Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2015 Oct 1.
Published in final edited form as: Dev Dyn. 2014 Jan 28;243(10):1249–1261. doi: 10.1002/dvdy.24108

Pax3 isoforms in sensory neurogenesis: expression and function in the ophthalmic trigeminal placode

Jason S Adams 1,2, Sterling N Sudweeks 1, Michael R Stark 1,*
PMCID: PMC4069251  NIHMSID: NIHMS562499  PMID: 24375872

Abstract

Background

In the trigeminal placode, Pax3 is classified as necessary but not sufficient for sensory neuron differentiation. One hypothesis is that different Pax3 isoforms regulate cellular differentiation uniquely. Pax3 is known to sometimes activate and sometimes repress gene transcription, and its activity can be dependent on the isoforms present. Pax3 isoforms had not previously been characterized in chick sensory neurogenesis.

Results

RT-PCR analysis revealed three well-expressed Pax3 splice variants: Full-length (flPax3), Pax3V1 and Pax3V2. Each was characterized for its effect on neurogenesis by misexpression in placodal ectoderm. The differences observed were more apparent under conditions of enhanced neurogenesis (via Notch inhibition), where flPax3 and Pax3V1 caused failed differentiation, while Pax3V2 misexpression resembled the neuronal differentiation seen in controls. qPCR analysis revealed a progressive increase in Pax3 expression, but no significant change in relative isoform expression. Interestingly, Notch inhibition led to a significant increase in Pax3 expression.

Conclusions

We can conclude that: 1) flPax3 and Pax3V1 inhibit neuronal differentiation; 2) Pax3V2 is permissive for neuronal differentiation; 3) while absolute levels change over time, relative splice form expression levels are largely maintained in the trigeminal placode domain; and 4) Pax3 expression generally increases in response to Notch inhibition.

Keywords: Pax3, Notch signaling, splice variants, isoforms, alternative splicing, transactivation domain, trigeminal placode

Introduction

Ophthalmic trigeminal (opV) placodes are areas of ectoderm located dorsolateral to the midbrain and rostral hindbrain. Cells from the opV placode delaminate and aggregate deep within the mesenchyme, contributing neurons to the opV ganglion (D’Amico-Martel & Noden 1983). The opV placodes contribute only sensory neurons to the ganglion, making this a good model to study sensory neurogenesis and neuronal differentiation. Pax3 is the earliest marker expressed specifically in the opV placode (Stark et al., 1997). Pax3 mRNA expression in the placode begins at the 4 somite stage (ss), and robust protein expression is seen in some cells as early as the 7ss, presumably just prior to the onset of specification and commitment to a neuronal fate at 8ss (Baker et al., 1999). As cells differentiate over a protracted period, they delaminate from the epithelium and migrate to condense with nearby neural crest cells to form the ganglion. During this process, placodal cells express additional molecular markers indicative of their future fate. While still in the ectoderm, FGFR4 is transiently upregulated, and the pre-neuronal markers Neurogenin2 (Ngn2) and Brn3a are expressed, along with several components of the Notch/Delta signaling pathway (Stark et al., 1997; Perez et al., 1999; Begbie et al., 2002; Lassiter et al., 2010). FGF, Wnt, and Notch signaling have all been evaluated for their role in sensory neuron differentiation using the chick opV placode model (Lassiter et al., 2007, 2009, 2010; Canning et al., 2008). Pax3, being expressed in the early placode and continuing its expression through late stages of neuronal differentiation, has also been studied for its role in opV sensory neurogenesis. It has been shown that Pax3 expression coincides with opV placodal cell specification and commitment, which evidence provides a possible role of Pax3 in the specification and commitment of placodal cells (Baker et al., 1999). Another study demonstrated that functional Pax3 is necessary for neurogenesis in the opV ganglion, as the ophthalmic nerve is reduced or absent in Splotch mice (Tremblay et al., 1995). Finally, blocking Pax3 target genes with a Pax3-Engrailed fusion protein prevented neurogenesis, while Pax3 misexpression was shown to be sufficient to activate expression of the early placodal and proneural markers FGFR4 and Ngn2, but is not sufficient for neuronal differentiation (Dude et al., 2009). In fact, aside from Notch signaling where a downregulation is necessary for neuronal differentiation, Pax3 and the other molecular components studied in the opV placode have all been described as necessary but not sufficient for sensory neuron differentiation.

The Pax3 transcription factor consists of an N-terminal inhibitory domain, a conserved paired domain (PD), a conserved octapeptide domain (OD), a homeodomain (HD), and a C-terminal transactivation domain (TAD) that is serine, threonine, and proline rich (Fig. 1; Chalepakis et al., 1994a; Chalepakis et al., 1994b; Seo et al., 1998; Barr et al., 1999). Pax3 is expressed in different tissue types. In the early embryo it is expressed in the dorsal neural tube, dermomyotome, limb precursor muscle cells, neural crest cells, and the trigeminal placodes (Goulding et al., 1991; Bober et al., 1994; Stark et al., 1997). Pax3 functions in many different ways depending on the cell type in which it is expressed. It is necessary for normal neurogenesis and myogenesis as seen in the abnormal tissue phenotypes within the Splotch mouse mutant and Waardenburg’s syndrome in humans (Auerbach, 1954; Waardenburg, 1951; Epstein et al., 1991; Tassabehji et al., 1993). Pax3 has been functionally associated with the events of cellular proliferation, differentiation, positional identity, and migration (Epstein et al., 1993; Evans & Lillycrop, 1996; Maroto et al., 1997; Mayanil et al., 2001; Baker & Bronner-Fraser, 2000; Streit, 2004; Wu et al., 2008; Collins et al., 2009).

Figure 1. Predicted chicken Pax3 genomic structure.

Figure 1

Open in a new tab

The chicken Pax3 gene has nine predicted exons with the corresponding cDNA as illustrated. Arrows mark predicted exon boundaries, with the corresponding base pair number indicated below each arrow. The paired domain is represented by the solid grey region of cDNA, the octapeptide domain is represented by the solid black region of cDNA, and the homeodomain is represented by the diagonal lines within the cDNA. The transactivation domain has not been characterized in chick, but is presumed to be in the region immediately downstream of the homeodomain.

For us, it was intriguing that Pax3 misexpression in cranial ectoderm could cause cells to upregulate some early opV placodal markers, but not be sufficient for cellular delamination and neuronal differentiation (Dude et al., 2009). As we more carefully evaluated the broad literature on Pax3 function, we became aware that differential splicing of Pax3 occurred in some scenarios, and that different variants displayed unique properties – some acting as transcriptional activators and others as transcriptional repressors (Chalepakis et al., 1994a; Kioussi et al., 1995; Vogan et al., 1996; Mayanil et al., 2001; Kwang et al., 2002; Relaix et al., 2003; Blake et al., 2005; Hsieh et al., 2006). Alternative splicing of Pax3 produces the potential of multiple isoforms that can stabilize binding to suboptimal recognition sequences or recognize specific DNA sequences of different target genes (Vogan & Gros, 1997). Thus, Pax3 potentially regulates the expression of multiple targets through different DNA-binding sites, and this regulation can be optimized through alternative splicing. Alternative splicing of Pax3 has been characterized in human cells and mouse embryos (Goulding et al., 1991; Barber et al., 1999), but not in the chick. The human and mouse Pax3 isoforms are either missing one or more exons, or they are truncated, resulting in a lack of various protein domains (Pritchard et al., 2003; Tsukamoto et al., 1994; Chalepakis et al., 1994a; Parker et al., 2004). Our interests were to determine: 1) whether different splice variants were present in the early chick embryo, 2) whether they were present specifically in the opV placode, and 3) whether they influenced cellular differentiation uniquely from one another when misexpressed in the region of the opV placode.

The data presented here show that Pax3 splice variants are present in the early chick embryo and in opV placodal ectoderm during early development, with quantitative expression between the splice variants differing during the assessed developmental stages. Misexpression of each isoform within cranial ectoderm was performed, and neuronal differentiation was assessed. The data supports variant Pax3V2 as having unique properties when compared to other variants, properties that allow specified cells to progress through neuronal differentiation.

Results

Identification of Pax3 alternative splice variants

The coding sequence of the Pax3 gene in chick (Gallus gallus) is 1455 base pairs (bp) encoding 484 amino acids (NM_204269, NP_989600.1). The genomic structure of human PAX3 has been characterized (Barr et al., 1993; Lalwani et al., 1995), and this was used in addition to the genomic data in the NCBI GenBank to produce a predicted genomic map of chick Pax3 (Fig. 1; NC_006096.3). The full-length Pax3 in chick appears to be most similar to the mammalian PAX3d splice variant described by Barber et al., (1999), as it contains nine exons, and the chick amino acid C-terminus (AFHYLKPDIA) is identical to the mouse and human PAX3d (Fig. 1; Barber et al., 1999; Parker et al., 2004; Blake & Ziman 2005; NP_989600.1). The chick full-length splice variant will be referred to as flPax3 through the rest of this study.

In order to identify possible Pax3 splice variants and their temporal expression patterns in the chick, mRNA from 6-8ss, 16-18ss, and 35-37ss was isolated and used for PCR amplification. Two primer pair sets (P1:P2 and P3:P4) were designed to amplify distinct but overlapping regions of the Pax3 gene (Fig.2A). The P1:P2 primers amplified two PCR products, which were gel extracted and sequenced (Fig. 2B). Sequencing confirmed that the longer product (615bp) was the same as the flPax3 sequence from bp 263 to bp 878 (NM_204269). The shorter PCR product was the result of a 135bp deletion between positions 451 and 586 of the Pax3 sequence. This splice variant (Pax3V1) contains a deletion of exon 4 that encompasses part of the PD and the OD (Fig. 3). The Pax3V1 splice variant is most similar to a splotch allele (Sp) mutant in mouse that also includes a deletion of exon 4 (Epstein et al., 1993).

Figure 2. PCR amplification of Pax3 splice variants.

Figure 2

Open in a new tab

To identify and characterize Pax3 splice variants in tissues of the developing chick embryo, total RNA isolated from whole chick embryo, chick ectoderm, and chick trigeminal ganglion at different developmental stages was reverse transcribed into cDNA and amplified using primer pairs P1, P2 (for exons 2-6) or P3, P4 (for exons 5-8) as shown in A. (B) Two PCR products were identifiable from the P1, P2 amplification: the 5′ fragments of flPax3, (630bp) and Pax3V1 (495bp). Sequence analysis revealed that Pax3V1 resulted from splicing out exon 4. (C) Two aberrant (no Pax3 homology) and two correct PCR products were identifiable from the P3, P4 amplification. All bands were evaluated by sequence analysis and only the bands associated with the variants described here proved to be Pax3 amplicons. The correct products were identified as the 3′ fragments of flPax3 (705bp) and Pax3V2 (324bp). Sequence analysis revealed that Pax3V2 resulted from splicing out exons 6 and 7.

Figure 3. Predicted isoforms of Pax3 in chick.

Figure 3

Open in a new tab

From top to bottom: The nine exons of chicken Pax3. The flPax3 isoform with the several protein domains depicted. The Pax3V1 isoform (deletion of exon 4). The Pax3V2 isoform (deletion of exons 6 and 7). ID, inhibitory domain; PD, paired domain; OD, octapeptide domain; HD, homeodomain; TAD, transactivation domain.

The second primer pair (P3:P4) amplified four PCR products, which were gel extracted and sequenced (Fig. 2C). The longest product (705bp) was the same as the flPax3 sequence, and the smallest product contained a 381bp deletion within the sequence. This deletion was found between positions 792 and 1173 of the Pax3 coding sequence. This splice variant (Pax3V2) contains a deletion of exons 6 and 7 corresponding to a small region of the HD and a large portion of the TAD (Fig. 3). The middle two bands were sequenced and identified as aberrant PCR products. The Pax3V2 splice variant resembles the mouse Pax3g and Pax3h splice variants that have deletions of exon 8 resulting in a deletion of part of the TAD (Pritchard et al., 2003; Parker et al., 2004). The two splice variants identified, Pax3V1 and Pax3V2, contain exon deletions that do not appear to introduce a premature stop codon. Figure 3 shows the predicted isoforms of Pax3 (flPax3, Pax3V1 and Pax3V2) with their corresponding deletions, all of which were detected in mRNA isolated from each of the tissue samples collected.

Isoform misexpression: flPax3 and Pax3V1 are strong inhibitors of neurogenesis

To determine whether each Pax3 isoform influences cellular differentiation uniquely in the opV trigeminal placode, constitutively expressing constructs were electroporated into the chick opV placode region. Each construct was electroporated at 6-8ss (before ectodermal commitment of opV placodal cells), at 10-12ss (as the delamination process begins), or at 14-16ss, (which marks the beginning of neuronal differentiation). The embryos were incubated for 24-32 hours and tissue sections were analyzed through immunohistochemistry to detect Pax3 and Islet1 protein expression, while GFP marked electroporated cells. Pax3 protein expression was seen in GFP+ cells and endogenous opV placodal cells, providing a landmark for proper targeting (Stark et al., 1997). Islet1 marks trigeminal neurons and is an early marker for neuronal differentiation (Begbie et al., 2002; Fedtsova et al., 2003, Lassiter et al., 2007; Dude et al., 2009). In the unmanipulated embryo, Pax3+ opV cells are maintained for a time in the ectoderm, eventually becoming fated as neurons, delaminating from the ectoderm and migrating through the mesenchyme to the site of ganglion condensation. Islet1 expression is most obvious in placodal cells located in the mesenchyme and condensing ganglion, however a few ectodermal cells can sometimes be found expressing this early neuronal marker (Dude et al., 2009).

The results of the 6-8ss misexpression experiments are shown in figure 4, where GFP identifies targeted cells, Pax3 marks the opV placode (and is found in non-control targeted cells), and Islet1 identifies early specified sensory neurons. A significantly greater number of targeted cells remained in the ectoderm in embryos electroporated with flPax3 (n=11) and Pax3V1 (n=12) when compared to controls (n=10; p=0.0374; p=0.0002, respectively), and in embryos electroporated with Pax3V2 (n=10) when compared to Pax3V1 electroporated embryos (p=0.0216; Fig. 4Q). Conversely, the number of targeted cells that had moved into the mesenchyme was significantly and dramatically reduced in flPax3 and Pax3V1, while Pax3V2-electroporated embryos showed a less dramatic but still significant reduction in number of targeted cells in the mesenchyme when compared to pCIG controls (p<0.0001; p<0.0001; p=0.0002, respectively; Fig. 4R). In addition, the total number of targeted and untargeted Islet1+ mesenchyme cells was significantly reduced in embryos electroporated with flPax3, Pax3V1, and Pax3V2 compared to controls (p<0.0001; p<0.0001; p<0.0001, respectively). Interestingly, a significantly greater reduction in Islet1+ mesenchyme cells was observed in embryos electroporated with flPax3 and Pax3V1 compared to embryo electroporated with Pax3V2 (p=0.0471; p=0.0509, respectively; Fig. 4S). This trend also persisted when counting only the target (GFP+) opV placode-derived (Pax3+) Islet1+ cells in the mesenchyme (Fig. 4T). Therefore, misexpression of flPax3 and Pax3V1 resulted in reduced neuronal differentiation, with targeted cells remaining in the ectoderm. Misexpression of Pax3V2 also resulted in reduced neuronal differentiation, although the reduction is less dramatic, and fewer cells are stalled in the ectoderm.

Figure 4. Early (6-8ss) Pax3 isoform misexpression and neuronal differentiation.

Figure 4

Open in a new tab

Sections through the opV placodes from embryos electroporated in ovo at 6-8ss, then incubated for 32 hours. The sections were stained for Pax3 and Islet1, while GFP marked targeted cells. (A-D) pCIG misexpression; (E-H) flPax3 misexpression; (I-L) Pax3V1 misexpression; (M-P) Pax3V2 misexpression. Significantly more targeted cells remained in the ectoderm in flPax3 and Pax3V1 embryos when compared to controls (D, H, L, Q). Significantly fewer Pax3+/GFP+ mesenchyme cells were seen in all electroporated isoforms compared to the control (B, F, J, N, R). Similarly, significantly fewer Islet1+ cells (targeted or untargeted) were observed (C, G, K, O, S). Pax3+/Islet1+ cells were rarely found in the mesenchyme after flPax3 or Pax3V1 misexpression. Pax3V2 cells were more numerous, but still reduced compared to controls (D, H, L, P, T). (Q-T) Graphical representation of in ovo misexpression data. Error bars represent the standard error of the mean (SEM). (*) p-value <0.05 (Tukey-Kramer test) between the experimental groups and the control group; (^) p-value <0.05 (Tukey-Kramer test) between two experimental groups (i.e. Pax3V2:Pax3V1).

To evaluate the effects of misexpression later in development, embryos were electroporated at 14-16ss, which marks the beginning of neuronal differentiation and delamination. The embryos were analyzed 24 hours after electroporation as previously described with GFP, Pax3, and Islet1 expression shown in figure 5. As with younger embryos, there was a significant increase in the number of targeted cells remaining in the ectoderm in embryos electroporated with flPax3 (n= 7; p=0.0052) and Pax3V1 (n=11; p=0.0022), while embryos electroporated with Pax3V2 (n=10) were more similar to control embryos (n=9; p=0.1381; Fig. 5Q). A significant reduction in the number of Islet1+ cells in the mesenchyme was seen in embryos electroporated with the Pax3V1 construct when compared to the pCIG controls and when compared to Pax3V2-electroporated embryos (p=0.0024; p=0.0090, respectively; Fig. 5S). Similarly, a significant reduction in the number of targeted (GFP+) Pax3+ cells expressing Islet1 was seen in the mesenchyme of embryos electroporated with flPax3, Pax3V1, and Pax3V2 compared to the pCIG controls (p<0.0001; p<0.0001; p=0.0007, respectively; Fig. 5T). In addition, there were significantly fewer GFP+/Pax3+/Islet1+ cells in the mesenchyme of flPax3 and Pax3V1 electroporated embryos compared to Pax3V2-electroporated embryos (p=0.0007; p<0.0001, respectively; Fig. 5T).

Figure 5. Pax3 isoform misexpression and neuronal differentiation at 14-16ss.

Figure 5

Open in a new tab

Sections through the opV placode of embryos electroporated at 14-16ss and incubated for 24 hours. The sections were stained for Pax3 and Islet1, while GFP marked targeted cells. (A-D) pCIG misexpression; (E-H) flPax3 misexpression; (I-L) Pax3V1 misexpression; (M-P) Pax3V2 misexpression. As with younger stages, significantly more targeted cells remained in the ectoderm in flPax3 and Pax3V1 embryos when compared to controls (D, H, L, Q). Similar trends were also observed in the number of Pax3+/GFP+ (R) and Islet1+ (S) mesenchyme cells, although at these later stages more untargeted cells have differentiated normally. Importantly, a significant decrease in the number of targeted Islet1+ cells in the mesenchyme was seen for all electroporated isoforms when compared to the control, especially with the flPax3 and Pax3V1 isoforms (D, H, L, P, T). (Q-T) Quantitative representation of in ovo misexpression at 14-16ss. Error bars represent the SEM. (*) p-value <0.05 (Tukey-Kramer test) between the experimental groups and the control group; (^) p-value <0.05 (Tukey-Kramer test) between two experimental groups.

Two additional experiments were performed with similar results: 1) Embryos were electroporated at 10-12ss, a time when opV placodal cells first begin to delaminate from the epithelium. The results of this experiment (data not shown) were similar to those described above for the embryos electroporated at 14-16ss. 2) Each Pax3 construct was electroporated caudal to the trigeminal placodal region, in and around the area of the otic placode at 6-8ss and 14-16ss. This region was analyzed for GFP, Pax3, and Islet1 expression. There was no noticeable neurogenesis in targeted cells, and it was concluded that none of the Pax3 isoforms were sufficient to promote ectopic neurogenesis (data not shown).

Notch inhibition reveals that Pax3V2 is permissive for neurogenesis

It has been shown that Pax3 directly binds to the promoter of the Hes1 gene, regulating its transcription (Nakazaki et al., 2008). Hes1 is also a known component of the Notch signaling pathway (Jarriault et al., 1998), and Notch signaling has been demonstrated to regulate sensory neurogenesis in the opV placode (Lassiter et al., 2010). Activation of the receptor, Notch-1, was shown to enhance expression of Pax3 protein in myogenic precursor cells, and overexpression of the Notch inhibitor, Numb, decreases Pax3 expression (Conboy & Rando, 2002). This Pax3/Notch relationship led us to investigate cell fate outcomes after misexpression of Pax3 and its isoforms concomitantly with Notch inhibition. Additionally, Notch inhibition leads to robust neurogenesis in the trigeminal placode (Lassiter et al., 2010), an effect that could magnify the readout of placode cell differentiation potential after Pax3 isoform misexpression. In other words, we wanted to observe the effects of each isoform under conditions of enhanced neurogenesis.

DAPT is a gamma-secretase inhibitor that prevents Notch signaling. In the opV trigeminal placode, applying DAPT leads to an increase in sensory neurogenesis (Lassiter et al., 2010). That study employed a collagen gel explant strategy for culturing chick embryo heads in the presence of DAPT. Here we used the EC culture technique developed by Chapman et al., (2001), modified for our application. These modifications included the addition of chemicals within the culture media and the placement of an additional ring underneath the yolk membrane, allowing for the transfer of chick embryos at any point during the incubation period without harming the embryo. The ability to safely transfer chick embryos between culture media during the experimental time course allows for embryo incubation conditions to change over time, thereby controlling the timing of incubation in the presence of experimental chemicals such as DAPT. Furthermore, using the double ring method allowed for the electroporation of explanted embryos with better results. To test the effectiveness of this method, DAPT was added to the culture media and cell fate outcomes were compared to those obtained using the head explant cultures in Lassiter et al., (2010). A similar increase in neurogenesis was observed (data not shown). This approach was then used to combine Pax3 electroporation with DAPT treatment. Embryos were electroporated with flPax3, Pax3V1, or Pax3V2 constructs at 5-7ss and incubated on the culture media for 12 hours to 13-15ss, corresponding to the stage of Notch inhibition performed by Lassiter et al., 2010. The embryos were transferred to another culture dish with media containing either DAPT or DMSO and incubated for an additional 24 hours. The embryos were then analyzed by immunohistochemistry for GFP, Pax3, and Islet1 expression similar to the analysis performed by Lassiter et al., (2010) (Figs. 6). Quantitative comparisons between control embryos (pCIG; n=10), flPax3 (n=14), Pax3V1 (n=11), and Pax3V2 (n=13) electroporated embryos revealed that the number of targeted cells expressing Islet1 in the mesenchyme was significantly reduced in embryos electroporated with flPax3 and Pax3V1 constructs when exposed to DAPT (p=0.0210; p=0.0140, respectively; Fig. 6Q), while Pax3V2 electroporated embryos were quantitatively similar to control embryos (p=0.0924). Therefore, flPax3 and Pax3V1 misexpression prevented differentiation, even when Notch signaling was blocked, while Pax3V2 misexpression did not prevent DAPT-enhanced neurogenesis.

Figure 6. Pax3 isoform misexpression with Notch inhibition demonstrates unique properties of Pax3V2.

Figure 6

Open in a new tab

Sections through the opV placode of embryos electroporated at 5-7ss, cultured for 12 hours without DAPT, and then cultured for 24 hours in the presence of DAPT. Sections were stained for Pax3 and Islet1, while GFP marked targeted cells. (A-D) pCIG misexpression; (E-H) flPax3 misexpression; (I-L) Pax3V1 misexpression; (M-P) Pax3V2 misexpression. (Q) The graph represents the total number of Pax3+/ GFP+/ Islet1+ cells in the mesenchyme. The first group of cell counts was from chick embryos electroporated at 6-8ss and incubated in ovo until embryos reached 24-28ss before being collected, as reported in figure 4. The second and third group of cell counts were collected from chick embryos electroporated at 5-7ss and incubated on culture media with DMSO (embryo sections not shown) or DAPT (panels A-P above), respectively, to approximately the 24-28ss. The number of Pax3+/Islet1+/GFP+ mesenchyme cells in DAPT-treated embryos after Pax3V2 misexpression is significantly different than that observed after flPax3 misexpression. Error bars represent the SEM. (*) p-value <0.05 (Tukey-Kramer test) between the experimental groups and the control group; (^) p-value <0.05 (Tukey-Kramer test) between two experimental groups.

Quantitative analysis of Pax3 and splice variant expression

Having observed a difference in differentiation potential after Pax3V2 misexpression compared to the other Pax3 isoforms, we next wanted to assess the quantitative expression (comparison of ddCt – see Livak and Schmittgen, 2001) of flPax3 and Pax3V2 during normal opV placodal development. This would allow us to determine whether a change in splice variant expression coincides with a differentiation stage of opV placodal cells.

Ectodermal tissue of the opV placode collected at 6-8ss, 10-15ss, 20-25ss, and 32-37ss, as well as 32-37ss tissue from opV ganglia was collected and assayed for Pax3 splice variant expression levels by quantitative RT-PCR. When comparing the quantitative fold expression change of flPax3 and PaxV2 a similar pattern was shown during the different time points and tissues examined (Fig. 7). Both are present in the ectoderm at the time of opV placodal cell commitment (6-8ss), and their ectodermal expression trends upward by 10-15ss, when the placode is well-established and differentiation is occurring. This increased expression becomes significant at 20-25ss, when opV cellular delamination and neuronal differentiation is at its peak. Then at 32-37ss the concentration of flPax3 and Pax3V2 in the ectoderm decreases as most Pax3+ cells have delaminated and migrated to form the opV ganglion. At 32-37ss, the concentration of flPax3 and Pax3V2 is the highest in the ganglia compared to any of the other stages where ectoderm was collected (Fig. 7), likely due to enrichment of expressing cells (no dilution of Pax3-negative ectoderm cells), and to the inclusion of neural crest cells, which also contribute to the trigeminal ganglion and express Pax3 at lower levels. This corresponds to past results showing a decline of Pax3 expression in opV placodal ectoderm by 35ss, with expression of Pax3 being maintained in placodal cells that have moved to the ganglion (Stark et al., 1997).

Figure 7. Quantitative expression analysis of flPax3 and Pax3V2 by real-time PCR.

Figure 7

Open in a new tab

qPCR was performed using the splice variant-specific primers described in the methods, and illustrated here where flPax3 was amplified using the primer pair PF1/PR1 and Pax3V2 was amplified using the primer pair PF1/PR2. Shown is the overall average fold change between flPax3 and Pax3V2 expression in placode cell development. Placodal ectoderm was analyzed at 6-8ss, 10-15ss, 20-25ss, and 32-37ss. Differentiating ganglion tissue (32-37ss) was also assessed, as was 20-25ss ectoderm of DAPT-treated embryos. A similar pattern is observed throughout, where flPax3 expression is relatively greater than Pax3V2 expression at approximately the same ratio. Expression of both splice variants increases from 6-8ss to 10-15ss, is maintained through 20-25ss, and then drops in the ectoderm as most cells have migrated to form the condensing ganglion. Interestingly, DAPT exposure results in a significant increase in ectodermal flPax3 and Pax3V2 expression. Isoform expression in the ganglion is also higher, though this may be expected in a more pure neuronal population.

Statistical analysis revealed that expression of flPax3 increased significantly between stages 6-8ss and 20-25ss in the ectoderm (p=0.0185), and then its expression decreased significantly from stage 20-25ss to 32-37ss in the ectoderm (p=0.0002). Though the same pattern was seen with the Pax3V2 splice variant, the changes were not statistically significant (p=0.8143; p=0.1146, respectively). The quantitative expression of both flPax3 and Pax3V2 was significantly less in the ectoderm than in the ganglia at 32-37ss (p < 0.0001).

To determine whether DAPT inhibition of Notch signaling in the opV placode would change the expression of flPax3 or Pax3V2, chick embryos were collected at 10-14ss and transferred to culture media containing DAPT. The embryos were incubated at 37°C for 12 hours and collected at 20-25ss to compare this tissue to the ectoderm tissue collected from chick embryos of the same stage that were not cultured with DAPT. Notch inhibition had an interesting effect on the quantity of flPax3 and Pax3V2 expression. Placodal tissue exposed to DAPT showed significantly higher flPax3 (p<0.0145) expression levels when compared to non-treated ectoderm of the same region. While expression levels of Pax3V2 also trended upward, the difference was not statistically significant (p<0.4784). The increase in flPax3 expression may imply that Notch signaling inhibits Pax3 transcription, and that downregulation of Notch signaling would allow for increased Pax3 expression. This is counter to the results reported by Conboy & Rando (2002), where Pax3 was enhanced by Notch activity.

Lastly, the quantitative fold difference between flPax3 and Pax3V2 was similar, independent of the developmental stage or of the DAPT treatment (Fig. 7). At the earlier stage (6-8ss), the relative expression of flPax3 was not significantly different from Pax3V2 when compared to the later stages (similar fold difference, p>0.05). The fold difference between the two mRNA transcripts increased moderately by 10-15ss and leveled off through 32-37ss. There was a moderate increase in the expression of flPax3 when compared to Pax3V2 in the ganglia at 32-37ss, though no significant difference was shown. These data show that flPax3 is expressed approximately 1.4 to 1.9 times greater than its splice variant Pax3V2 during different embryonic stages and in the different tissue types studied.

We next aimed to determine whether another splice variant with a TAD deletion could be identified. In human and mouse, isoforms have been shown to include a deletion of exon 8, spanning the TAD (Parker et al., 2004; Pritchard et al., 2003). The identification of such an isoform in chick might reveal an additional TAD deletion with similar function to Pax3V2. Characterizing the quantitative expression over time could reveal important information. Using specific PCR primers, we identified an exon 7/8-deletion splice variant, Pax3V3, from whole chick embryo and placodal cDNA samples. However, quantitative analysis revealed very low expression levels of this splice variant compared to flPax3 and Pax3V3, with no detectable change in expression over time (data not shown).

Discussion

Several past publications have identified Pax3 as an essential component in many developmental processes, including muscle cell differentiation (Tajbakhsh et al., 1997; Relaix et al., 2003; Bajard et al., 2006), neural crest cell migration (Serbedzija & McMahon, 1997; Mansouri et al., 2001), somite cell survival (Borycki et al., 1999), and opV sensory neurogenesis (Baker et al., 1999; Dude et al., 2009). Dude et al., (2009) concluded that activation of Pax3 target genes is required for sensory neurogenesis in the opV placode, and that some opV-specific genes were upregulated in response to Pax3 misexpression, but Pax3 was not sufficient for neuronal differentiation. Follow up observations, including those presented here; showed that Pax3 misexpression within the opV placode domain actually inhibits differentiation. To better understand how Pax3 both activates important target genes in the opV placode, and can inhibit neurogenesis, we aimed to know whether certain Pax3 isoforms could differentially regulate this process.

One isoform, Pax3V1, contains a deletion that encompasses part of the paired domain and the entire octapeptide domain. The paired domain is necessary and sufficient to bind DNA (Treisman et al., 1991), though the C-terminal end of this domain does not appear to be as essential since mutations of this region have been shown not to affect DNA binding (Apuzzo et al., 2004). The octapeptide domain of Pax5 was shown to bind a co-repressor, providing evidence that the octapeptide domain is involved in protein to protein interactions (Eberhard et al., 2000). Substitution of a Pax3 non-specific domain in place of the Pax3 linker region showed that the linker region is also important for the interaction between the paired domain and homeodomain (Fortin et al., 1998). Despite the evidence that this region would play an important role in Pax3 function, misexpression of Pax3V1 (which has a partial deletion in the paired domain and a complete deletion of the octapeptide domain) gave similar results to that seen after flPax3 misexpression, where Pax3V1+ cells remained in the ectoderm and did not express the differentiation marker Islet1. We had expected a functional difference between the Pax3V1 and Pax3V2 isoforms due to the partial deletion in the binding domain and complete deletion of the octapeptide in Pax3V1; however, as this was not seen in the results, our focus became concentrated on the function of the other Pax3 isoform.

The second isoform, Pax3V2, contains a partial deletion of the C-terminal region of the homeodomain and a large deletion of the transactivation domain. The homeodomain recognizes DNA and contains residues that interact with DNA (Biarrane et al., 2009). The transactivation domain is essential, but not sufficient for transcriptional activity (Lechner & Dressler, 1996), and it participates in protein to protein interactions with various cofactors (Eberhard et al., 2000; Murakami et al., 2006). Misexpression experiments using the Pax3V2 isoform showed that even though many Pax3V2+ cells remained in the ectoderm, more delaminated from the ectoderm than Pax3 or Pax3V1 cells, and the migrating cells were typically Islet1+. However, when the Pax3V2 isoform was misexpressed in competent ectoderm caudal to the opV placode, Islet1 expression was not enhanced, showing that the Pax3V2 isoform is not sufficient for sensory neurogenesis. These results hinted that the Pax3V2 isoform is a permissive molecule for sensory neurogenesis in the opV placode. Follow-up experiments under conditions of enhanced neurogenesis (by blocking Notch signaling with DAPT) further supported this hypothesis, as many more targeted cells expressed Islet1 in the Pax3V2 embryos compared to flPax3 or Pax3V1 embryos. One might argue that Pax3V2 is non-functional. The presence of this isoform in both whole embryo and placodal cDNA as detected by our PCR strategies demonstrate that it occurs in relative abundance, and likely functions in some way in the cell. A similar isoform exists in mammals (Parker, et al., 2004; Pritchard et al., 2003). Reporter assays testing this isoform showed that partial deletion of the TAD inactivates transcription of Pax3 targets (Pritchard et al., 2003), while others have shown that the transactivation domain is involved in protein to protein interactions important for gene regulation (Murakami et al., 2006; Hsieh et al., 2006). If Pax3V2 does act as a transcriptional repressor, one possible model in the opV placode is that individual cells primed for differentiation temporarily upregulate Pax3V2 at sufficient levels to inactivate certain Pax3 targets, thereby permitting differentiation. However, this is not a likely scenario for known targets, since misexpression of flPax3 results in the upregulation of FGFR4 and Ngn2. A proper balance of Pax isoform expression is likely, however, since flPax3 misexpression also disrupts development in the Pax2-expressing otic placode (Dude et al., 2009).

From this data, we next set out to test the hypothesis that the levels of Pax3V2 expression increased relative to flPax3 expression as individual cells shifted toward differentiation. A quantitative PCR strategy was employed and we observed a sustained increase in both flPax3 and Pax3V2 expression in the ectoderm from 6-8ss, to 10-15ss, and to 20-25ss, followed by a significant decrease at 32-37ss. The fold difference between flPax3 and Pax3V2 was similar during these stages, as flPax3 was 1.4 to 1.9 times more abundant. After DAPT treatment, the fold difference was again similar, with the concentrations of both variants increasing. Therefore, the quantitative PCR results showed that flPax3 and Pax3V2 do not change in their relative expression levels throughout the stages of opV placode differentiation indicating that a tissue-wide change in splice variant expression does not account for cellular progression toward a differentiated state. The question remains as to whether individual cells transiently modulate relative splice variant expression at a critical point of differentiation, such as after FGFR4 and Ngn2 expression and prior to upregulation of Islet1. As suggested above, correctly modulating the relative levels of Pax3 isoforms in individual cells may be important. A single cell qPCR analysis could reveal transient changes, however identifying the cells at the proper stage of differentiation would require specific reporter tools not currently available.

The observation that Pax3 expression increased after Notch inhibition may be important, particularly in light of published results showing that in myogenic precursor cells, activation of Notch-1 was shown to enhance expression of Pax3 protein, and overexpression of the Notch inhibitor, Numb, decreases Pax3 expression (Conboy & Rando, 2002). We had assumed that Pax3 expression is upstream of most other events, and certainly precedes any changes in Notch regulation since prior work showed that Pax3 regulates the Notch effector gene Hes1 (Nakazaki et al., 2008; Ichi et al., 2011). The result reported here indicates that, in the opV placode, Notch inhibition acts to either enhance Pax3 expression, or adds more Pax3-expressing placode cells to the pool. The suggestion that Notch may upregulate Pax3 is an interesting new discovery in the complex gene regulatory network of sensory neurogenesis.

This study describes the characterization of four Pax3 splice variants that are expressed in the chick opV placode during neuronal differentiation. Two of these isoforms, flPax3 and Pax3V1, cause failure of neuronal differentiation when misexpressed in placodal ectoderm, while a third isoform, Pax3V2, was permissive for neurogenesis. The fourth splice variant was found to be expressed only at very low levels in the opV placode. By examining expression levels by qPCR, flPax3 was found to be expressed approximately 1.4 to 1.9 fold higher than Pax3V2 throughout placode development, with no evidence of a shift in the ratio of expression at any time. However, this assay was performed with mRNA isolated from the entire placode domain, and does not reflect what might be occurring in individual cells. Therefore, we cannot rule out the possibility that a brief shift in splice variant expression ratios occurs at the onset of neurogenesis. Characterizing such an event would likely require a live-cell marker for the brief event of delamination/differentiation, followed by single cell PCR, or another method of detecting specific splice variants at the single-cell level. FGFR4 has been shown to be transiently upregulated in delaminating cells in the chick and quail embryo (Stark et al., 1997), so it is possible that an FGFR4 reporter could serve this purpose. The observation that Pax3 expression increases after Notch inhibition is a new observation, and is being further investigated for a possible dual role for Pax3; expressed early for one purpose, and upregulated prior to neuronal differentiation (in response to reduced cellular Notch signaling) for another. We can conclude that: 1) the flPax3 and Pax3V1 isoforms inhibit neuronal differentiation; 2) the Pax3V2 isoform is permissive for neuronal differentiation; 3) relative splice form expression levels are largely maintained when measured in the whole embryo or in the trigeminal placode domain at several stages; and 3) Pax3 expression generally increases in response to Notch inhibition.

Experimental Procedures

Tissue Isolation

Two methods were used, both allowing for efficient isolation of tissue for subsequent RNA isolation. Tissue was isolated from: 1) whole chick embryos at 6-8ss, 16-18ss, and 35-36ss, 2) from whole-head tissue dissected from chick embryos at 8ss and 20ss, and 3) from ectoderm dissected from chick embryos at 6-8ss, 16-18ss, and 35ss as well as the trigeminal ganglion at 32-37ss. To facilitate separation of the ectoderm and ganglia from the mesenchyme at the two later embryonic stages, pre-warmed 1mg/ml dispase diluted in DMEM was added to the tissue and incubated at 38°C for five minutes. The enzyme was neutralized by adding 0.1% bovine serum albumin to this solution and placed on ice for ten minutes before rinsing with PBS (McCabe and Bronner-Fraser, 2008).

The second method involved chick embryos cultured with 200μM N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycine t-Butyl Ester (DAPT) at 10-15ss and incubated at 37°C for 12 hours. The cultured ectoderm of the trigeminal placode was dissected by cutting the heads of the chick embryos caudal to the otic placode and through the optic vesicles or cups. Separation of the ectoderm and the ganglia from the mesenchyme was facilitated by incubating the dissected heads with ice-cold 3U/ml dispase I in DMEM, 20mM Hepes, pH 8.0 for 15-20 minutes. The tissue was then transferred to ice-cold 0.05% Trypsin with EDTA for five minutes and then incubated at 37°C for at least 20 minutes. Trypsin was neutralized with 10% fetal bovine serum in DMEM for five minutes at room temperature and then washed in PBS.

RNA isolation and RT-PCR

Total RNA was isolated from all tissue samples using 2-mercaptoethanol and purified using the PureLink Micro-to-Midi Total RNA Purification System kit (Invitrogen).

Reverse transcription of RNA was performed using a Superscript Double-Stranded cDNA Synthesis Kit from Invitrogen with oligo primers. Forward and reverse primers used for initial PCR amplification of the splice variants are diagramed in figure 2 as follows: P1 = 5′-CTGCGTCTCCAAGATCCTCT-3′; P2 = 3′-AAAAGCCATCAGTTGGTTGG-5′; P3 = 5′-AGCAACTGGAAGAGCTGGAA-3′; P4 = 3′-TGACAGGGTCCATGCTGTAG-5′.

Isoform cloning

As each splice variant was identified by RT-PCR as a fragment of the full-length Pax3 cDNA sequence, misexpression constructs for each isoform were assembled using convenient restriction sites within the Pax3 coding sequence. The Pax3V1 construct was produced by cutting coding sequence from position 451 to 586, eliminating the region corresponding to exon 4. The Pax3V2 construct was similarly cloned by cutting coding sequence from position 792 to 1173, eliminating the region corresponding to exons 6 and 7. Constructed clones were then shuttled from the pGEM-T Easy vector (Promega) into the pCIG-GFP (gift from McMahon lab) expression vector using the EcoR1 restriction site. Cloning was performed using a T4 DNA ligase in the Promega Ligafast Rapid DNA Ligation System.

Electroporation

The empty vector pCIG, Pax3, Pax3V1, and Pax3V2 plasmid constructs were individually misexpressed at two different developmental stages, 8ss and 16ss via electroporation in ovo. Electroporation of the individual construct was performed with a BTX pulse electroporator (10V, 10msec, 7 pulses). After electroporation, the eggs were incubated for 30-36 hours and collected.

Embryos of 5-7ss were explanted from their egg and cultured similarly to the Easy Chick (EC) method as described by Chapman et al., (2001). Briefly, this method included cracking an egg into a Petri dish and carefully removing the thick albumin away from the chick embryo using a Kimwipe. Rings made from filter paper the size of a quarter with a small circle punched out from the middle were placed on top of the yolk with the embryo being in the middle of the small circle. The filtered ring was cut out of the yolk and the embryo was staged. The excess yolk was removed using a saline wash or manually removed by blunt forceps and a second filtered ring was placed below the embryo, sandwiching the embryo between the two filtered rings that contacted only the extraembryonic tissue, revealing the embryo in the center of the ring. This was a variation of the EC culture allowing for subsequent removal of the cultured embryo onto a new media. The explanted embryos were immediately placed into a Petri dish with simple saline and electroporation of the empty vector pCIG, Pax3, Pax3V1, or Pax3V2 construct was performed with a BTX pulse electroporator (10V, 10msec, 7 pulses). The electroporated embryos were placed on top of pre-warmed culture media (37°C) that had previously been poured into 35mm Petri dishes. The cultured embryos were incubated at 37°C in a humidified chamber until the desired stage was reached.

Tissue culture

After electroporation, embryos were cultured in a six-well plate on an agar-albumen substrate (Darnel & Schoenwolf, 2000) and incubated at 37°C in a humidified chamber for 12 hours. At this point, the ringed embryos were transferred from the agar-albumen substrate to another well within a six-well plate with the agar-albumen substrate that contained either 200μM DAPT or 8μL/mL dimethylsulfoxide (DMSO). Embryos were incubated at 37°C in a humidified chamber for 24 hours before being collected in PBS and fixed.

Immunohistochemistry and analysis

Embryos were embedded in gelatin and cryosectioned. Sections of 12μm were mounted on Superfrost Plus glass slides and the gelatin removed in PBS at 37°C for ten minutes. Embryo sections were incubated overnight at 4°C with a dilution 1:300 Pax3 primary antibody (Developmental Studies Hybridoma Bank) and a dilution of 1:200 Islet1 primary antibody (Developmental Studies Hybridoma Bank) in BSA/PBS buffer (0.1% bovine serum albumen, with 0.1% Tween-20). The primary antibody was rinsed and washed twice for ten minutes in PBS at room temperature. The tissue was then covered with Alexa 633- and Alexa 546-conjugated goat anti-mouse IgG2a and IgG2b (Invitrogen), respectively. IgG2a was diluted 1:200 and IgG2b 1:1000 in BSA/PBS buffer. The tissue was incubated with the secondary antibodies for one hour at room temperature, upon which it was rinsed and washed twice for ten minutes in PBS. Antibody staining was seen using an Olympus BX-61 fluorescent microscope.

Cell counts were performed on randomly selected opV placodes using the Olympus Microsuite software as described by Lassiter et al., (2007). Cell counts for the electroporation experiments included cells expressing GFP, Pax3 antibody, Pax3 antibody and GFP, Islet1 antibody, and Pax3 antibody, GFP, and Islet1 antibody in the ectoderm and in the mesenchyme. One-way ANOVA and Tukey-Kramer test was used to analyze data using SAS software, version 9.2. One-way ANOVA was used to show whether all the means of the groups were equal or not. Tukey-Kramer test was performed to determine which of the means differed significantly in the analysis of variance. P-values of ≤0.05 were considered statistically significant.

Quantitative PCR

Complementary DNA samples were diluted to a concentration of 100ng/μl. Three to five replicates of each sample were performed using Bio-Rad’s SsoFast EvaGreen Supermix on a Bio-Rad CFX Connect Real-Time System cycler. Primers were designed to bind to regions where there was sequence specificity that was unique for each variant, i.e., either binding to regions that were only present in full length variants, or by binding to regions that spanned splice junctions. For flPax3:Pax3V2 qPCR runs, the same upstream primer (PF1) was used, and the downstream primers (PR1 and PR2) were unique to each isoform: flPax3 primer pair: PF1 = 5′-CAGCAGAGCAACTGGAAGAG-3′ and PR1 = 3′-GCTTCCTCCATCTAGCAC-5′. Pax3V2 primer pair: PF1 = 5′-CAGCAGAGCAACTGGAAGAG-3′ and PR2 = 3′-GTCCCATTACCTGAACTCG -5′. For flPax3:Pax3V3 qPCR runs, again the same upstream primer was used, and the downstream primers were unique to each isoform. flPax3 primer pair: forward 5′-GTGCTAGATGGAGGAAGCAG-3′ and reverse 3′-CCTTCAAACCCAGAGAGCAG-5′. Pax3V3 primer pair: forward 5′-GTGCTAGATGGAGGAAGCAG-3′ and reverse 3′-CATACCGCAAGGTGCCT-5′. The PCR cycling parameters were 95°C for 30 seconds, and 40 cycles of 95°C for five seconds, 53°C for five seconds, and followed by a melting curve from 65°C to 95°C at five second increments. Beta-actin mRNA expression was used to normalize the samples among the groups and used to calculate the delta Ct followed by the delta delta Ct (ddCt), which allowed for the comparison of fold expression changes between all samples (see Livak and Schmittgen, 2001). One-way ANOVA was used to show whether all the means of the groups were equal or not. Tukey-Kramer test was performed to determine which of the means differed significantly in the analysis of variance. P-values of ≤0.05 were considered statistically significant.

Key Findings.

  • Splice variants of Pax3 are expressed within the ophthalmic trigeminal placode in the chick embryo.

  • Misexpression of the isoform Pax3V2 permits sensory neurogenesis, while misexpression of the isoform flPax3 or Pax3V1 blocks sensory neurogenesis.

  • Notch inhibition increases the quantitative expression of Pax3.

Acknowledgments

We thank Ben Bikman for his contribution to this work. We also thank Michael Matthews, Matthew Snow, Bret Gardner, and the many other undergraduate students who contributed to this work. This research was supported by the following sources: NIH/NICHD #1R01HD046475; BYU graduate mentoring award.

REFERENCES

  1. Amstutz R, Wachtel M, Troxler H, Kleinert P, Ebauer M, Haneke T, Oehler-Janne C, Fabbro D, Niggli FK, Schafer BW. Phosphorylation regulates transcriptional activity of PAX3/FKHR and reveals novel therapeutic possibilities. Cancer Res. 2008;68:3767–3776. doi: 10.1158/0008-5472.CAN-07-2447. [DOI] [PubMed] [Google Scholar]
  2. Apuzzo S, Abdelhakim A, Fortin AS, Gros P. Cross-talk between the paired domain and the homeodomain of Pax3: DNA binding by each domain causes a structural change in the other domain, supporting interdependence for DNA Binding. J Biol Chem. 2004;279:33601–33612. doi: 10.1074/jbc.M402949200. [DOI] [PubMed] [Google Scholar]
  3. Auerbach R. Analysis of the developmental effects of a lethal mutation in the house mouse. Journal of Experimental Zoology. 1954;127:305–329. [Google Scholar]
  4. Bajard L, Relaix F, Lagha M, Rocancourt D, Daubas P, Buckingham ME. A novel genetic hierarchy functions during hypaxial myogenesis: Pax3 directly activates Myf5 in muscle progenitor cells in the limb. Genes Dev. 2006;20:2450–2464. doi: 10.1101/gad.382806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Baker CV, Stark MR, Marcelle C, Bronner-Fraser M. Competence, specification and induction of Pax-3 in the trigeminal placode. Development. 1999;126:147–156. doi: 10.1242/dev.126.1.147. [DOI] [PubMed] [Google Scholar]
  6. Baker CV, Bronner-Fraser M. Establishing neuronal identity in vertebrate neurogenic placodes. Development. 2000;127:3045–3056. doi: 10.1242/dev.127.14.3045. [DOI] [PubMed] [Google Scholar]
  7. Baker CV, Stark MR, Bronner-Fraser M. Pax3-expressing trigeminal placode cells can localize to trunk neural crest sites but are committed to a cutaneous sensory neuron fate. Dev Biol. 2002;249:219–236. doi: 10.1006/dbio.2002.0767. [DOI] [PubMed] [Google Scholar]
  8. Baldwin CT, Hoth CF, Macina RA, Milunsky A. Mutations in PAX3 that cause Waardenburg syndrome type I: ten new mutations and review of the literature. Am J Med Genet. 1995;58:115–122. doi: 10.1002/ajmg.1320580205. [DOI] [PubMed] [Google Scholar]
  9. Barber TD, Barber MC, Cloutier TE, Friedman TB. PAX3 gene structure, alternative splicing and evolution. Gene. 1999;237:311–319. doi: 10.1016/s0378-1119(99)00339-x. [DOI] [PubMed] [Google Scholar]
  10. Barr FG, Galili N, Holick J, Biegel JA, Rovera G, Emanuel BS. Rearrangement of the PAX3 paired box gene in the paediatric solid tumour alveolar rhabdomyosarcoma. Nat Genet. 1993;3:113–117. doi: 10.1038/ng0293-113. [DOI] [PubMed] [Google Scholar]
  11. Barr FG, Fitzgerald JC, Ginsberg JP, Vanella ML, Davis RJ, Bennicelli JL. Predominant expression of alternative PAX3 and PAX7 forms in myogenic and neural tumor cell lines. Cancer Res. 1999;59:5443–5448. [PubMed] [Google Scholar]
  12. Begbie J, Ballivet M, Graham A. Early steps in the production of sensory neurons by the neurogenic placodes. Mol Cell Neurosci. 2002;21:502–511. doi: 10.1006/mcne.2002.1197. [DOI] [PubMed] [Google Scholar]
  13. Blake JA, Ziman MR. Pax3 transcripts in melanoblast development. Dev Growth Differ. 2005;47:627–635. doi: 10.1111/j.1440-169X.2005.00835.x. [DOI] [PubMed] [Google Scholar]
  14. Bober E, Franz T, Arnold HH, Gruss P, Tremblay P. Pax-3 is required for the development of limb muscles: a possible role for the migration of dermomyotomal muscle progenitor cells. Development. 1994;120:603–612. doi: 10.1242/dev.120.3.603. [DOI] [PubMed] [Google Scholar]
  15. Canning CA, Lee L, Luo SX, Graham A, Jones CM. Neural tube derived Wnt signals cooperate with FGF signaling in the formation and differentiation of the trigeminal placodes. Neural Dev. 2008;3:35. doi: 10.1186/1749-8104-3-35. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cao Y, Wang C. The COOH-terminal transactivation domain plays a key role in regulating the in vitro and in vivo function of Pax3 homeodomain. J Biol Chem. 2000;275:9854–9862. doi: 10.1074/jbc.275.13.9854. [DOI] [PubMed] [Google Scholar]
  17. Chalepakis G, Jones FS, Edelman GM, Gruss P. Pax-3 contains domains for transcription activation and transcription inhibition. Proc Natl Acad Sci U S A. 1994a;91:12745–12749. doi: 10.1073/pnas.91.26.12745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Chalepakis G, Wijnholds J, Gruss P. Pax-3-DNA interaction: flexibility in the DNA binding and induction of DNA conformational changes by paired domains. Nucleic Acids Res. 1994b;22:3131–3137. doi: 10.1093/nar/22.15.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Chapman SC, Collignon J, Schoenwolf GC, Lumsden A. Improved method for chick whole-embryo culture using a filter paper carrier. Dev Dyn. 2001;220:284–289. doi: 10.1002/1097-0177(20010301)220:3<284::AID-DVDY1102>3.0.CO;2-5. [DOI] [PubMed] [Google Scholar]
  20. Collins CA, Gnocchi VF, White RB, Boldrin L, Perez-Ruiz A, Relaix F, Morgan JE, Zammit PS. Integrated functions of Pax3 and Pax7 in the regulation of proliferation, cell size and myogenic differentiation. PLoS One. 2009;4:e4475. doi: 10.1371/journal.pone.0004475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Conboy IM, Rando TA. The regulation of Notch signaling controls satellite cell activation and cell fate determination in postnatal myogenesis. Dev Cell. 2002;3:397–409. doi: 10.1016/s1534-5807(02)00254-x. [DOI] [PubMed] [Google Scholar]
  22. Czerny T, Schaffner G, Busslinger M. DNA sequence recognition by Pax proteins: bipartite structure of the paired domain and its binding site. Genes Dev. 1993;7:2048–2061. doi: 10.1101/gad.7.10.2048. [DOI] [PubMed] [Google Scholar]
  23. D’Amico-Martel A, Noden DM. Contributions of placodal and neural crest cells to avian cranial peripheral ganglia. Am J Anat. 1983;166:445–468. doi: 10.1002/aja.1001660406. [DOI] [PubMed] [Google Scholar]
  24. Darnell DK, Schoenwolf GC. Culture of avian embryos. Methods Mol Biol. 2000;135:31–38. doi: 10.1385/1-59259-685-1:31. [DOI] [PubMed] [Google Scholar]
  25. Dude CM, Kuan CY, Bradshaw JR, Greene ND, Relaix F, Stark MR, Baker CV. Activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the ophthalmic trigeminal placode. Dev Biol. 2009;326:314–326. doi: 10.1016/j.ydbio.2008.11. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Eberhard D, Jimenez G, Heavey B, Busslinger M. Transcriptional repression by Pax5 (BSAP) through interaction with corepressors of the Groucho family. EMBO J. 2000;19:2292–2303. doi: 10.1093/emboj/19.10.2292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Epstein DJ, Vekemans M, Gros P. Splotch (Sp2H), a mutation affecting development of the mouse neural tube, shows a deletion within the paired homeodomain of Pax-3. Cell. 1991;67:767–774. doi: 10.1016/0092-8674(91)90071-6. [DOI] [PubMed] [Google Scholar]
  28. Epstein DJ, Vogan KJ, Trasler DG, Gros P. A mutation within intron 3 of the Pax-3 gene produces aberrantly spliced mRNA transcripts in the splotch (Sp) mouse mutant. Proc Natl Acad Sci U S A. 1993;90:532–536. doi: 10.1073/pnas.90.2.532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Evans J, Lillycrop KA. Serum growth factor regulation of the paired-box transcription factor Pax-3 in neuronal cells. Neurosci Lett. 1996;220:125–128. doi: 10.1016/s0304-3940(96)13246-8. [DOI] [PubMed] [Google Scholar]
  30. Fedtsova N, Perris R, Turner EE. Sonic hedgehog regulates the position of the trigeminal ganglia. Dev Biol. 2003;261:456–469. doi: 10.1016/s0012-1606(03)00316-6. [DOI] [PubMed] [Google Scholar]
  31. Fode C, Gradwohl G, Morin X, Dierich A, LeMeur M, Goridis C, Guillemot F. The bHLH protein NEUROGENIN 2 is a determination factor for epibranchial placode-derived sensory neurons. Neuron. 1998;20:483–494. doi: 10.1016/s0896-6273(00)80989-7. [DOI] [PubMed] [Google Scholar]
  32. Fortin AS, Underhill DA, Gros P. Helix 2 of the paired domain plays a key role in the regulation of DNA-binding by the Pax-3 homeodomain. Nucleic Acids Res. 1998;26:4574–4581. doi: 10.1093/nar/26.20.4574. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Goulding MD, Chalepakis G, Deutsch U, Erselius JR, Gruss P. Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. EMBO J. 1991;10:1135–1147. doi: 10.1002/j.1460-2075.1991.tb08054.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hamburger V, Hamilton HL. A series of normal stages in the development of the chick embryo. Journal of Morphology. 1951;88:49–92. [PubMed] [Google Scholar]
  35. Hsieh MJ, Yao YL, Lai IL, Yang WM. Transcriptional repression activity of PAX3 is modulated by competition between corepressor KAP1 and heterochromatin protein 1. Biochem Biophys Res Commun. 2006;349:573–581. doi: 10.1016/j.bbrc.2006.08.064. [DOI] [PubMed] [Google Scholar]
  36. Ichi S, Boshnjaku V, Shen YW, Mania-Farnell B, Ahlgren S, Sapru S, Mansukhani N, McLone DG, Tomita T, Mayanil CS. Role of Pax3 acetylation in the regulation of Hes1 and Neurog2. Mol Biol Cell. 2011;22:503–512. doi: 10.1091/mbc.E10-06-0541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jarriault S, Le Bail O, Hirsinger E, Pourquie O, Logeat F, Strong CF, Brou C, Seidah NG, Isra l A. Delta-1 activation of notch-1 signaling results in HES-1 transactivation. Mol Cell Biol. 1998;18:7423–7431. doi: 10.1128/mcb.18.12.7423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Kioussi C, Gross MK, Gruss P. Pax3: a paired domain gene as a regulator in PNS myelination. Neuron. 1995;15:553–562. doi: 10.1016/0896-6273(95)90144-2. [DOI] [PubMed] [Google Scholar]
  39. Kwang SJ, Brugger SM, Lazik A, Merrill AE, Wu LY, Liu YH, Ishii M, Sangiorgi FO, Rauchman M, Sucov HM, Maas RL, Maxson RE., Jr Msx2 is an immediate downstream effector of Pax3 in the development of the murine cardiac neural crest. Development. 2002;129:527–538. doi: 10.1242/dev.129.2.527. [DOI] [PubMed] [Google Scholar]
  40. Lalwani AK, Brister JR, Fex J, Grundfast KM, Ploplis B, San Agustin TB, Wilcox ER. Further elucidation of the genomic structure of PAX3, and identification of two different point mutations within the PAX3 homeobox that cause Waardenburg syndrome type 1 in two families. Am J Hum Genet. 1995;56:75–83. [PMC free article] [PubMed] [Google Scholar]
  41. Lassiter RN, Dude CM, Reynolds SB, Winters NI, Baker CV, Stark MR. Canonical Wnt signaling is required for ophthalmic trigeminal placode cell fate determination and maintenance. Dev Biol. 2007;308:392–406. doi: 10.1016/j.ydbio.2007.05.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Lassiter RN, Reynolds SB, Marin KD, Mayo TF, Stark MR. FGF signaling is essential for ophthalmic trigeminal placode cell delamination and differentiation. Dev Dyn. 2009;238:1073–1082. doi: 10.1002/dvdy.21949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Lassiter RN, Ball MK, Adams JS, Wright BT, Stark MR. Sensory neuron differentiation is regulated by notch signaling in the trigeminal placode. Dev Biol. 2010;344:836–848. doi: 10.1016/j.ydbio.2010.05.514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  45. Mansouri A. The role of Pax3 and Pax7 in development and cancer. Crit Rev Oncog. 1998;9:141–149. doi: 10.1615/critrevoncog.v9.i2.40. [DOI] [PubMed] [Google Scholar]
  46. Marcelle C, Eichmann A, Halevy O, Breant C, Le Douarin NM. Distinct developmental expression of a new avian fibroblast growth factor receptor. Development. 1994;120:683–694. doi: 10.1242/dev.120.3.683. [DOI] [PubMed] [Google Scholar]
  47. Maroto M, Reshef R, Munsterberg AE, Koester S, Goulding M, Lassar AB. Ectopic Pax-3 activates MyoD and Myf-5 expression in embryonic mesoderm and neural tissue. Cell. 1997;89:139–148. doi: 10.1016/s0092-8674(00)80190-7. [DOI] [PubMed] [Google Scholar]
  48. Mayanil CS, George D, Freilich L, Miljan EJ, Mania-Farnell B, McLone DG, Bremer EG. Microarray analysis detects novel Pax3 downstream target genes. J Biol Chem. 2001;276:49299–49309. doi: 10.1074/jbc.M107933200. [DOI] [PubMed] [Google Scholar]
  49. McCabe KL, Bronner-Fraser M. Essential role for PDGF signaling in ophthalmic trigeminal placode induction. Development. 2008;135:1863–1874. doi: 10.1242/dev.017954. [DOI] [PubMed] [Google Scholar]
  50. Murakami M, Tominaga J, Makita R, Uchijima Y, Kurihara Y, Nakagawa O, Asano T, Kurihara H. Transcriptional activity of Pax3 is co-activated by TAZ. Biochem Biophys Res Commun. 2006;339:533–539. doi: 10.1016/j.bbrc.2005.10.214. [DOI] [PubMed] [Google Scholar]
  51. Nakazaki H, Reddy AC, Mania-Farnell BL, Shen YW, Ichi S, McCabe C, George D, McLone DG, Tomita T, Mayanil CS. Key basic helix-loop-helix transcription factor genes Hes1 and Ngn2 are regulated by Pax3 during mouse embryonic development. Dev Biol. 2008;316:510–523. doi: 10.1016/j.ydbio.2008.01.008. [DOI] [PubMed] [Google Scholar]
  52. Parker CJ, Shawcross SG, Li H, Wang QY, Herrington CS, Kumar S, MacKie RM, Prime W, Rennie IG, Sisley K, Kumar P. Expression of PAX 3 alternatively spliced transcripts and identification of two new isoforms in human tumors of neural crest origin. Int J Cancer. 2004;108:314–320. doi: 10.1002/ijc.11527. [DOI] [PubMed] [Google Scholar]
  53. Perez SE, Rebelo S, Anderson DJ. Early specification of sensory neuron fate revealed by expression and function of neurogenins in the chick embryo. Development. 1999;126:1715–1728. doi: 10.1242/dev.126.8.1715. [DOI] [PubMed] [Google Scholar]
  54. Phelan SA, Loeken MR. Identification of a new binding motif for the paired domain of Pax-3 and unusual characteristics of spacing of bipartite recognition elements on binding and transcription activation. J Biol Chem. 1998;273:19153–19159. doi: 10.1074/jbc.273.30.19153. [DOI] [PubMed] [Google Scholar]
  55. Pritchard C, Grosveld G, Hollenbach AD. Alternative splicing of Pax3 produces a transcriptionally inactive protein. Gene. 2003;305:61–69. doi: 10.1016/s0378-1119(02)01186-1. [DOI] [PubMed] [Google Scholar]
  56. Relaix F, Polimeni M, Rocancourt D, Ponzetto C, Schafer BW, Buckingham M. The transcriptional activator PAX3-FKHR rescues the defects of Pax3 mutant mice but induces a myogenic gain-of-function phenotype with ligand-independent activation of Met signaling in vivo. Genes Dev. 2003;17:2950–2965. doi: 10.1101/gad.281203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Relaix F, Montarras D, Zaffran S, Gayraud-Morel B, Rocancourt D, Tajbakhsh S, Mansouri A, Cumano A, Buckingham M. Pax3 and Pax7 have distinct and overlapping functions in adult muscle progenitor cells. J Cell Biol. 2006;172:91–102. doi: 10.1083/jcb.200508044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Seo HC, Saetre BO, Havik B, Ellingsen S, Fjose A. The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain. Mech Dev. 1998;70:49–63. doi: 10.1016/s0925-4773(97)00175-5. [DOI] [PubMed] [Google Scholar]
  59. Stark MR, Sechrist J, Bronner-Fraser M, Marcelle C. Neural tube-ectoderm interactions are required for trigeminal placode formation. Development. 1997;124:4287–4295. doi: 10.1242/dev.124.21.4287. [DOI] [PubMed] [Google Scholar]
  60. Streit A. Early development of the cranial sensory nervous system: from a common field to individual placodes. Dev Biol. 2004;276:1–15. doi: 10.1016/j.ydbio.2004.08.037. [DOI] [PubMed] [Google Scholar]
  61. Stuart ET, Kioussi C, Gruss P. Mammalian Pax genes. Annu Rev Genet. 1994;28:219–236. doi: 10.1146/annurev.ge.28.120194.001251. [DOI] [PubMed] [Google Scholar]
  62. Sun Y, Dykes IM, Liang X, Eng SR, Evans SM, Turner EE. A central role for Islet1 in sensory neuron development linking sensory and spinal gene regulatory programs. Nat Neurosci. 2008;11:1283–1293. doi: 10.1038/nn.2209. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Tassabehji M, Read AP, Newton VE, Patton M, Gruss P, Harris R, Strachan T. Mutations in the PAX3 gene causing Waardenburg syndrome type 1 and type 2. Nat Genet. 1993;3:26–30. doi: 10.1038/ng0193-26. [DOI] [PubMed] [Google Scholar]
  64. Treisman J, Harris E, Desplan C. The paired box encodes a second DNA-binding domain in the paired homeo domain protein. Genes Dev. 1991;5:594–604. doi: 10.1101/gad.5.4.594. [DOI] [PubMed] [Google Scholar]
  65. Tremblay P, Kessel M, Gruss P. A transgenic neuroanatomical marker identifies cranial neural crest deficiencies associated with the Pax3 mutant Splotch. Dev Biol. 1995;171:317–329. doi: 10.1006/dbio.1995.1284. [DOI] [PubMed] [Google Scholar]
  66. Tsukamoto K, Nakamura Y, Niikawa N. Isolation of two isoforms of the PAX3 gene transcripts and their tissue-specific alternative expression in human adult tissues. Hum Genet. 1994;93:270–274. doi: 10.1007/BF00212021. [DOI] [PubMed] [Google Scholar]
  67. Vogan KJ, Epstein DJ, Trasler DG, Gros P. The splotch-delayed (Spd) mouse mutant carries a point mutation within the paired box of the Pax-3 gene. Genomics. 1993;17:364–369. doi: 10.1006/geno.1993.1333. [DOI] [PubMed] [Google Scholar]
  68. Vogan KJ, Underhill DA, Gros P. An alternative splicing event in the Pax-3 paired domain identifies the linker region as a key determinant of paired domain DNA-binding activity. Mol Cell Biol. 1996;16:6677–6686. doi: 10.1128/mcb.16.12.6677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Vogan KJ, Gros P. The C-terminal subdomain makes an important contribution to the DNA binding activity of the Pax-3 paired domain. J Biol Chem. 1997;272:28289–28295. doi: 10.1074/jbc.272.45.28289. [DOI] [PubMed] [Google Scholar]
  70. Waardenburg PJ. A new syndrome combining developmental anomalies of the eyelids, eyebrows and nose root with pigmentary defects of the iris and head hair and with congenital deafness. Am J Hum Genet. 1951;3:195–253. [PMC free article] [PubMed] [Google Scholar]
  71. Wu M, Li J, Engleka KA, Zhou B, Lu MM, Plotkin JB, Epstein JA. Persistent expression of Pax3 in the neural crest causes cleft palate and defective osteogenesis in mice. J Clin Invest. 2008;118:2076–2087. doi: 10.1172/JCI33715. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES