Wnt3 and Wnt3a are required for induction of the mid-diencephalic organizer in the caudal forebrain
© Mattes et al; licensee BioMed Central Ltd. 2012
Received: 27 January 2012
Accepted: 4 April 2012
Published: 4 April 2012
A fundamental requirement for development of diverse brain regions is the function of local organizers at morphological boundaries. These organizers are restricted groups of cells that secrete signaling molecules, which in turn regulate the fate of the adjacent neural tissue. The thalamus is located in the caudal diencephalon and is the central relay station between the sense organs and higher brain areas. The mid-diencephalic organizer (MDO) orchestrates the development of the thalamus by releasing secreted signaling molecules such as Shh.
Here we show that canonical Wnt signaling in the caudal forebrain is required for the formation of the Shh-secreting MD organizer in zebrafish. Wnt signaling induces the MDO in a narrow time window of 4 hours - between 10 and 14 hours post fertilization. Loss of Wnt3 and Wnt3a prevents induction of the MDO, a phenotype also observed upon blockage of canonical Wnt signaling per se. Pharmaceutical activation of the canonical Wnt pathways in Wnt3/Wnt3a compound morphant embryos is able to restore the lack of the MDO. After blockage of Wnt signaling or knock-down of Wnt3/Wnt3a we find an increase of apoptotic cells specifically within the organizer primordium. Consistently, blockage of apoptosis restores the thalamus organizer MDO in Wnt deficient embryos.
We have identified canonical Wnt signaling as a novel pathway, that is required for proper formation of the MDO and consequently for the development of the major relay station of the brain - the thalamus. We propose that Wnt ligands are necessary to maintain the primordial tissue of the organizer during somitogenesis by suppressing Tp53-mediated apoptosis.
KeywordsForebrain patterning Thalamus development Zona limitans intrathalamica ZLI
Zona limitans intrathalamica
The thalamic complex consists of the anteriorly located pre-thalamus and the posterior located thalamus . The prosomeric model would describe these two areas as main dorsal components of the prosomere 3 (P3) and prosomere 2 (P2) respectively . Between these two neural segments there is an intervening ventricular ridge - the zona limitans intrathalamica (ZLI). The anatomical ZLI border zone contains a small cell population, which releases signaling molecules. This signaling center orchestrates thalamus development by controlled release of the morphogen Sonic hedgehog and thus, we termed it the middiencephalic organizer (MDO, formerly known as the ZLI organizer; ). Lack of the Shh-positive MDO leads to gross malformation of the caudal forebrain and loss of the entire thalamus. Local abrogation of Shh signaling in small cell clones blocks acquisition of thalamic neuronal cell fate in vertebrates [4–6]. Thus the MDO determines the size of the thalamic complex and orchestrates the neuronal development of the central relay station of the brain.
A further important diffusible, external cue during neural development is Wnt signaling. Patterning of the vertebrate anterior neural tube requires the function of this pathway at multiple stages . Canonical Wnt signaling regulates anteroposterior patterning in the forebrain and midbrain, is required for development of the dorsal telencephalon  and the eyes , and allows the establishment of the midbrain-hindbrain boundary (MHB) organizer [10, 11]. Several lines of experimental evidence have demonstrated that other signaling pathways counteract Wnt signaling during neural development, independent from direct antagonists, such as sFRPs or Dkk1. Indeed, Shh and Wnt signaling are mutually antagonistic during some events in embryonic development, such as spinal cord patterning . Despite the recognized importance of Wnt signaling for central nervous system (CNS) development, its functional relevance during diencephalon formation and how Wnt signaling and Shh signaling interact there remains unknown. Receptors, ligands and modifiers of the Wnt signaling pathway are expressed during early stages of caudal forebrain regionalization [13, 14]. Recently, we showed that Wnt signaling is required for cell adhesion in the thalamus and thalamic neurogenesis . However, the early function of Wnts during MDO establishment has to be determined.
Here we show that blockage of the canonical Wnt signaling pathway leads to malformation of the MDO. By a Morpholino-based knock-down approach we identified Wnt3 and Wnt3a as the responsible ligands and hence are required to maintain the primordium of the MDO. Lack of canonical Wnt signaling per se or knock-down of Wnt3/Wnt3a leads similarly to an increase of apoptosis specifically within the organizer primordium. Consistently, blockage of Tp53-mediated apoptosis is able to rescue the MDO. Furthermore, abrogation of the repressive factors Fezf2 and Irx1b leads to restoration of the organizer. In summary, we propose that canonical Wnt signaling triggered by Wnt3/Wnt3a is necessary to suppress Tp53-mediated apoptosis and thus maintain the organizer tissue during development.
Next, we analyzed the expression of components of the Wnt signaling pathway, wnt8b and axin2 in wnt3/wnt3a morphant embryos. We observed a reduction of wnt8b expression in the central area of the MDO (Figure 4I, J). Consequently, the Wnt signaling target gene axin2 shows a reduced expression in the caudal forebrain (Figure 4K, L). These results suggest that Wnt3 and Wnt3a are required for proper MDO formation. All analyzed marker genes show a consistent alteration - the central area of the MDO shows a down-regulation, whereas the dorsal tip seems to be less affected by the knock-down and displays a residual robust expression of MDO markers such as shh and wnt8b (Figure 4D, H, J, L). We wondered whether MDO fate is induced within tip cells of compound morphant embryos or if cells from the basal plate migrate dorsally to form the MDO spot. Therefore, we performed a time-lapse analysis using the Shh::RFP transgenic zebrafish line with strong expression of the transgene in the basal plate prior MDO formation . At 27 hpf, we detected Shh::RFP expression in the ventral MDO with a progressively dorsal expansion over the next 12 h (Figure 4M). In Wnt3/Wnt3a-deficient embryos we observed induction of dorsal MDO shh expression independently of basal plate contact (Figure 4N). This is in agreement with the so-called bucket-brigade induction model of the MDO . In this model cells progressively adopt MDO fate from ventral to dorsal without changing their dorso-ventral position. Importantly, activation of the canonical Wnt pathway using the GSK3ß inhibitor BIO between 10 and 28 hpf is sufficient to restore MDO formation in Wnt3/Wnt3a deficient embryos (Figure 4O, P).
From the performed knock-down experiments, we conclude that canonical Wnt signaling between 10 and 14 hpf, by Wnt3/Wnt3a, is required for shh induction at the MDO.
To elucidate this aspect further, we mapped the expression of markers of the prethalamus relative to markers of the thalamus. Interestingly, we found the lhx5 positive prethalamus abuts the irx1b positive thalamus and the MDO anlage is lacking in compound morphant embryos at 28 hpf (Figure 6A-B'). Likewise, analysis of the MDO and thalamus primordia in these embryos showed that the otx2-positive MDO is severely decreased, whereas the size of the irx1b and otx2-positive thalamus is unaltered (Figure 6D-E'). This suggests that Wnt3/Wnt3a are required for establishment of the MDO primordium. Consistently, blockage of Wnt signaling with IWR1 between 10 and 28 hpf leads to a similar phenotype (Figure 6C, C', F, F'). This suggests that Wnt3/Wnt3a function is required to maintain the anlage of the MDO, but not for maintenance of the primordia of the prethalamus and thalamus.
Wnt3 and Wnt3a are the principal, but not sole Wnt ligands during MDO formation
In Wnt signaling-deficient embryos, we find a persistent spot of Shh-positive cells in the dorsal most tip of the organizer. There are two possibilities to explain this phenotype. First, the dorsal diencephalic roof plate is a rich source of several Wnt ligands: in addition to Wnt3a, we find expression of Wnt8b, Wnt1 and others suggesting that there is a compensation mechanism operating at the dorsal MDO. Indeed, in a few embryos treated with IWR1 or overexpressing the Wnt antagonist Dkk1 (Figure 1), we observed a total block of organizer formation. However, these treatments also led to gross malformation of the embryo making it difficult to identify a specific Wnt-related function. A further explanation could be the third signaling pathway important for thalamus formation, the Fgf pathway. Fgf ligands, such as Fgf8, are strongly expressed at the dorsal area of thalamic anlage - in the epithalamus. Here, Fgf signaling is required for the formation of the rostral thalamus and influences expression of thalamic transcription factors such as Gbx2 [32, 33]. This could suggest that Fgf signaling is required independently to maintain MDO fate, a possibility that requires future analysis.
Wnt signaling during thalamus development
Wnt signaling is important to set up the initial anteroposterior pattern of the entire neuraxis. Subsequently, Wnt signaling becomes important in individual brain regions. In the caudal forebrain, the thalamus is an area that shows enriched expression of ligands, receptors and mediators of the canonical Wnt signaling pathway. Wnt3 and Wnt3a mark the MDO and the dorsal part of the thalamus in fish, an expression pattern that is conserved in the vertebrate lineage as recent work has demonstrated that both ligands are similarly expressed in the embryonic thalamus of the chick  and mouse . During thalamic complex development, however, a comprehensive picture of the function of Wnt signaling is still lacking and only recently individual aspects have begun to be elucidated. Inhibition of canonical Wnt signaling by Dkk-1 transforms the thalamus into pre-thalamus during the early regionalization phase . Furthermore, it has been shown that the pre-thalamus marker Lhx5 can activate the expression of the extracellular Wnt inhibitor sFRP1a and sFRP5 . These data suggest that canonical Wnt signaling is required for thalamus development, whereas the development of the pre-thalamus requires inhibition of canonical Wnt signaling.
The canonical Wnt signaling pathway plays a pivotal role in mediating the clustering of cells. The key effector of the Wnt pathway, β-catenin, promotes adhesiveness by binding to the transmembrane adhesion molecule cadherin [36, 37]. Recently, a member of this group, the Protocadherin 10b (Pcdh10b, formerly known as OL-protocadherin) has been shown to modulate cell adhesion in the thalamic complex . Stabilization of ß-catenin leads to a broadening of the expression domain of pcdh10b whereas inhibition of Wnt signaling blocks pcdh10b expression. Hence, alteration of pcdh10b expression in the thalamus leads to an intermingling of thalamic cells with the neighboring brain areas, predominantly with the pretectum. Furthermore, Wnt signaling seems to play a crucial role in thalamic neurogenesis as post-mitotic neurons express Wnt specific target genes such as lef1  and these markers have been shown to be activated by Wnt signaling during late thalamic maturation .
The foregoing descriptions notwithstanding, our knowledge of the requirement for Wnt signaling for the formation of the MDO is still fragmented. Reduced Wnt signaling activity in the Lrp6 -/- knockout mouse led to a reduction of the MDO and thalamus , and the expression of thalamic transcription factors, such as Gbx2, is severely down-regulated in these mice, suggesting that Lrp6-mediated Wnt signaling is required for proper thalamus development. However, organizer markers, such as Wnt3 and Shh, are similarly down-regulated. These data support our observation that lack of Wnt signaling leads to a malformation of the organizer tissue in zebrafish. Interestingly, we identified a narrow window of four hours during somitogenesis, which is sufficient to maintain the organizer anlage in zebrafish. This time point correlates with the expression dynamic of both ligands as co-expression of Wnt3 and Wnt3a in the organizer can only be observed between the 10 and the 16 somite stages (Figure 2). In light of our data the defects observed in the Lrp6-/- mouse thalamus may be interpreted as a dual phenotype, (i) disruption of the MDO and (ii) misspecification of thalamic cells - both due to a lack of Wnt signaling.
Fez and Irx are able to suppress MDO competence in pre-thalamus and thalamus
Interestingly, we find that the expression of pre-thalamic markers, such as fezf2 (Figure 5) and lhx5 (Figure 6), as well as thalamic markers, such as irx1b and otx2 (Figure 6), are not affected by the abrogation of Wnt signaling during somitogenesis. Although the MDO area disappears, the size of the surrounding territories is maintained. This suggests that development of the primordia of pre-thalamus, MDO and thalamus are largely independent at this early stage. Indeed, cell lineage restriction operates at the borders of the organizer [13, 40]. However, by simultaneous knockdown of Fezf2 function in the pre-thalamus or Irx1b function in the thalamus in Wnt deficient embryos, we were able to rescue the formation of the organizer. We found that the territory of the caudal forebrain and midbrain is similarly small in Wnt3/Wnt3a double morphant embryos compared to the triple morphant embryos. Therefore, we propose a comparable increased rate of apoptosis. However, we found that both pre-thalamus and thalamus are competent to form the MDO organizer. However, they lose their competence for organizer induction by expression of the transcription factors, Fez or Irx. Indeed, both transcription factors have been characterized by their pivotal repressive function during neural development [41–43]. For example, Irx2 restricts the MHB organizer primordium by suppression of the competence in the cerebellum to adopt MHB organizer fate . Consequently, a dominant-negative version of Irx2 leads to ectopic induction of the organizer. Although the MHB organizer is characterized by the expression of several Wnt ligands, the relation between Wnt signaling and Irx function is unclear during organizer formation.
Thus, we may conclude that canonical Wnt signaling is required for maintenance of the organizer primordium and, subsequently, for the formation of the entire thalamic complex.
Wnt signaling and apoptosis
Wnt signaling has been suggested as a crucial survival factor in many contexts. In Drosophila, patches of cells that are deficient in Wg signal transduction are progressively eliminated by apoptosis [44, 45]. In vertebrates, Wnt signaling has been suggested as an important external trigger for proliferation of stem cell and cancer cells . During the development of the central nervous system, stabilization of ß-catenin in neural precursors leads to enlarged brains with increased cerebral cortical surface area and folds, suggesting that Wnt signaling can regulate cerebral cortical size by controlling the generation of neural precursor cells . Consistently, reduction of ß-catenin signaling leads to reduction of central nervous tissue as the neuronal precursor population is not maintained . Here, we show that blockade of canonical Wnt signaling leads to specific cell death in the MDO. Recently, it has been suggested that Morpholino oligomeres per se may induce Tp53mediated apoptosis [48, 49]. However, we provide evidence that apoptosis observed in compound morphant embryos is due to a specific loss of Wnt3/Wnt3a function. First, we observed locally enriched apoptosis within the organizer tissue, but the surrounding areas are unaffected. Second, we observed a similar apoptotic phenotype after treatment with the small molecule inhibitor IWR1 and the organizer is similarly reduced in embryos with ectopically induced Dkk1 expression. Third, we were able to restore the organizer in double morphant embryos by treatment with the Wnt agonist BIO and we rescue the organizer in embryos treated with IWR1 by simultaneously blocking Tp53-mediated apoptosis. Taking these arguments together, we conclude that Wnt3 and Wnt3a are required for protecting the organizer tissue from Tp53-mediated apoptosis. Consistently, our findings are supported by a recent observation in cancer cells suggesting that Tp53-mediated apoptosis acts in a negative feedback loop with Wnt signaling .
In summary, we show that canonical Wnt signaling is required for regionalization of the caudal forebrain. Alteration of the canonical Wnt signaling pathway leads to apoptosis of the MDO primordium and subsequently to a mis-specification of the entire thalamic complex (Figure 9). We suggest that Wnt3 and Wnt3a are the crucial Wnt ligands, which are required between 10 h and 14 h to maintain the MDO anlage by protecting the cells from Tp53mediated apoptosis. Thus, by determining MDO fate and thalamic compartition, Wnt3 and Wnt3a control the development of the organizer of the major relay station in the brain - the thalamus.
Maintenance of fish
Breeding zebrafish (Danio rerio) were maintained at 28°C on a 14 h light/10 h dark cycle . To prevent pigment formation, embryos were raised in 0.2 mM 1-phenyl-2-thiourea (PTU, Sigma, St. Louis, MO 63103 USA) after 24 hpf. The data we present in this study were acquired from analysis of KIT wild-type zebrafish AB2O2 as well as the transgenic zebrafish line Shh::RFP , Nkx2.2::GFP and masterblind mutant line carrying a mutation in axin1 .
Transient knock-down of gene expression was performed as described in . We used the following Morpholino-antisense oligomeres (MO, Gene Tools, Philomath, OR 97370 USA) at a concentration of 0.5 mM: wnt3 MO (5'-GATCTCTTACCATTCGTCCTGC-3'), 0.25 mM wnt3a MO , irx1b MO , fezf2 MO , and Tp53 MO . The injection of MO oligomers was performed into the yolk cell close to blastomeres at one-cell or two-cell stage.
To manipulate Wnt signaling in-vivo, we used BIO ; (2'Z,3'E)-6-Bromo-indirubin-3'oxime, TOCRIS Bioscience, Minneapolis, MN 55413 USA) or IWR-1 ; (Sigma) as pharmacological agonist and antagonist of the Wnt signaling pathway. For Wnt signaling analyzes, embryos were dechorionated and incubated with 4 μM of BIO in 1% dimethyl sulfoxide (DMSO), 40 μM IWR-1 in 0.2% DMSO or with 1% DMSO only at given time points. Heparin-coated acrylic beads (Adar Biotech, Rehovot 76360 Israel) were prepared as described previously . The beads were coated with recombinant Wnt3a protein (R&D Systems, Minneapolis, MN 55413 USA) and implanted dorsally into the region of the presumptive MDO of wild-type embryos at the 10 hpf. HS-Dkk1-GFP DNA  was injected into one-cell stage embryos. A 15-minute heat shock treatment at 42°C was performed at 10 hpf. All treated embryos were incubated at 28°C and fixed at 28 hpf.
Prior to staining, embryos were fixed in 4% paraformaldehyde/PBS at 4°C overnight for further analysis. Whole-mount mRNA in situ hybridizations were performed as described in . The expression pattern and/or antisene RNA probes have been described for wnt3 (formerly known as wnt3l) and wnt3a , shh (shh-a) ), shh-b , ptc1 , axin2 , lhx5 , irx1b , otx1l and otx2 , neurog1 , fezf2 .
SDS-PAGE/Western blot analysis was performed with polyclonal antibodies to detect Wnt3 (GTX105679, Acris Antibodies, San Diego, CA 92121 USA) and Wnt3a (ab28472, Abcam, Cambridge, CB4 0FL UK) and a monoclonal antibody against PCNA (sc-56, Santa Cruz Biotechnology, Santa Cruz, CA, USA) as loading control, respectively.
Prior to imaging, embryos were de-yolked, dissected and mounted in 70% (v/v) glycerol/PBS on slides with cover slips. Images were taken on an Olympus SZX16 microscope equipped with a DP71 digital camera by using the imaging software Cell A. For confocal analysis, embryos were embedded for live imaging in 1.5% low-melting-point agarose (Sigma) dissolved in 1× Ringer's solution containing 0.016% tricaine at 48 hpf. Confocal images stacks were obtained using the Leica TCS SP5 confocal laser-scanning microscope. We collected a series of optical planes (z-stacks) to reconstruct the imaged area. Rendering the volume in three dimensions provided a view of the image stack at different angles. The step size for the z-stack was usually 1 to 2 μm and was chosen upon calculation of the theoretical z-resolution of the 40× objective. Images were further processed using Imaris 6 (Bitplane AG, CH-8048, Zurich Switzerland).
We would like to thank Daniela Peukert for technical help and Andrew Lumsden (MRC Centre of Developmental Neurobiology, King's College London) for critical reading of the manuscript. BM, SW, DP and SS are funded by the Emmy-Noether grant 847/2 of the Deutsche Forschungsgemeinschaft (DFG). The Medical Research Council, UK, supports JP and CH.
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