [rev. in 3, 4]. In Xenopus, BMP antagonists secreted from the Spemann organizer initially induce anterior neural tissue [5, 6, rev. in 7, 8]. A subsequent caudalizing step specifies these neural induced cells to posterior hindbrain and spinal cord fates 9, 5, 10. These classic experiments suggested that a morphogen gradient could act to caudalize the posterior nervous system. Morphogens are secreted diffusible molecules that can induce changes in cell fates over distance in a concentration-dependent manner. Morphogen gradients have been suggested to regulate body axis formation during early vertebrate development [rev. in 11]. A secreted morphogen gradient could explain the induction of specific neural cell fates in a concentration-dependent manner along the vertebrate AP axis. The signaling molecules that caudalize the vertebrate nervous system are retinoic acid, FGF, and Wnt [rev. in 12-14]. These signaling molecules could all be candidates for morphogens regulating neural AP axis formation. Experimental evidence in Xenopus and chick embryos supports a paradigm in which canonical Wnt signaling acts as a morphogen to form a "low to high" gradient along the AP axis that concomitantly acts with BMP antagonism (neural induction) to pattern the developing nervous system 15, 16. This paradigm suggests that Wnt activity is at its highest levels in the posterior, inducing the spinal cord, at intermediate levels to induce the hindbrain and midbrain in the trunk, and at low activity in the most anterior forebrain regions. Our present results suggest that this paradigm needs to be re-evaluated. We show that Xenopus laevis embryos knocked down for zygotic canonical Wnt activity by ectopic Dkk1 protein surprisingly express normal levels of spinal cord markers, and these embryos have remarkably normal spinal cord morphology. These embryos display the typical Wnt knockdown phenotype, having expanded anterior neural-ectodermal tissues such as forebrain and cement gland, while simultaneously losing hindbrain fates. Thus, while the forebrain is expanded and the hindbrain is lost, the more posterior spinal cord is still intact. If a gradient of high Wnt activity is required to induce the most caudal embryonic regions, then the posterior spinal cord region should have the highest sensitivity to the loss of Wnt activity and not the lowest. In a complementary manner, we show that the knockdown of Zic transcription factor activity gives a similar phenotype. Zic transcription factors mediate Wnt signaling to induce different posterior neural cell fates 17-23. We show that knockdown of the Wnt-mediating downstream Zic1 transcription factor protein inhibits hindbrain fates, while expanding the forebrain. However, similar to the inhibition of canonical Wnt signaling, expression of the most posterior spinal cord markers is not decreased, but increased by Zic1 protein knockdown. In the "classic" paradigm for neural patterning, BMP antagonists and neural caudalizers were proposed to jointly act in specifying posterior cell fates along the vertebrate AP axis. We show in cultured ectodermal ex vivo explants that the more posterior spinal cord markers are not induced in the presence of BMP antagonists and neural caudalizers, while hindbrain markers are robustly induced. Counterintuitively, BMP alone is an efficient inducer of spinal cord marker expression and an inhibitor of hindbrain marker expression in embryos and ectodermal explants. We further found that BMP induction of spinal cord markers is FGF-dependent. We propose that the interplay between canonical Wnt activity, BMP activation, and BMP antagonism needs to be re-assessed concerning neural AP patterning. A simple high-to-low gradient of Wnt morphogen activity appears incompatible for determining the AP differences in spinal cord versus hindbrain cell fate specification. Low–moderate BMP signaling levels also appear crucial for spinal cord induction. These findings challenge the present paradigm of neural AP pattern formation in the developing vertebrate nervous system. Results Canonical Wnt signaling is required for hindbrain, but not spinal cord formation It has been a generally accepted paradigm that canonical Wnt signaling acts in a morphogen gradient to pattern neural tissue [rev. in 3] from the most posterior spinal cord (high Wnt) to the most anterior forebrain (low Wnt). Our previous studies showed that Wnt3a in the dorsal–lateral mesoderm induces hindbrain, neural crest, and primary neuron cell fates in the adjacent neural plate 24. Wnt induces these cell fates via direct transcriptional activation of the TALE-class homeobox protein, Meis3 24, yet Meis3 knockdown has only minor effects on spinal cord development 25. This observation does not necessarily support the idea of a Wnt gradient in neural AP patterning. To rigorously address the role of Wnt signaling in posterior neural patterning, the canonical Wnt-inhibitor Dkk1 protein was ectopically expressed in one-cell stage embryos. As seen by sqRT–PCR in neurula stage embryos (Fig 1A), expression of anterior forebrain and cement gland markers (xanf1, otx2, xag1) is increased, but expression of hindbrain markers is reduced (krox20, hoxb3). In an interesting and surprising manner, expression levels of the more posterior spinal cord markers (hoxb9, cdx1, cdx2, cdx4, hoxd10) are barely modulated by Wnt activity inhibition. The hoxb4 gene that is expressed in the hindbrain/spinal cord border region has an intermediate level of inhibition. Similar results were also seen by zygotic ectopic expression of the Nxfz8 protein (Fig 1B), a dominant-inhibitory Frizzled-receptor protein that inhibits canonical Wnt signaling 26. When examining embryos by in situ hybridization, the differences between spinal cord and hindbrain markers are striking (Fig 1C). Expression of krox20 in the hindbrain is highly perturbed, but expression of the spinal cord-specific hoxb9 and cdx4 genes looks normal. Even hoxd10 expression in the most posterior spinal cord region looks quite normal. Mesodermal (dorsal and ventral) markers are expressed at normal levels in gastrula and neurula stage embryos injected with dkk1 mRNA into one blastomere at the one-cell stage (Appendix Fig S1A–D) or one blastomere/two-cell stage (Appendix Fig S2A and C). Mesoderm fates do not seem to be perturbed at gastrula or neurula stages. At gastrula stages (Appendix Fig S1A and C), the expression of ventral (vent1, vent2, wnt8), lateral (osr1, osr2), dorsal (xnot, chordin-chd), and pan-mesodermal (xbra) markers is at normal levels. At later neurula stages, dorsal-notochord (xnot, chd, xbra) and dorsal–lateral skeletal muscle (myod, muscle actin-MA) marker expression is normal (Appendix Figs S1B and D, and S2A and C). In these embryos, anterior neural markers are expanded, hindbrain markers are inhibited, and spinal cord markers are expressed normally (Appendix Figs S1D, and S2A and C). In situ hybridization was performed on embryos that were injected with dkk1 mRNA into one blastomere at the two-cell stage. These embryos had asymmetric expression of the hindbrain-specific krox20 marker, but symmetric expression of spinal cord markers (hoxb9, cdx4) and dorsal mesoderm (chd, MA) markers (Appendix Fig S2A and C). We also sectioned the trunk regions of later tailbud stage embryos that were injected with dkk1 mRNA into one blastomere at the two-cell stage; these embryos had normal spinal cord, notochord, and somite formation (Appendix Fig S2E). These results strongly show that the effects of Wnt inhibition appear specific to the neural plate and not a result of mesoderm perturbation. Figure 1. Inhibition of canonical Wnt signaling perturbs hindbrain, but not spinal cord cell fates One-cell stage embryos were injected animally with mRNA encoding dkk1 (35/45 pg) protein. Total RNA was isolated from 10 control embryos at neurula stage 17 (lane 2) and 10 embryos from each dkk1-injected group (lanes 3–4). Various neural AP markers were examined by sqRT–PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with the zygotic-expressing Nxfz8/pCS2 encoding plasmid (100/200 pg). Total RNA was isolated from 10 control embryos at neurula stage 17 (lane 2) and 10 embryos from each Nxfz8-injected group (lanes 3–4). Various neural AP markers were examined by sqRT–PCR: cement gland and forebrain (xag1, xanf1, otx2), hindbrain (krox20, hoxb3), hindbrain/spinal cord border (hoxb4), and spinal cord (hoxb9, cdx1-2-4, hoxd10). Ef1α serves as a control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). Embryos from the same experiment as in (A) underwent in situ hybridization. Expression of the hindbrain marker krox20 (upper two panels) was reduced by Dkk1 (45 pg) in 83% of the embryos (n = 18). Expression of spinal cord markers (hoxb9, cdx4, hox10) was normal, being unchanged in 89% of the embryos (n = 18 for each marker). Control embryos (CE). Download figure Download PowerPoint To further extend this point, four of the animal blastomeres fated for ectoderm were injected with dkk1 mRNA at the eight-cell stage. These embryos also expressed lower levels of hindbrain markers (krox20, hoxb3) with normal levels of forebrain (xanf1), spinal cord (hoxb9, cdx2, cdx4), and dorsal mesoderm (chd—notochord, myod—muscle) markers (Appendix Fig S3A). As shown in previous studies 24, we lineage traced embryos injected animally at either the one- or eight-cell stage, and β-gal expression was localized to the neural plate at stages 17–18 (Appendix Fig S3C). If Wnt was working in a high-to-low gradient from posterior to anterior, we would not expect this observed robust expression of spinal cord markers. We would expect spinal cord marker genes to be more sensitive to the loss of Wnt activity than hindbrain markers, but the opposite result is seen. The Zic1 protein has been previously shown to act with Wnt signaling to mediate induction of neural crest, hindbrain, and primary neuron cell fates 17-23. We determined how dose-dependent Zic1-MO knockdown modulated neural AP cell fates. As can be seen by sqRT–PCR in neurula stage embryos (Fig 2A), forebrain marker expression (xanf1, otx2) is increased and hindbrain markers are reduced (krox20, hoxb1, hoxb3). Expression levels of spinal cord markers (hoxb9, cdx1, cdx4) are strongly increased by Zic1 protein knockdown. The panneural soxd1 marker is unaffected by the loss of Zic1 protein activity. Similar to the Zic1 morphant embryos, ectopic expression of the Zic5 dominant-negative protein (Fig 2B) inhibited hindbrain marker expression (krox20), while enhancing spinal cord marker expression (cdx1, cdx4). When examining embryos by in situ hybridization, marker expression differences along the neural AP axis are clear (Fig 2C). Expression of the sox2 panneural marker is unaltered by Zic1 knockdown, but forebrain-specific xanf1 expression is expanded versus the control (Fig 2C). Expression of a representative hindbrain marker such as krox20 is eliminated in comparison with the control (Fig 2C). Interestingly, expression of a representative spinal cord marker, cdx4, is laterally expanded and increased (Fig 2C; similar to Fig 2A and B). It is not clear if the spinal cord markers are anteriorly expanded, but expression of these genes is not perturbed by the knockdown of the Wnt mediator, Zic1. These observations complement the results seen with Dkk1 protein (Fig 1), suggesting that Wnt signaling is required for hindbrain formation, but is not crucial for the specification of more posterior spinal cord fates. Figure 2. Inhibition of Zic protein activity differentially affects spinal cord and hindbrain cell fates One-cell stage embryos were injected animally with the Zic1-MO (25/40 ng). Total RNA was isolated from 10 control embryos at neurula stage 18 (lane 2) and 10 embryos from each Zic1-MO-injected group (lanes 3–4). Neural AP markers were examined by sqRT–PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4), and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNA encoding Zic5 dominant-negative (0.5 ng) protein. Total RNA was isolated from five control embryos at neurula stage 18 (lane 1) and five embryos from the injected group (lanes 2). Neural AP markers were examined by sqRT–PCR: hindbrain (krox20) and spinal cord (cdx1-4). Ef1α is the control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 3). One-cell stage embryos were injected animally with the Zic1-MO (25 ng). Embryos underwent in situ hybridization. Expression of the panneural sox2 marker was normal, the forebrain marker xanf1 was expanded in 80% of the embryos, the hindbrain representative marker krox20 was reduced in 80% of the embryos, and the representative spinal cord marker cdx4 was expanded in 90% of the embryos. N = 23 embryos were per marker. Download figure Download PowerPoint BMP can caudalize neural tissue to spinal cord fates Our interest in hindbrain patterning led us to perform experiments on the induction of posterior neural markers in animal cap (AC) explants co-expressing noggin and the Meis3 neural caudalizing protein 25, 24, 27. Meis3 typically induces expression of an array of posterior markers in ACs in the absence of neural induction (Fig 3A, lane 4). When co-injecting noggin and Meis3 in ACs, neural pattern is altered in a number of ways. The caudalizing Meis3 protein inhibits noggin induction of anterior neural marker 27 expression (Fig 3A, lane 5 versus 6). In these same explants, the combination of noggin and Meis3 enhances hindbrain marker expression as might be expected if a neural inducer and caudalizer are co-expressed (Fig 3A, lane 4 versus 5). However for spinal cord markers, the opposite is true; noggin represses spinal cord marker expression (Fig 3A, lane 4 versus 5), despite Meis3 protein significantly repressing noggin anteriorizing activity in the same ACs (Fig 3A, lane 5 versus 6). This observation suggests that BMP activity may be required for Meis3 to induce spinal cord markers in the ACs. To further address this point, Meis3 was co-expressed with a fourfold less noggin RNA concentration (Fig 3A, lane 7). In these explants, anterior markers were still repressed by Meis3, but spinal cord markers were re-expressed (Fig 3A, lane 5 versus 7). This observation suggests that at the lower noggin concentration, higher endogenous BMP levels are required for the activation or maintenance of spinal cord marker expression in ACs. Figure 3. BMP activity regulates neural AP patterning One-cell stage embryos were injected animally with mRNAs encoding the noggin (5/20 pg) and/or meis3 (0.5 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 17. Total RNA was isolated from seven control embryos (lane 2), and from eighteen ACs from each group (lanes 3–8). Neural AP markers were examined by sqRT–PCR: forebrain (xanf1, otx2), hindbrain (krox20, hoxb1, hoxb3), spinal cord (hoxb9, cdx1-4), and panneural (soxd). Ef1α is the control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein. Total RNA was isolated from five control embryos at neurula stage 17 (lane 1) and five embryos from the injected group (lanes 2). Neural and mesodermal markers were examined by sqRT–PCR: forebrain (otx2), hindbrain (krox20, hoxb3), spinal cord (hoxb9, hoxc10, cdx4), panneural (nrp1, ncam), and mesodermal (muscle actin—MA). ODC is the control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNA encoding bmp4 (0.2 ng) protein Embryos underwent in situ hybridization. Expression of the hindbrain marker hoxb3 was reduced/eliminated by bmp4 (0.2 ng) in 68% of the embryos (n = 19). Expression of the spinal cord marker cdx4 was slightly increased/normal, in 100% of the embryos (n = 19). Download figure Download PowerPoint We next addressed this same question in vivo. One-cell (Fig 3B) and eight-cell (Appendix Fig S3B) stage embryos were injected with RNA encoding BMP4 protein in the animal hemisphere (fated for ectoderm). Previous studies have shown that ectopic BMP ventralizes embryonic mesoderm 28, 29. However, in these present experiments, the RNA was injected at much lower concentrations (five- to 10-fold lower) and was targeted to the ectoderm, and not the mesoderm. As can be seen by sqRT–PCR in neurula stage embryos (Fig 3B), panneural and forebrain marker expression (ncam, nrp1, otx2) is unaltered by the ectopic BMP levels; thus, the dose is not high enough to inhibit neural induction. However, hindbrain markers are strongly reduced (krox20, hoxb3), while spinal cord marker expression is increased (hoxb9, cdx4, hoxd10). Dorsal mesoderm fate does not seem to be perturbed by these low BMP levels, since muscle actin (MA) expression is normal in these embryos. When examining embryos by in situ hybridization, the differences between spinal cord and hindbrain markers are clear (Fig 3C); hoxb3 expression is eliminated, while cdx4 expression looks normal or expanded. Embryos injected with BMP4 into four animal blastomeres at the eight-cell stage expressed a similar pattern of marker expression like Fig 3A (Appendix Fig S3B), with strongly reduced expression of hindbrain markers (krox20, hoxb3), but normal expression levels of spinal cord markers (hoxb9, hoxc10, cdx2, cdx4), dorsal mesoderm markers (chd, myod), and the anterior cement gland marker xag. In situ hybridization was performed on embryos that were injected with bmp4 mRNA into one blastomere at the two-cell stage. These embryos had asymmetric expression of the hindbrain-specific krox20 marker, but symmetric expression of spinal cord markers (hoxb9, cdx4) and dorsal mesoderm (chd, MA) markers (Appendix Fig S2B and D). We also sectioned the trunk regions of later tailbud stage embryos that were injected with bmp mRNA into one blastomere at the two-cell stage; these embryos had normal spinal cord, notochord, and somite formation (Appendix Fig S2E). Thus, the effects of ectopic BMP levels appear specific to the neural plate and not a result of mesoderm perturbation. These results show that BMP acts to promote spinal cord formation, while repressing hindbrain cell fates, suggesting that BMP is acting to balance caudal cell fates along the neural AP axis. BMP can activate spinal cord markers independently of mesoderm in ectodermal explants To address a more direct role for BMP signaling in activating spinal cord markers, we examined whether it can activate spinal cord marker expression in AC explants. We found that ectopic BMP4 can specifically activate expression of spinal cord markers such as hoxb9, cdx1-2-4, and hoxc10-d10 in a dose-dependent manner (Fig 4A). Hindbrain (krox20) markers are never induced (Fig 4A). Induction of these neural markers appears to be direct in the ectoderm and uncoupled from mesoderm induction. Expression of the lateral mesoderm (kidney) BMP-target markers osr1-2 30 is not induced by BMP4 at these concentrations (Fig 4A). Additionally, muscle actin expression is not induced (Fig 4A). These results suggest that BMP does not indirectly mediate expression of spinal cord markers via mesoderm induction, but directly activates their expression in the ectoderm. Supporting this conclusion, ectodermal cytokeratin (E13) expression is not reduced, but constant in the explants (Fig 4A). Figure 4. BMP activity caudalizes ectoderm to spinal cord fates One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (5–50 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–7). Neural, epidermal, and mesodermal markers were examined by sqRT–PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10/d10, cdx1-2-4), epidermal cytokeratin (E13), lateral mesoderm (osr1-2), and dorsal–lateral mesoderm muscle actin (MA). ODC is the control for quantitating the RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with mRNAs encoding the bmp4 (50/150 pg) and/or meis3 (0.7 ng) proteins. AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to neurula stage 16. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–8). Neural and mesodermal markers were examined by sqRT–PCR: hindbrain (krox20), spinal cord (hoxb9, hoxc10, cdx2), and lateral mesoderm (osr1). Ef1α is the control for quantitating the RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). One-cell stage embryos were injected animally with increasing concentrations of bmp4 encoding mRNA (75–200 pg). AC explants were removed from control and injected embryos at blastula stage 9, and explants were cultured to gastrula stage 11–11.5. Total RNA was isolated from five control embryos (lane 2), and from eighteen ACs from each group (lanes 3–6). Marker expression was determined by sqRT–PCR. Xenopus brachyury (xbra) expression was induced by BMP4 in a dose-dependent manner. Marker expression was determined by sqRT–PCR. Neither dorsal (chd, goosecoid-gsc) nor ventral–lateral (osr1) mesoderm markers were induced by BMP4. Sox2/3 neural markers are expressed in the ACs and are unchanged by BMP expression. EF1α is the control for quantitating RNA levels. -RT–PCR was performed on total RNA isolated from control embryos (lane 1). Download figure Download PowerPoint In a reciprocal experiment to Fig 3A (noggin/Meis3 co-expression), Meis3 is co-expressed with BMP4 in ACs. In these ACs, krox20 expression (hindbrain) is inhibited by BMP (Fig 4B, compare lanes 4–8), but spinal cord marker expression (hoxb9, cdx2, hoxc10) is highly enhanced by BMP (Fig 4B, compare lanes 4–8). Lower concentrations of BMP4 that alone do not induce spinal cord expression (Fig 4B, lane 5) act additively with Meis3 to enhance spinal cord marker expression (Fig 4B, compare lanes 4–5, 7). Higher concentrations of BMP that can induce spinal cord marker expression also additively enhance spinal cord marker expression with Meis3 (Fig 4B, compare lanes 4, 6, 8). Mesoderm is not induced in these explants, as determined by osr1 expression. Studies in other vertebrate species suggested that co-expression of the brachyury (Bra) and Sox proteins suffices to induce neural precursor cells that will form the spinal cord [31, rev. in 32]. A number of studies suggest that neuromesodermal precursors (NMPs) are present in the vertebrate embryo posterior that will give rise to somite or spinal cord tissues 33, 34, 31, 35, 36. The co-expression of Bra/Sox p
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