Neural tube derived Wnt signals cooperate with FGF signaling in the formation and differentiation of the trigeminal placodes
© Canning et al.; licensee BioMed Central Ltd. 2008
Received: 05 March 2008
Accepted: 15 December 2008
Published: 15 December 2008
Neurogenic placodes are focal thickenings of the embryonic ectoderm that form in the vertebrate head. It is within these structures that the precursors of the majority of the sensory neurons of the cranial ganglia are specified. The trigeminal placodes, the ophthalmic and maxillomandibular, form close to the midbrain-hindbrain boundary and many lines of evidence have shown that signals emanating from this level of the neuraxis are important for the development of the ophthalmic placode.
Here, we provide the first evidence that both the ophthalmic and maxillomandibular placodes form under the influence of isthmic Wnt and FGF signals. Activated Wnt signals direct development of the Pax3 expressing ophthalmic placodal field and induce premature differentiation of both the ophthalmic and the maxillomandibular placodes. Similarly, overexpression of Fgf8 directs premature differentiation of the trigeminal placodes. Wnt signals require FGF receptor activity to initiate Pax3 expression and, subsequently, the expression of neural markers, such as Brn3a, within the cranial ectoderm. Furthermore, fibroblast growth factor signaling via the mitogen activated protein kinase pathway is required to maintain early neuronal differentiation within the trigeminal placodes.
We demonstrate the identity of inductive signals that are necessary for trigeminal ganglion formation. This is the first report that describes how isthmic derived Wnt signals act in concert with fibroblast growth factor signaling. Together, both are necessary and sufficient for the establishment and differentiation of the ophthalmic and maxillomandibular placodes and, consequently, the trigeminal ganglion.
The sensory neurons of the head develop differently from those of the trunk. In the trunk, these neurons are derived exclusively from the neural crest, while in the head, sensory neurons are generated from neural crest and focal thickenings of the embryonic ectoderm, the neurogenic placodes [1, 2]. A ganglion in which this complexity is readily observed is the trigeminal. The neurons of this ganglion arise from two distinct placodes, the ophthalmic and the maxillomandibular, which form alongside the midbrain-hindbrain junction. They also have a neural crest cell component [1, 3, 4]. Trigeminal neurons are generated initially by the ophthalmic placode and, subsequently, by the maxillomandibular placode. The neural crest derived neurons are generated at significantly later stages. The trigeminal ganglion is of great importance. It conveys somatosensory information from the face, but there is relatively little understanding of how its early development is controlled, particularly with respect to the development of the ophthalmic and maxillomandibular placodes.
The ophthalmic placode forms early, at the six somite stage in chick, and is marked by the robust expression of Pax3 . This placode lies adjacent to the midbrain-hindbrain boundary and evidence suggests that its development is under the influence of signals emanating from the midbrain. The placodal cells extend anterior-laterally in the ectoderm and then delaminate into the underlying mesenchyme as neuronal differentiation begins. The maxillomandibular placode forms slightly later, by stage 12 in chick .
An in-depth analysis was carried out to address the competence and specification of Pax3 expression in relation to the ophthalmic trigeminal placode . The findings are summarized as follows. At the three somite stage, head ectoderm rostral to the first somite is competent to express Pax3 when grafted into cranial ectoderm, as shown by quail-chick transplantations. Different regions, comprising forebrain level, midbrain level, rhombomere 2–3 and otic level quail ectoderm exhibited different levels of competence when grafted into the midbrain region of chick hosts. Competence to express Pax3 was highest in anterior cranial ectoderm (forebrain, midbrain and rhombomere 2–3). Previous work has outlined a role for neural tube-ectoderm interactions in relation to Pax3 expression , and Baker et al. , in explant studies, confirmed that this interaction was direct. Foil barriers implanted between the neural folds and adjacent ectoderm, from two to nine somites, lead to a complete loss of Pax3 expression in the developing ophthalmic placode. The loss of Pax3 expression was coupled with a subsequent loss of developing neurons. Inductive cues from the neuroepithelium were proposed, therefore, to be involved in controlling ectodermal Pax3 expression. Lassiter et al.  undertook further studies to describe the role of canonical Wnt signaling within the cranial ectoderm itself and the role this signaling pathway plays in maintaining placodal fate. Furthermore, an RT-PCR screen was recently carried out to identify candidate secreted factors that might play a role in trigeminal placode development . This particular strategy involved screening for receptors expressed directly within the cranial ectoderm, including those of the fibroblast growth factor (FGF) and Wnt family, some of which have been previously characterized .
However, the search for neural tube derived signals regulating the development and differentiation of the trigeminal ganglion is ongoing. Here we present evidence that Wnt and FGF signaling are both necessary and sufficient to regulate the earliest ophthalmic placode marker, Pax3. Neural tube derived Wnt signals have a temporal requirement in the establishment of the early trigeminal placode but are no longer required once the Pax3 domain is specified. Ectopic activation of Wnt1 or FGF8 at the midbrain-hindbrain boundary is sufficient to direct differentiation of both the ophthalmic and maxillomandibular branches of the trigeminal ganglion. Under their influences, precursor pools are increased and neuronal differentiation occurs prematurely, albeit normally. Additionally, we describe how FGF signaling, via the mitogen-activated protein kinase (MAPK) pathway, maintains further differentiation of the developing placodal branches. This is the first instance whereby neural tube derived Wnt signals in conjunction with FGF signaling have been shown to influence the establishment and differentiation of the trigeminal ganglion.
Materials and methods
Electroporation of DNA constructs
Fertilized White Leghorn eggs were incubated at 38°C in a humid atmosphere. pCAGGSmWnt1 (2 μg/μl) , pCAGGSmFgf8b (2 μg/μl)  or dominant negative Wnt1(2 μg/μl)  were electroporated together with pCAGGSeGFP (1 μg/μl) using an IntracelTSS20 at the following settings; 20 volts, 4 pulses, 50 millisecond duration, space 950 milliseconds. At the five to six somite stage, DNA was targeted to the neural tube at the presumptive midbrain region, and at HH10 (Hamburger and Hamilton stage 10), DNA was targeted to the neural tube, encompassing midbrain and anterior hindbrain.
In situ hybridization and immunohistochemistry
Wholemount in situ hybridization using digoxygenin labeled probes was performed according to . Double in situ hybridization using digoxygenin and fluorescein (FITC) labeled probes was carried out as previously described . Embryos were flat mounted as previously described . Following in situ hybridization, embryos were paraffin embedded and processed to generate 20 μm sections using a microtome (Leica). For antibody staining, embryos were fixed in 4% paraformaldehyde overnight, followed by dehydration and subsequent rehydration through a methanol:PBSTx (phosphate-buffered saline plus 1% Triton X-100, 1% serum) series and blocked for 1 hour in PBSTx. The following antibodies were used: anti-neurofilament (RMO-270, Zymed, San Francisco, USA,; 1:300), anti-Islet 1 (clone 39.4D5, Developmental Studies Hybridoma Bank, DSHB, University of Iowa, Iowa City, USA; 1/10), anti-HNK1 (clone VC1-1, Sigma, MI, USA; 1/500), anti-double phosphorylated Erk1/2 (dpErk1/2, Cell Signaling, Boston, MA, USA; 1/200) and anti-green fluorescent protein (anti-GFP; Calbiochem, an affiliate of Merck, Germany,; 1/500). Embryos were washed in PBSTx five to six times, blocked again in PBSTx and incubated with appropriately species matched horseradish peroxidase (Dako, Glostrup, Denmark; 1:400), Alexa488, or Alexa594 conjugated (Molecular Probes/Invitrogen, CA, USA) secondary antibodies. All antibody incubations were carried out at 4°C overnight. horseradish peroxidase detection was performed using DAB tablets (Sigma). Confocal analysis was performed using a Zeiss LSM 510 Meta confocal microscope, and images were processed using Image J software (NIH). Embryos were fixed in 4% paraformaldehyde then incubated overnight in 30% sucrose and embedded in optimal cutting temperature (OCT) compound. Cryostat cut sections (16 μm) were blocked and incubated with antibodies as above.
Western blot analysis
Proteins from explants were extracted with modified RIPA buffer (20 mM Tris, pH 8.0, 1 mM EDTA, 0.1% NP-40, 10% glycerol, 1 mM Na3VO4, 1 mM PMSF, 1× Proteinase inhibitor cocktail) (Roche). Samples were separated by SDS-PAGE on an 8% bis-acrylamide gel and transferred to PVDF membrane (Hybond, GE Healthcare, NJ, USA). The membrane was blocked in 5% skimmed milk and incubated overnight in 1× TBS/0.1% Tween 20/5% milk containing a dilution of double phosphorylated Erk1/2 (dpErk1/2; 1/2000; Cell Signalling), or nuclear specific dephosphorylated β-catenin antibodies (1/500; Calbiochem). Blots were then stripped (stripping buffer: 2% SDS, 100 mM β-mercaptoethanol, 50 mM Tris, pH 6.8) and probed with anti-Actin (1/2000; Chemicon), which served as a loading control. Peroxidase conjugated secondary antibodies (1/1000) were detected using chemiluminescence (ECL+, Amersham).
Collagen explant assays
Explant tissues were isolated using sharp tungsten needles to remove cranial ectoderm and underlying mesenchyme from surrounding neural tissue at the five to six somite and ten somite stages. Cranial ectoderm was isolated at the level of the presumptive midbrain to hindbrain level (five to six somites) or at the midbrain to rhombomere 2 level (HH10). Collagen solution was made by adding 100 μl of 10× MEM (Sigma) and 100 μl bicarbonate buffer (0.1 M NaOH, 240 mM NaHCO3) to 0.8 ml collagen (PurCol, Nutacon, The Netherlands). Explants were cultured alone in 75% (v/v) Optimem: 25% (v/v) Leibovitz medium (Invitrogen), or supplemented with PD184352 (5 μM; a gift from Professor Philip Cohen, University of Dundee), SU5402 (50 μM; Calbiochem), 0.5 μg/ml Wnt3A (R and D Systems, Minneapolis, MN, USA), 0.1 μg/ml FGF8b (R and D Systems) or 0.5 μg/ml SFRP2 (R and D Systems). In each experimental setup, all explants used for an individual figure were cultured and processed simultaneously such that experimental conditions were comparable.
Axon outgrowth assay
Cranial ectoderm explants were dissected at HH10 and grown for 48 hours on cover slips that were coated with 0.1% poly L Lysine followed by 5 μg/ml laminin. Explants were either grown in recombinant Wnt3A plus FGF8b, or grown alone . Explants were subsequently fixed and processed for anti-neurofilament staining as above.
Elevated Wnt signals from within the neural tube result in increased numbers of Pax3 positive cells within the ophthalmic branch of the trigeminal placode
To determine if the earliest Pax3 positive placodal cells respond to emerging isthmic signals, Wnt1 was overexpressed in the neuroepithelium of 6, 8 and 10 somite embryos and incubated for 6 or 16 hours. Overexpression of Wnt1 in the neural tube at the 6 somite stage, when Pax3 expression begins to be detected in the adjacent ectoderm, resulted in increased Pax3 expression corresponding to the early ophthalmic placode (Figure 1E; n = 8/8). Similar results were observed when Wnt1 was overexpressed at the 8 or 10 somite stage, when Pax3 positive cells are specified to a placodal fate. When these embryos were incubated for 6 hours, until they reached 12 and 14 somites, respectively, the Pax3 expressing domain within the ophthalmic placode was larger (n = 7/8, 8 somites; n = 10/10, 10 somites; Figure 1F, G, respectively). Notably, the isthmic Fgf8 domain had not yet responded to activated Wnt signaling by this time point; we could only detect ectopic expression of Fgf8 9 hours post-electroporation of Wnt1 (CAC and CMJ, unpublished results). These results suggest that the earliest expansion of the trigeminal placode may be responding to Wnt signals alone, although we do not rule out the role of related FGF and Wnt family members also expressed within the midbrain-hindbrain region. Overexpression of Wnt1 at HH10, a time when the Pax3 domain is committed to placodal fate, expands the population of Pax3 positive precursor cells within the ophthalmic branch, when analyzed at HH15 (Figure 1H, J). These results demonstrate that overexpression of Wnt1 within the midbrain over a significant period of time – from the time the earliest Pax3 positive cells emerge until they are committed to form the placode-results in an increased domain of Pax3 expression in the cranial ectoderm. Therefore, Wnt signals are involved in the early development of the trigeminal placodes and appear to act simply by expanding the number of cells expressing the earliest known marker, Pax3. Wnt signals are also involved in neural crest migration. However, overexpression of Wnt1 at HH7 (Additional file 1) and concomitant DiI labeling of the neural folds resulted in an expanded Pax3 expression domain that is not overlapping with labeled migrating crest.
Attenuating midbrain Wnt signals results in a reduction of the number of Pax3 positive cells in the emerging ophthalmic placode
Wnt activity is dependent on FGF signaling to initiate Pax3 expression within the cranial ectoderm, and for subsequent neuronal differentiation
Overexpression of Wnt1 and ectopic activation of isthmic Fgf8 expression results in a hyperdifferentiated trigeminal ganglion
Elevated Fgf8 signals result in premature differentiation of both the ophthalmic and maxillomandibular branches of the trigeminal placode
FGF signaling via the MAPK pathway directs differentiation of the trigeminal placodes
Three well characterized pathways are activated downstream of the FGF receptors, namely the MAPK pathway, the phosphoinositide 3-kinase-AKT and phospholipase Cγ pathways. To test whether MAPK signaling is required for the onset of neuronal differentiation within placodal ectoderm, HH10 explants were treated with a MEK antagonist (PD184352, 5μM) and cultured until they reached approximately HH16. As a control for the activity of the MEK antagonist, HH10 midbrain to rhombomere 2 explants that normally express Fgf8 (Figure 8D; n = 4/4) lost this expression when cultured overnight in the presence of PD184352 (Figure 8E). These results are identical to our previous findings using SU5402 . PD184352 was then used to assess whether neuronal differentiation of the trigeminal placodes was dependent on MAPK activity. The presence of PD184352 resulted in a loss of Islet1 expression (Figure 8G; n = 12/14) compared to HH10 ectoderm controls (Figure 8F). These results demonstrate that FGF signaling, via the MAPK pathway, is necessary to direct an early differentiation event within the trigeminal placodes. Western blot analysis of midbrain to rhombomere 2 and cranial ectoderm explants at HH10 also detected dpErk, which was downregulated in the presence of either SU5402 (Figure 8H) or PD184352 (a selective MEK antagonist; data not shown). Similar stage explants were also positive for the accumulation of nuclear β-catenin. Cryosections of HH15 embryos were also positive for dpERK immunofluorescence within the cranial ectoderm (Figure 8I).
Our aim is to understand the precise origin and identity of the signals and mechanisms that govern the earliest development of the trigeminal placodes and their differentiation into sensory neurons. The trigeminal ganglion develops as a relatively simple two-dimensional structure. First, the placodal precursors marked by Pax3 expression are specified in the cranial ectoderm and grow anterior-laterally adjacent to the midbrain-hindbrain boundary and rhombomere 1 . Secondly, these cells begin to delaminate into the underlying mesenchyme in a manner that has recently been described to occur independent of epithelial to mesenchymal transition . Surprisingly, the molecular mechanisms underlying the development of the early ophthalmic and maxillomandibular trigeminal placodes, and further differentiation of the ganglion are poorly understood. A recent finding described how canonical Wnt signaling was involved in the maintenance of cell fate within the developing ophthalmic placode . However, one of the conclusions from this study was that although canonical Wnt signaling was necessary for maintenance of placodal cell fate, it was not sufficient. Here we present data demonstrating that isthmic Wnt signals cooperate with FGF signaling and that, acting together, these are necessary and sufficient for the establishment of the trigeminal placodes. Furthermore, FGF signals maintain differentiation of both ophthalmic and maxillomandibular neurons.
Isthmic Wnt signals and FGF activity are necessary and sufficient for the formation and differentiation of the trigeminal ganglion
Our observations extend those made by Lassiter et al. , whereby Wnt signaling within the cranial ectoderm was deemed necessary but not sufficient for the maintenance of placodal cell fate. In their study, overexpression of activated β-catenin in the ectoderm itself was not sufficient to promote placodal differentiation. Here, we demonstrate that activation of either Wnt1 or FGF8 signals from within the neural tube is sufficient to direct premature differentiation of the trigeminal placodes and ganglion. In addition, our gain of function studies reveal for the first time that the maxillomandibular lobe of the trigeminal also responds to elevated Wnt and FGF signaling in a manner similar to the ophthalmic branch. It should be noted that while Wnt1 or FGF8 was activated separately within the neural tube in ovo, it resulted in the ectopic activation of the other. It was impossible, therefore, to uncouple individual Wnt and FGF ligand activity in this experimental setting. Explants assays were therefore employed to address these issues. We demonstrate that Wnt signals depend on FGF activity to induce the expression of the early ophthalmic placode marker Pax3. Loss of function studies show the absolute requirement for neural tube derived Wnt signals in the establishment of early placode development. Additionally, the FGF pathway, via MAPK signaling, is required for the maintenance of early neuronal differentiation. We reveal that Wnt and FGF signals are required to control multiple aspects of trigeminal placode development and early differentiation. Similar requirements for Wnt and FGF signaling have been described for the initiation and maintenance of the otic placodes [18–24]. Together, these findings support that the stereotypical positioning of cranial placodes is directed by localized inductive interactions.
Wnt and FGF signaling are necessary for early development of the trigeminal placode and FGF signaling controls later maintenance of trigeminal differentiation
Of particular note is the temporal mode in which Wnt and FGF signals function within this system. Both Wnt and FGF signals are required to initiate the onset of Pax3 expression in cranial ectoderm explants. Misexpression of Wnt1 within the neural tube also results in an increased number of Pax3 positive cells within the ophthalmic placode. Surprisingly, perturbing neural tube derived Wnt signals after HH10 did not affect the development of the ophthalmic placode, despite the fact that we were able to detect a loss of isthmic Fgf8 expression as previously described . This result was supported by explant analyses where the presence of a Wnt antagonist after HH12 did not alter Pax3 expression. A recent report describes how hindbrain derived Wnt and FGF signals cooperate to regulate otic placode induction in Xenopus . In their findings the authors describe how Wnt signals are required early for the induction of Pax8 but not for its subsequent maintenance, a function that depends on FGF signaling. An additional report describes how FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated, multipotent state, but act separately to determine cell lineage specification . Thus, it seems that in a given context Wnt and FGF signals can act together to direct a particular cellular response, but may act independently to regulate further downstream events. We demonstrate an earlier than anticipated role for isthmic-derived Wnt signals in regulating the development of the emerging ophthalmic placode but show that this is ultimately dependent on FGF signaling. Subsequently, as early neurons begin to differentiate, Wnt signals are no longer required but FGF activity via the MAPK cascade is essential. These results complement those observations made by Lassiter et al. , whereby blocking Wnt signaling within the cranial ectoderm led to a loss of Pax3 expression, coincident with downstream neuronal differentiation defects. At the time when the Pax3 placodal domain is being established, perturbation of neural tube derived Wnt signals greatly reduces the size of the trigeminal placode. This non-cell autonomous effect may act indirectly, and possibly via FGF ligands, to regulate development of the placode within the adjacent ectoderm. Indeed, Fgf8 transcripts are reduced in conjunction with a reduction in Pax3 placodal expression in loss of function experiments. Similarly, the presence of SFRP2 in midbrain to rhombomere 2 explants diminishes Wnt1 expression, which is itself known to regulate the maintenance of isthmic Fgf8 expression. This knock-on effect may result in a mechanism by which both signals maintain differentiation in the ectoderm. Our findings that isthmic Wnt signals are dependent on FGF activity during early placode development may explain some of the differences observed when comparing our analyses with those of Lassiter et al. . Perhaps, when Wnt3A is added alone in explant culture, it can act upon additional signaling pathways, such as FGF or platelet-derived growth factor. The latter was recently shown to be involved in placode development . Platelet-derived growth factor ligands alone, however, were not sufficient to induce Pax3 expression in pre-placodal ectoderm, implicating the early requirement for additional pathways. An emerging trend is the occurrence of cross-talk between signaling pathways such as Wnt and FGF . Thus, integration of multiple pathways may underlie the complex signaling events that are known to be ongoing in the vicinity of the isthmic organizer.
For the first time, we provide evidence that FGF signaling is necessary to maintain early neuronal differentiation within placodal tissue. In depth analyses have been carried out by a number of groups to study the expression of FGF receptors (FGFR1-4) in the chick brain [5, 9, 28, 29]. Both FGFR2 and FGFR4 are detected within the cranial ectoderm and mesoderm. We demonstrate that MAPK activity is required within this tissue to maintain neurogenic markers, such as Islet1 and Brn3a, and that FGF8b can enhance the expression of neuronal markers. Thus, these results reveal a new role for isthmic FGF signaling in promoting trigeminal placode differentiation.
Previously, FGF signals have been implicated in the early development of the mesencephalic trigeminal nucleus in chick . It is intriguing to consider how many aspects of the trigeminal system are directly or indirectly influenced by Wnt and FGF signaling, whether acting sequentially or simultaneously. The molecular pathway regulating the early development of mesencephalic trigeminal neurons has not yet been dissected, but it is interesting to note that these neurons are born under the influence of isthmic FGF8 signals and lie within a territory at the midline that is also occupied by dorsal Wnt1 and Wnt3A transcripts . This implies a regulatory mechanism that may be involved in the development of the mesencephalic trigeminal nucleus.
This study describes how Wnt and FGF signals act together to regulate multiple aspects of trigeminal placode development. We demonstrate that Wnt and FGF signals are required for the onset of Pax3 expression in the ophthalmic placode. FGF signaling via the MAPK pathway is subsequently involved in the maintenance of early neuronal differentiation events. It will be intriguing to further investigate how Wnt and FGF signaling is integrated in the context of the isthmic organizer to direct common and distinct aspects of trigeminal ganglion differentiation and additionally in the context of the entire trigeminal system.
Here we describe the isthmic organizer region as a source of secreted factors that control the early development and differentiation of the trigeminal placodes. Isthmic derived Wnt1 cooperates with FGF signals to initiate the onset of the ophthalmic placodal marker Pax3. FGF signals are subsequently required to direct the early differentiation events of the ophthalmic and maxillomandibular placodes. The MAPK pathway is essential for the maintenance of Islet1 expression, one of the earliest neuronal markers detected within the placodes. In summary, we present for the first time the requirement for isthmic derived Wnt signals and FGF activity to establish and promote differentiation of the trigeminal placodes.
double phosphorylated Erk1/2
fibroblast growth factor
green fluorescent protein
Hamburger and Hamilton stage
mitogen-activated protein kinase
phosphate-buffered saline plus 1% Triton X-100, 1% serum
secreted frizzled-related protein.
We thank Anna Philpott, Norris Ray Dunn, Tom Keeble and Maxine Lam for comments. We thank Kate Osborne for help with confocal microscopy, and Lee Hui Ling and Keith Rogers (A*STAR BSF) for help with histology. This work was carried out in the laboratory of CMJ (CAC, LL and SL), and funded through the Institute of Medical Biology, Agency of Science Technology and Research (A*STAR), Singapore. AG is funded by the Wellcome Trust.
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