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• W2105652093 abstract "Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA—miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs—in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties. Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by the function of a microRNA—miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs—in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties. miR-9 function in neural progenitors is context dependent miR-9 loss induces proliferation in the hindbrain and apoptosis in the forebrain The primary function of miR-9 in neurogenesis is to downregulate hairy1 mRNA levels miR-9/hairy1 affects proliferation via cyclinD/p27 and apoptosis via p53 During neurogenesis, proliferating neural cells (neural progenitor or neural stem cells), located in the ventricular zone (VZ), undergo self-renewal to replenish the progenitor population or, alternatively, engage in asymmetric divisions associated with the generation of neurons (Götz and Huttner, 2005Götz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Crossref PubMed Scopus (1570) Google Scholar). The process of neurogenesis is tightly coupled with the process of regional specification, which dictates the identity of neurons born in different areas of the central nervous system (CNS) (Gaspard and Vanderhaeghen, 2010Gaspard N. Vanderhaeghen P. Mechanisms of neural specification from embryonic stem cells.Curr. Opin. Neurobiol. 2010; 20: 37-43Crossref PubMed Scopus (90) Google Scholar). Neural stem cells themselves have different positional identity and can give rise to tumors with different signatures depending on their origin (Lee da et al., 2010Lee da Y. Yeh T.H. Emnett R.J. White C.R. Gutmann D.H. Neurofibromatosis-1 regulates neuroglial progenitor proliferation and glial differentiation in a brain region-specific manner.Genes Dev. 2010; 24: 2317-2329Crossref PubMed Scopus (87) Google Scholar, Palm and Schwamborn, 2010Palm T. Schwamborn J.C. Brain tumor stem cells.Biol. Chem. 2010; 391: 607-617Crossref PubMed Google Scholar). However, how regional specificity is integrated with the fundamental cellular decisions that drive neurogenesis is not well understood. Both intrinsic and external factors are thought to contribute to the correct execution and the transition from the transcriptional programs of neural stem cells to differentiated neurons in a region-specific manner (Falk et al., 2008Falk S. Wurdak H. Ittner L.M. Ille F. Sumara G. Schmid M.-T. Draganova K. Lang K.S. Paratore C. Leveen P. et al.Brain area-specific effect of TGF-beta signaling on Wnt-dependent neural stem cell expansion.Cell Stem Cell. 2008; 2: 472-483Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, Jessell, 2000Jessell T.M. Neuronal specification in the spinal cord: inductive signals and transcriptional codes.Nat. Rev. Genet. 2000; 1: 20-29Crossref PubMed Scopus (1657) Google Scholar, Lee and Pfaff, 2003Lee S.K. Pfaff S.L. Synchronization of neurogenesis and motor neuron specification by direct coupling of bHLH and homeodomain transcription factors.Neuron. 2003; 38: 731-745Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, Marklund et al., 2010Marklund U. Hansson E.M. Sundstrom E. de Angelis M.H. Przemeck G.K. Lendahl U. Muhr J. Ericson J. Domain-specific control of neurogenesis achieved through patterned regulation of Notch ligand expression.Development. 2010; 137: 437-445Crossref PubMed Scopus (47) Google Scholar). MicroRNAs are a class of small noncoding RNAs, which have been shown to play key roles in many developmental processes including stem cell proliferation and differentiation (Gangaraju and Lin, 2009Gangaraju V.K. Lin H. MicroRNAs: key regulators of stem cells.Nat. Rev. Mol. Cell Biol. 2009; 10: 116-125Crossref PubMed Scopus (602) Google Scholar, Kosik, 2006Kosik K.S. The neuronal microRNA system.Nat. Rev. Neurosci. 2006; 7: 911-920Crossref PubMed Scopus (680) Google Scholar, Stefani and Slack, 2008Stefani G. Slack F.J. Small non-coding RNAs in animal development.Nat. Rev. Mol. Cell Biol. 2008; 9: 219-230Crossref PubMed Scopus (1143) Google Scholar). They are particularly attractive for their potential to coordinate the response of many target genes, thereby acting as point of information integration. Knockout of the essential component of microRNA-processing Dicer has shown that microRNAs are indispensable for proper neural development in zebrafish (Giraldez et al., 2005Giraldez A.J. Cinalli R.M. Glasner M.E. Enright A.J. Thomson J.M. Baskerville S. Hammond S.M. Bartel D.P. Schier A.F. MicroRNAs regulate brain morphogenesis in zebrafish.Science. 2005; 308: 833-838Crossref PubMed Scopus (1085) Google Scholar) and mouse (De Pietri Tonelli et al., 2008De Pietri Tonelli D. Pulvers J.N. Haffner C. Murchison E.P. Hannon G.J. Huttner W.B. miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex.Development. 2008; 135: 3911-3921Crossref PubMed Scopus (291) Google Scholar), although the key miRs and their precise molecular targets have not been fully examined. miR-9 is a highly conserved microRNA, which is expressed primarily in the CNS (Kapsimali et al., 2007Kapsimali M. Kloosterman W.P. de Bruijn E. Rosa F. Plasterk R.H.A. Wilson S.W. MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system.Genome Biol. 2007; 8: R173Crossref PubMed Scopus (331) Google Scholar, Wienholds et al., 2005Wienholds E. Kloosterman W.P. Miska E. Alvarez-Saavedra E. Berezikov E. de Bruijn E. Horvitz H.R. Kauppinen S. Plasterk R.H.A. MicroRNA expression in zebrafish embryonic development.Science. 2005; 309: 310-311Crossref PubMed Scopus (1345) Google Scholar). In vertebrates the function of miR-9 has been studied in fish and mice with loss and gain-of-function approaches. In the fish, miR-9 has been shown to be necessary to define the mid-hindbrain boundary (MHB), a non-neurogenic boundary zone with organizer properties (Leucht et al., 2008Leucht C. Stigloher C. Wizenmann A. Klafke R. Folchert A. Bally-Cuif L. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary.Nat. Neurosci. 2008; 11: 641-648Crossref PubMed Scopus (256) Google Scholar). However, with respect to the role of miR-9 in neuronal differentiation and proliferation, the results obtained by the loss-of-function experiments in different systems have not always been consistent. In the anterior hindbrain, where miR-9 is expressed, a decrease in neuronal differentiation was reported, which, however, was not accompanied by an increase in progenitor proliferation (Leucht et al., 2008Leucht C. Stigloher C. Wizenmann A. Klafke R. Folchert A. Bally-Cuif L. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary.Nat. Neurosci. 2008; 11: 641-648Crossref PubMed Scopus (256) Google Scholar). This is similar to the result obtained in the embryonic mammalian forebrain, where miR-9 knockdown caused a reduction of early-born Cajal-Retzius neurons but did not have an effect on progenitors (Shibata et al., 2008Shibata M. Kurokawa D. Nakao H. Ohmura T. Aizawa S. MicroRNA-9 modulates Cajal-Retzius cell differentiation by suppressing Foxg1 expression in mouse medial pallium.J. Neurosci. 2008; 28: 10415-10421Crossref PubMed Scopus (172) Google Scholar). In another study, miR-9 knockdown caused a reduction in neural progenitors derived from mouse ES cells, accompanied by a slight increase in GFAP+ astrocytes, although the effects on proliferation were not directly tested (Krichevsky et al., 2006Krichevsky A.M. Sonntag K.-C. Isacson O. Kosik K.S. Specific microRNAs modulate embryonic stem cell-derived neurogenesis.Stem Cells. 2006; 24: 857-864Crossref PubMed Scopus (595) Google Scholar). However, the opposite result was obtained in neural stem cells derived from adult mammalian forebrain, where miR-9 knockdown caused a small increase in proliferating cells (1.37-fold) but did not change differentiation (Zhao et al., 2009Zhao C. Sun G. Li S. Shi Y. A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination.Nat. Struct. Mol. Biol. 2009; 16: 365-371Crossref PubMed Scopus (466) Google Scholar). Finally, in neural progenitors derived from human ES cells, loss of miR-9 has been shown to suppress proliferation, albeit by a small degree. In this system, loss of miR-9 promoted migration of neural progenitors (Delaloy et al., 2010Delaloy C. Liu L. Lee J.A. Su H. Shen F. Yang G.Y. Young W.L. Ivey K.N. Gao F.B. MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors.Cell Stem Cell. 2010; 6: 323-335Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). From these studies the emerging theme is that in most systems, miR-9 is necessary for neuronal differentiation, but the effect on proliferation is highly variable. Differences in the results obtained may be partly due to different model systems or experimental methodology; however, these discrepancies also raise the possibility that the function of miR-9 in neurogenesis and proliferation is highly context dependent. Here, we have undertaken a systematic analysis of miR-9 expression and function along the anterior-posterior (AP) axis during X. tropicalis development and uncovered an unexpected regional specificity. In the forebrain, miR-9 is expressed in both neural progenitors and developing neurons, whereas in the more posterior regions of the brain (mid- and hindbrain), it is restricted to neural progenitors only. Using loss-of-function experiments, we demonstrate that even though miR-9 is required for neuronal differentiation, regardless of the position along the AP axis, it regulates neural progenitors in a region-specific manner—it limits progenitor proliferation and promotes neuronal fate throughout the neural tube; in addition, in the forebrain it is important for progenitor survival. We have identified several genes that contain miR-9 binding sites in their 3′UTR and respond to miR-9 in vitro and in vivo. However, functional analysis showed that hairy1 is the single key target that mediates the effects of miR-9 in the forebrain and the hindbrain. hairy1 is a member of the Hes family of genes, and we show that, unlike other Hes genes, it is primarily expressed in neurogenic rather than boundary areas of the CNS (Baek et al., 2006Baek J.H. Hatakeyama J. Sakamoto S. Ohtsuka T. Kageyama R. Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system.Development. 2006; 133: 2467-2476Crossref PubMed Scopus (175) Google Scholar). Finally, we provide a molecular explanation for the regional-specific effects: miR-9 regulates proliferation by feeding into the network controlling cyclinD1/p27 expression in both areas, through Wnt signaling in the hindbrain and Fgf8 signaling in the forebrain, but affects apoptosis via the mdm2/p53 pathway specifically in the forebrain. These findings suggest that the positional embryonic origin of neural progenitors is an important parameter that dictates their response to the same microRNA and that in the case of miR-9 the specificity of response is generated downstream of a key target, hairy1. They show a regional diversity in the properties of neural progenitors and highlight the importance of taking into account the positional origin of stem cells in designing rational strategies to manipulate their proliferative potential. First, we examined miR-9 expression during the development of X. tropicalis using in situ hybridization (miR-9 LNA probe). miR-9 expression was evident in the prospective forebrain region in the anterior neural plate at stage 18/19. At stage 23/24 mature miR-9 was also detected in the developing eye and retina but later on its expression in the neural tube expanded to the more posterior parts of the brain, including the mid- and hindbrain at stage 30–36 (Figure 1A ). There are four predicted miR-9 encoding loci in the genome of X. tropicalis, which give rise to nearly identical mature miR-9 after processing (see Figure S1A available online). Expression of the individual transcripts was similar to the expression of mature miR-9 (Figure 1B; Figure S1B); however, miR-9a-1 was expressed at higher levels than the others. Transcripts were present in the forebrain, the eye, and in the mid- and hindbrain, but no expression was detected in the MHB (Figure 1B; Figure S1B, marked with asterisk), in agreement with reports in the zebrafish (Leucht et al., 2008Leucht C. Stigloher C. Wizenmann A. Klafke R. Folchert A. Bally-Cuif L. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary.Nat. Neurosci. 2008; 11: 641-648Crossref PubMed Scopus (256) Google Scholar), and no expression was evident in the spinal cord. We could not detect a signal for miR-9b, consistent with previous results (Walker and Harland, 2008Walker J.C. Harland R.M. Expression of microRNAs during embryonic development of Xenopus tropicalis.Gene Expr. Patterns. 2008; 8: 452-456Crossref PubMed Scopus (21) Google Scholar). During neural development progenitors divide in the VZ, and daughters that exit the cell cycle, migrate laterally to the marginal zone where they differentiate (Figure 1C). Sections showed that miR-9 transcripts have widespread expression in the forebrain but were restricted to the VZ in the more posterior areas (Figure 1D; Figure S1C). These spatial differences became even more apparent later during development (stage 36, Figure S1D). To determine whether miR-9 was also present in post-mitotic neurons in the forebrain or expressed only in progenitors along the AP axis, we used fluorescent in situ hybridization (FISH) for miR-9a-1 combined with immunostaining for Sox3 (marker for neural progenitors) at stages 30 and 36. We found that in the forebrain, miR-9 was transcribed in both Sox3-positive and Sox3-negative cells, whereas it appeared to be restricted to the Sox3-positive domain in the hindbrain (Figure 1E; Figure S1E). This suggests that miR-9 expression differs along the AP axis within a single species and raises the question whether it has the same function in different populations of neural progenitors. In order to gain insight about miR-9's role during neural development, we decided to examine its loss-of-function phenotype. We used an anti-miR-9 specific morpholino (miR-9 MO), which interferes with both the processing of miR-9 precursors and inhibits the activity of the mature miRNA (Kapsimali et al., 2007Kapsimali M. Kloosterman W.P. de Bruijn E. Rosa F. Plasterk R.H.A. Wilson S.W. MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system.Genome Biol. 2007; 8: R173Crossref PubMed Scopus (331) Google Scholar, Martello et al., 2007Martello G. Zacchigna L. Inui M. Montagner M. Adorno M. Mamidi A. Morsut L. Soligo S. Tran U. Dupont S. et al.MicroRNA control of Nodal signalling.Nature. 2007; 449: 183-188Crossref PubMed Scopus (174) Google Scholar) (see Figure S2A for schematic). Injection of miR-9 MO led to an almost complete knockdown of mature miR-9 at early tadpole stage compared to wild-type (WT) embryos, whereas miR-9 levels were increased in embryos injected with miR-9-2 precursor (Figure 2A ), as shown using semiquantitative RT-PCR. Knockdown was also confirmed using in situ hybridization and real-time PCR for the mature form of miR-9 (Figures S2B and S2C). Next, we injected miR-9 MO in one cell of a two-cell stage embryo and compared the injected to the control side at stage 30 at the forebrain and hindbrain level (Figure 2B). Depletion of miR-9 negatively affected neuronal differentiation, as indicated by the decreased expression of N-tubulin (n = 14/25) and NeuroD1 (n = 18/24) (Figure 2C, arrows). The number of Myt1-positive cells (a transcription factor expressed in post-mitotic neurons; Bellefroid et al., 1996Bellefroid E.J. Bourguignon C. Hollemann T. Ma Q. Anderson D.J. Kintner C. Pieler T. X-MyT1, a Xenopus C2HC-type zinc finger protein with a regulatory function in neuronal differentiation.Cell. 1996; 87: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar) was also reduced in the miR-9 MO-injected side (Figure 2D), but not when control MO was used (Figure S2D). Quantification of the results showed that miR-9 depletion caused a reduction of the number of Myt1-positive cells to about 51% of the control in the forebrain (n = 7 embryos; p < 0.001), and 53% of the control in the hindbrain (n = 9 embryos; p < 0.001) (Figure 2E). These results indicate that miR-9 is required for neuronal differentiation, regardless of the position along the AP axis. We hypothesized that miR-9 depletion could interfere with the onset of the neurogenic program by preventing cell-cycle exit, resulting in an increase in the number of progenitors. To test this we measured the area occupied by Sox3-positive neuronal progenitors per section in miR-9 MO-injected embryos. As expected, in the hindbrain there was an increase of the progenitor domain by 28% compared to the control (n = 9; p < 0.001) (Figures 3A and 3B ). However, in the forebrain the Sox3-positive area was not increased, and if anything it was slightly decreased by 14% compared to the control (n = 7; p = 0.008). In the hindbrain some Sox3-positive cells were found further away from the ventricle (data not shown), thus found in positions where differentiated cells would normally reside. To find out if there was a corresponding increase in the number of cells undergoing mitosis, we examined the number of phospho-histone H3 (pH3)-positive cells in both areas. miR-9 knockdown led to an almost 2-fold increase in the number of pH3-positive cells in the hindbrain, but there was no apparent change in the forebrain (Figures 3C and 4D , p < 0.001). Injection of control MO had no effect on either Sox3 or pH3 expression (Figures S2E and S2F). To examine whether the increase in the Sox3-positive and pH3-positive cells was due to a change in cell proliferation, we performed double labeling for pH3 and Sox3 and found that the labeling index (pH3+/Sox3+ cells in the hindbrain) is increased upon miR-9 knockdown (Figure 3E, p < 0.01). The increased rate of proliferation of the hindbrain progenitors was also confirmed using BrdU labeling of the proliferating progenitors (Figures 3F and 3G, p < 0.001). These observations suggest that miR-9 function in the hindbrain is important for limiting progenitor proliferation and promoting the onset of the neurogenic program and raises interesting questions about how (and why) that differs in the forebrain. One possibility for the decrease of differentiated neurons in the forebrain is increased apoptosis. Indeed, TUNEL analysis showed that miR-9 MO caused an increase in apoptosis in the forebrain, which was specific for that area, and it was not observed in the hindbrain (Figures 4A and 4B, p < 0.001). Apoptotic cells were present throughout the forebrain but were most frequent in the VZ (Figure 4A, arrows). No increase in apoptotic cells was apparent when control MO was used (Figure S2G). An important question is whether the cells undergoing apoptosis represent neuronal progenitors or differentiating neurons. Because miR-9 knockdown caused only a modest reduction of the progenitor domain but a significant decrease in the number of neurons (see Figure 2), one may hypothesize that it is the forebrain neurons that undergo apoptosis in the absence on miR-9. Alternatively, miR-9 depletion could reduce the survival of the forebrain progenitors, which would be consistent with the location of the majority of the apoptosing cells (see above). In order to distinguish between these possibilities, we blocked cell death by injecting a pan-caspase inhibitor together with miR-9 MO or control MO. Cell death was efficiently prevented, as evident by the reduction of the number of apoptotic cells compared to injecting miR-9 MO alone (Figure S2H). Coinjection of caspase inhibitor together with miR-9 MO led to an expansion of the Sox3-positive area in the forebrain, compared to miR-9 MO alone (Figures 4C and 4D), whereas the number of differentiating neurons was still reduced (Figures 4E and 4F). Effectively, preventing apoptosis made the miR-9 loss-of-function phenotype in the forebrain more similar to the one observed in the hindbrain. Taken together, this suggests that miR-9 is necessary for the transition of progenitors to neurons across the AP axis, and in addition it is required for the survival of progenitors in the forebrain. To understand how the differences in miR-9 loss-of-function phenotype along the AP axis arise at molecular level, we set to determine the potential miR-9 targets in X. tropicalis in relation to the phenotype we observed. One possibility was that miR-9 might regulate two or more regionally restricted targets, which in turn mediate functional specificity in different areas of the CNS. Alternatively, miR-9 specificity of function might be generated downstream of one primary target, which is expressed along the AP axis but has different functions in different axial levels (Figure S3A). As a starting point we used bioinformatic analysis using the overlap of the targets predicted by the algorithms PicTar (Krek et al., 2005Krek A. Grun D. Poy M.N. Wolf R. Rosenberg L. Epstein E.J. MacMenamin P. da Piedade I. Gunsalus K.C. Stoffel M. et al.Combinatorial microRNA target predictions.Nat. Genet. 2005; 37: 495-500Crossref PubMed Scopus (3919) Google Scholar) and TargetScan (Lewis et al., 2003Lewis B.P. Shih I.H. Jones-Rhoades M.W. Bartel D.P. Burge C.B. Prediction of mammalian microRNA targets.Cell. 2003; 115: 787-798Abstract Full Text Full Text PDF PubMed Scopus (4247) Google Scholar) to identify more than 500 potential miR-9 targets based on target site conservation in mammals (data not shown). This data set was further refined using GO analysis (Figure S3B) conservation of the seed in Xenopus (data not shown), luciferase reporter assay in HeLa cells (Figures S3D and S3E), and whole-mount in situ hybridization expression screen (Figure S3F). We decided to focus on the members of the hes (hairy and enhancer of split) family, which have been shown to play crucial roles in maintaining neural progenitors (Baek et al., 2006Baek J.H. Hatakeyama J. Sakamoto S. Ohtsuka T. Kageyama R. Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system.Development. 2006; 133: 2467-2476Crossref PubMed Scopus (175) Google Scholar, Ohtsuka et al., 2001Ohtsuka T. Sakamoto M. Guillemot F. Kageyama R. Roles of the basic helix-loop-helix genes Hes1 and Hes5 in expansion of neural stem cells of the developing brain.J. Biol. Chem. 2001; 276: 30467-30474Crossref PubMed Scopus (339) Google Scholar) Among them, Hes1 was present in all three GO categories, its Xenopus homolog hairy1 showed a prominent effect in the reporter assays, and was also expressed in the CNS, which is why we decided to examine it further. The X. tropicalis hairy1 is most closely related to the mammalian Hes1 based on sequence conservation (72%) (Jouve et al., 2000Jouve C. Palmeirim I. Henrique D. Beckers J. Gossler A. Ish-Horowicz D. Pourquié O. Notch signalling is required for cyclic expression of the hairy-like gene HES1 in the presomitic mesoderm.Development. 2000; 127: 1421-1429Crossref PubMed Google Scholar; data not shown). miR-9 binding site is highly conserved in the vertebrate homologs of Hes1, with 100% sequence homology in the seed-complementary region (Figure 5A ). In order to test whether miR-9 regulates hairy1 in vitro, we tested Xenopus hairy1 (xHairy1) and mouse Hes1 (mHes1) using luciferase-based reporter assay. Both xhairy1 3′UTR (xHairy1-WT) and mHes1 3′UTR were significantly repressed by synthetic miR-9 precursors, whereas this effect was absent when a mutant reporter lacking the seed-complementary sequence (xHairy1_Mut) was used. In order to validate the specificity of the repression, we used a target-protector approach to block miR-9 binding site (Choi et al., 2007Choi W.Y. Giraldez A.J. Schier A.F. Target protectors reveal dampening and balancing of Nodal agonist and antagonist by miR-430.Science. 2007; 318: 271-274Crossref PubMed Scopus (431) Google Scholar). A hairy1 target protector morpholino (hairy1 TP) was designed to overlap with the seed-complementary sequence on hairy1 and extend further in the 3′ direction to confer specificity (Figure 5C). Next, we examined the efficiency and specificity of hairy1 TP. Luciferase reporter assays confirmed that hairy1 TP is able to partially alleviate the repression of miR-9 on the hairy1 luciferase reporter when introduced in vitro together with miR-9 mimics, but not of a reporter carrying the 3′UTR of other miR-9 targets such as hairy2, TLX, and Onecut1 (Plaisance et al., 2006Plaisance V. Abderrahmani A. Perret-Menoud V. Jacquemin P. Lemaigre F. Regazzi R. MicroRNA-9 controls the expression of Granuphilin/Slp4 and the secretory response of insulin-producing cells.J. Biol. Chem. 2006; 281: 26932-26942Crossref PubMed Scopus (308) Google Scholar) (Figure 5D). These results show that miR-9 is able to repress hairy1 in vitro. To gain insight into the miR-9-hairy1 interaction, we compared their expression in vivo. Hairy1 has been cloned from Xenopus before (Palmeirim et al., 1997Palmeirim I. Henrique D. Ish-Horowicz D. Pourquie O. Avian hairy gene expression identifies a molecular clock linked to vertebrate segmentation and somitogenesis.Cell. 1997; 91: 639-648Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar), but here we described its expression in the nervous system in detail. During early brain development (stages 21–26), hairy1 is expressed in a broad region in the forebrain (data not shown) but later becomes restricted to the roof plate and an intermediate patch of progenitors, which represents the zona limitans intrathalamica (ZLI)—a boundary region between the thalamus and the prethalamus (Figure 5Eb). In this region hairy1 expression overlaps with the known marker of the ZLI Shh (Ishibashi and McMahon, 2002Ishibashi M. McMahon A.P. A sonic hedgehog-dependent signaling relay regulates growth of diencephalic and mesencephalic primordia in the early mouse embryo.Development. 2002; 129: 4807-4819PubMed Google Scholar) and is immediately adjacent to the expression of Irx3, which marks the thalamic region in chick and mouse (Kiecker and Lumsden, 2004Kiecker C. Lumsden A. Hedgehog signaling from the ZLI regulates diencephalic regional identity.Nat. Neurosci. 2004; 7: 1242-1249Crossref PubMed Scopus (179) Google Scholar) (Figure S4A). Conversely, in the more posterior areas, hairy1 transcripts are present ventrally in the midbrain but are absent from the mid- and hindbrain boundary, contrary to the expression of Hes1 in the mouse and the hairy-related genes her5/9 in zebrafish. In the hindbrain hairy1 expression is restricted to distinct domains—in a ventral region adjacent to the floor plate and in an intermediate region of progenitors (Figure 5Ec). Mammalian Hes1 is also expressed at high levels in the ZLI and in an intermediate zone of progenitors in the hindbrain (Baek et al., 2006Baek J.H. Hatakeyama J. Sakamoto S. Ohtsuka T. Kageyama R. Persistent and high levels of Hes1 expression regulate boundary formation in the developing central nervous system.Development. 2006; 133: 2467-2476Crossref PubMed Scopus (175) Google Scholar), but in addition it is also expressed throughout the VZ in the telencephalon and in the boundary regions such as MHB, the roof plate, and the floor plate. The zebrafish her5 is also expressed in boundary regions such as the MHB (Geling et al., 2003Geling A. Itoh M. Tallafuss A. Chapouton P. Tannhäuser B. Kuwada J.Y. Chitnis A.B. Bally-Cuif L. bHLH transcription factor Her5 links patterning to regional inhibition of neurogenesis at the midbrain-hindbrain boundary.Development. 2003; 130: 1591-1604Crossref PubMed Scopus (74) Google Scholar). Thus, Xenopus tropicalis hairy1 shows similarities and differences with" @default.
• W2105652093 created "2016-06-24" @default.
• W2105652093 creator A5010244124 @default.
• W2105652093 creator A5077496202 @default.
• W2105652093 creator A5086456495 @default.
• W2105652093 date "2011-01-01" @default.
• W2105652093 modified "2023-09-29" @default.
• W2105652093 title "MicroRNA-9 Reveals Regional Diversity of Neural Progenitors along the Anterior-Posterior Axis" @default.
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