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Keywords:

  • Pax3;
  • Pax6;
  • placode;
  • trigeminal

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cranial sense organs and a subset of cranial sensory neurons are generated from placodes, thickenings of the ectoderm. Pax3 has been known as a marker for ophthalmic trigeminal placode specification, and also an important regulator of trigeminal placode neuron differentiation. In this study, I show that Pax6 is initially expressed in the preplacodal region at the level of ophthalmic trigeminal placode, and that this expression gradually regresses in a medial-to-lateral direction as Pax3 expression expands in the same direction. Misexpression studies revealed that Pax6 represses Pax3 expression indirectly as a transcriptional activator in a cell-autonomous manner. Pax3-misexpression represses Pax6 expression in an indirect fashion, suggesting that unknown factor(s) downstream of Pax3 may repress Pax6 expression, and thereby allow an expansion of Pax3-positive ophthalmic trigeminal placode region. These results indicate that the mutual repression between Pax3 and Pax6 has important roles in the specification and the positioning of the ophthalmic trigeminal placode.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Cranial sense organs and a subset of cranial sensory neurons are generated from placodes, thickenings of the ectoderm. Fate map studies have shown that placodal tissues originate from the ectoderm surrounding the neural plate and the neural crest (Streit 2002; Bhattacharyya et al. 2004; Xu et al. 2008). This horse-shoe-like ectodermal domain can be identified by the co-expression of genes, which encode components of Eya-Six-Dach transcriptional complex, and this domain is termed as pre-placodal region (PPR; see Streit 2004, 2007 for reviews). Streit and colleagues have shown the importance of fibroblast growth factor and other signaling that emanated from surrounding tissues in the formation of PPR (Listou et al. 2005). Placodal cells originated from PPR form patches of placodes, such as nasal, lens, otic and several neurogenic placodes, including trigeminal placode.

Emerging evidence indicates that the character and the position of each placode are determined under the influence of surrounding tissues. A classical example is lens. Thus, lens placode is induced from the potent PPR by the inductive cues emanated from the juxtaposing optic vesicle and other nearby tissues (See Chow & Lang 2001; for a review). Transplantation experiments revealed that the competence to differentiate into lens is restricted to the anterior PPR (Grainger 1992), and such competence seems to be acquired by the expression of a paired-homeodomain transcription factor, Pax6 (reviewed in Kondoh et al. 2004). Genetic studies revealed the requirement of Pax6 in lens and olfactory placode formation (Fujiwara et al. 1994).

Ophthalmic trigeminal placode, which generates trigeminal sensory neurons, is originated from the PPR nearby the developing midbrain. Previous studies indicated that platelet-derived growth factor and Wnt ligands provided by the midbrain and midbrain/hindbrain boundary region induce Pax3 expression expanding from a medial to ventro-lateral direction (Lassiter et al. 2007; Canning et al. 2008; McCabe & Bronner-Fraser 2008). Pax3 function is important for the promotion of further differentiation of trigeminal placode/sensory neurons (Dude et al. 2009). Thus, Pax3 expression has been considered as a very early sign of ophthalmic trigeminal placode specification of the PPR cells. It is still unclear how only a part of PPR at the level of the midbrain is specified as the ophthalmic trigeminal placode.

In this study, I have found (i) that Pax6 is initially expressed in PPR at the midbrain level, (ii) that, as Pax3 expression expands ventro-laterally in PPR, Pax6 expression regresses to form Pax3/Pax6 boundary, (iii) that misexpression of Pax6 in the prospective ophthalmic trigeminal placode region represses Pax3 expression and subsequent differentiation of the trigeminal sensory neurons, and (iv) that Pax3 misexpression indirectly represses Pax6 expression. These results indicate that the mutual repression of Pax3 and Pax6 sets the ventro-lateral margin of the ophthalmic trigeminal placode, and thereby contributing the positioning of it.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Experimental animals

Fertilized chicken eggs were obtained from a local chicken farm. Embryos were staged according to Hamburger & Hamilton (1951).

In situ hybridization

In situ hybridization in whole-mount preparation and on section was performed as described previously (Wakamatsu & Weston 1997; Wakamatsu 2009).

Template cDNAs for antisense probes were kindly provided by Drs Kosuke Kataoka (L-Maf), Martyn Goulding (Pax3), Clare Baker (FGFR4), Shigeru Sato courtesy of Atsushi Kuroiwa (Six1). Quail Brn3a and RALDH3 cDNAs were PCR-amplified from E4 quail cDNA pool.

Immunological staining

Immuno-fluorescent staining of sections and cultured NIH3T3 cells was performed as previously described (Wakamatsu & Weston 1997; Wakamatsu et al. 1997). Anti-Pax6 polyclonal antibody was described previously (Inoue et al. 2000). Anti-Pax3 and anti-Pax6 mouse monoclonal antibodies were obtained from DSHB. Anti-δ-crystallin rabbit polyclonal antibody (Kondoh et al. 1984) was provided by Dr Hisato Kondoh. 16A11 anti-HuC/D mouse IgG was purchased from Invitrogen.

Expression vectors

Expression vectors of chick Pax3, Pax6, Pax6-En, and Pax6-VP16 were provided by Dr Harukazu Nakamura (Matsunaga et al. 2000, 2001). Chick Pax6 coding sequence was also transferred to pIRES-mRFP1. Co-expression of Pax6 and mRFP1 proteins in individual cells was confirmed by transfecting NIH3T3 cells (not shown). pEGFP-N1 was purchased from BD Clontech. pCAGGS-mRFP1 was described previously (Wakamatsu et al. 2011).

Electroporation into PPR

Electroporation into PPR of stage 7–8 chicken embryos was performed basically as described (Funahashi et al. 1999; Wakamatsu et al. 2004; Suzuki et al. 2006). A rod-like positive electrode was inserted underneath the embryos. DNA solution (approximately 1 μL of 5 μg/μL in PBS containing 0.025% Fast Green) was overlaid onto PPR, and a tungsten needle as a negative electrode was placed over PPR. Electroporation was performed with CUY21 electroporator (BEX, condition: 3 V, 25 ms duration, 225 ms interval, and three pulses).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Dynamic expression of Pax3/6 defines the ventro-lateral border of trigeminal placode

I found that at around Hamburger and Hamilton stage 8.0–8.5, when Pax3 protein expression was undetectable in PPR, Pax6 protein was expressed in the anterior PPR (Figs 1, 2). This Pax6 expression had little overlap with Pax7 expression in the premigratory neural crest, indicating PPR-restricted expression of Pax6 (Fig. 1). Posterior margin of this PPR expression of Pax6 appeared to coincide with the anterior margin of Pax2 expression, a posterior PPR marker (Fig. 1). At the midbrain level of stage nine embryos, Pax3 protein expression was first observed in the dorsal part of the midbrain-level PPR (not shown), and at later stages such as stage 11, this expression expanded ventro-laterally (Fig. 2) as previously described by Canning et al. 2008 at the mRNA level. Six1 expression confirmed the placodal identity of this Pax3-domain (Fig. 2). Coincidentally, Pax6 expression in PPR of this level regressed laterally, with small overlaps with Pax3 expression (Fig. 2, stage 11). At this stage, a few Pax3-positive, Pax6-negative cells emerged in the mesenchyme underlying PPR. Such cells were likely to be delaminated trigeminal sensory neurons, as these cells would also express HuC/D as a neuronal marker (not shown, but see McCabe et al. 2009). As development proceeded (such as stage 14, Fig. 2), the boundary of Pax3 and Pax6 expressions in PPR became sharp, and the number of delaminated Pax3-positive cells increased. Nearby sections were processed for in situ hybridizations with Six1 and Brn3a probes, and a similar pattern was observed (Fig. 2), further confirming the identity of Pax3-positive cells in the mesenchyme as the trigeminal placode neurons.

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Figure 1.  Distributions of Pax2, 6, and 7 proteins in a chick embryo at stage 8 (3–4 somite stage). Transverse sections are stained with anti-Pax2, Pax6, Pax7 antibodies and counter-stained the nuclei with 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI). Pax6 expression is restricted to the anterior pre-placodal region (PPR) (arrowheads), and Pax2 expression is in contrast restricted to the posterior PPR (arrows). Pax7 expression indicates premigratory neural crest cells.

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image

Figure 2.  Ventro-lateral expansion of Pax3 protein expression in the pre-placodal region (PPR) coincides with a downregulation of Pax6 protein expression in the same direction. Initially Pax6 is expressed in the entire PPR at the midbrain level (stage 8.5, 5–6 somites), but, as Pax3 expression begins medially and expands laterally, Pax6 expression regresses laterally (stage 11). Nearby the Pax3/6 boundary, Pax3-positive cells, Pax6 positive cells, and double-positive cells intermingle at this stage (see the high magnification view on the right. White arrowheads indicate double-positive cells.). At stage 14, the boundary becomes sharp and no double-positive cells are detected. Many Pax3-positive cells have delaminated (green arrowheads), and these cells are likely Brn3a-positive, Six1-positive trigeminal placode neurons (black arrowheads).

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These observations suggested that, while the trigeminal sensory neurons were generated from the Pax3 domain of the midbrain level PPR, Pax6 expression in the same level preceded Pax3 expression, and that, as Pax3 expression expanded ventro-laterally, Pax6 expression regressed. Thus, Pax3/6 border appeared to define the ventro-lateral edge of the ophthalmic trigeminal placode.

Pax6 represses Pax3 expression and subsequent trigeminal placode development

Based on the expression studies described above, it was hypothesized that the mutual repression between Pax3 and Pax6 might contribute to define the position of the ophthalmic trigeminal placode. Thus, a plasmid vector carrying an expression cassette of chick Pax6-IRES-mRFP1 was constructed, and was electroporated into the right side PPR at the level of the prospective ophthalmic trigeminal placode. Transfection of both Pax6-IRES-mRFP1 and IRES-mRFP1 did not cause obvious morphological abnormality (Fig. 3A, A″), and an intense expression of Pax6 protein was confirmed in the Pax6-IRES-mRFP1-transfected cells (Fig. 3B–B″′). While embryos transfected with IRES-mRFP1 bilaterally expressed Pax3 in the trigeminal placode 10–12 h after electroporation (n = 10/10; Fig. 3F), Pax3 expression was considerably reduced in the ipsi-lateral Pax6-IRES-mRFP1-transfected side, compared with the contra-lateral untransfected side (n = 9/10; Fig. 3G). This repression of Pax3 seemed to be cell-autonomous as revealed by the immunostaining analysis on sections (Fig. 3D–E). Concomitantly, the expression domain of FGFR4, a downstream target of Pax3 (Dude et al. 2009), was also significantly reduced 18 h after electroporation (n = 5/5; Fig. 3H), and Brn3a expression aberrantly decreased 24 h after electroporation (n = 6/6; Fig. 3J). These results suggested that Pax6 expression has to be downregulated for the expansion of Pax3 expression domain and the following differentiation of the trigeminal placode neurons.

image

Figure 3. Pax6 misexpression impairs ophthalmic trigeminal placode development. Embryos were transfected either with IRES-mRFP1 as controls, or chick Pax6-IRES-mRFP1 at stage 7, and allowed to develop for 10 h (A–E), 12 h (F, G), 18 h (H), and 24 h (I–K). (A, A′) An embryo transfected with Pax6-IRES-mRFP1 on the right side of the PPR. Note that apparent morphological abnormalities are not observed. (B–B″) A section of Pax6-IRES-mRFP1-transfected embryo at the midbrain level. Expression of Pax6 protein in the transfected PPR is detected. (C–C″) Higher magnification of B–B″. Transfected cells express mRFP1 and Pax6 (arrowheads). (D–D″) Another section of Pax6-IRES-mRFP1-transfected embryo at the midbrain level, stained with anti-Pax3 antibody. (E–E″) Higher magnification of D–D″. Transfected cells expressing high levels of mRFP1 have lower Pax3 protein compared with the surrounding cells (arrowheads). (F) Pax3 mRNA expression in the ophthalmic trigeminal placode (inside of dots) of IRES-mRFP1-transfected control embryo. (G) Pax3 mRNA expression in the ophthalmic trigeminal placode (inside of dots) of Pax6-IRES-mRFP1-transfected embryo. Note that the level of Pax3 expression appears lower on the transfected right side. (H, H′) Dorso-lateral views of Pax6-IRES-mRFP1-transfected embryo, showing FGFR4 mRNA expression in the ophthalmic trigeminal placode. Compared to the expression on the contra-lateral control side (arrowheads in H), FGFR4 expression is almost missing on the transfected ipsi-lateral side (H′). (I) A dorsal view of IRES-mRFP1-transfected control embryo, showing Brn3a mRNA expression in trigeminal placode neurons. Expression level is comparable between untransfected and transfected (arrowheads) sides. (J) A dorsal view of Pax6-IRES-mRFP1-transfected embryo. Brn3a expression is severely reduced in the transfected side (arrowheads). (K–K″′) Sections of Pax6-IRES-mRFP1-transfected embryo, showing expression of δ-crystallin protein (K″) and L-Maf mRNA (K″′). While δ-crystallin and L-Maf are normally expressed in the developing lens placode (asterisks), no ectopic expression is detected in the transfected ectoderm (arrowheads).

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As mentioned above, Pax6 has been known to be required for formation of olfactory and lens placodes (Fujiwara et al. 1994). Thus, the suppression of trigeminal placode fate by Pax6 transfection might be a consequence of transformation to these more anterior placodes. I therefore examined the expression of RALDH3, as an olfactory placode marker, and L-Maf and δ-crystallin protein, as lens placode markers in Pax6-transfected embryos (Fig. 3K and data not shown). Since no ectopic expression of these markers was detected after Pax6 transfection (n = 5/5 for each probe), I concluded that the suppressed trigeminal placode development by Pax6-transfection was not caused by the transformation of the placode identity.

Pax6 protein has been considered as a transcriptional activator (e.g. Tang et al. 1998; Muta et al. 2002), although it may also function as a repressor in certain contexts (Duncan et al. 1998). To examine the mode of Pax6 action in the inhibition of the trigeminal placode development, expression vectors of a constitutive-activator and constitutive-repressor versions of Pax6 (Pax6-VP16 and Pax6-En, respectively, see Matsunaga et al. 2000 for details) were co-transfected into PPR of the prospective trigeminal placode region along with EGFP or mRFP1 expression vector. When Pax6-VP16 was transfected, Pax3 expression was repressed 10–12 h after transfection (n = 7/7; Fig. 4D). Consistently, the number of Brn3a-positive trigeminal neurons was significantly reduced in such embryos 24 h after transfection (n = 5/5; Fig. 4A). It is of note, however, that Pax6-VP16-transfected PPR cells expressed δ-crystallin, suggesting the transformation of the trigeminal placode into lens (n = 3/3; Fig 4E), although L-Maf expression was not induced (n = 3/3; Fig. 4C). I did not detect ectopic RALDH3 expression (n = 5/5; Fig. 4B), excluding a possible transformation into the olfactory placode. In contrast, Pax6-En-transfected embryos normally expressed Pax3 at the midbrain level (n = 10/10; Fig. 5A, B), further suggesting that Pax6 indirectly repressed Pax3 as an activator. Consistently, no ectopic δ-crystallin expression was observed (n = 3/3; Fig. 5B). It is of note that, in a few embryos transfected with Pax6-En, a posterior expansion of Pax3 expression domain was observed (n = 2/5; Fig. 5D, E).

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Figure 4. Pax6-VP16 expression prevents ophthalmic trigeminal placode development, and induces ectopic δ-crystallin expression. (A, A′) An example of Pax6-VP16-transfected embryo 24 h after transfection. Co-transfected EGFP indicates the transfected area (A ipsi-lateral side, arrowheads). Compared with the Brn3a expression in trigeminal sensory neurons in the untransfected side (A′ contra-lateral), Brn3a expression is severely diminished in the transfected side (A′ ispi-lateral). (B) RALDH3 expression in a Pax6-VP16-transfected embryo 18 h after transfection. Expression in the olfactory placode (arrowheads) is observed. (C) L-Maf expression in a Pax6-VP16-transfected embryo 18 h after transfection. Expression in the lens placode (arrowheads) is observed. (D–D″) Pax6-VP16 represses Pax3 expression in the ophthalmic trigeminal placode (arrowheads). 12 h after transfection. (E–E″) A neighboring section of (D). Pax6-VP16 induces ectopic expression of δ-crystallin expression (arrowheads).

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Figure 5. Pax6-En transfection affects Pax3 expression in the PPR. (A–A″) Pax6-En does not appear to affect Pax3 expression in the ophthalmic trigeminal placode on a transverse section (arrowheads), 12 h after transfection. (B–B″) A neighboring section of (A). Pax6-En does not induce δ-crystallin expression (arrowheads). (C) A dorsal view of Pax6-En-transfected embryo 12 h after transfection. Co-transfected EGFP indicates the transfected area. No morphological defect can be observed. (D) An example of EGFP-transfected embryo, showing Pax3 mRNA expression in the ophthalmic trigeminal placodes (dotted lines) 12 h after transfection. (E) An example of Pax6-En-transfected embryo, showing a posterior expansion of Pax3-domain (dotted lines) in the right side 12 h after transfection.

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Pax3 represses Pax6 expression in PPR

A previous paper revealed that Pax3 misexpression in the PPR expanded an expression of FGFR4, and promoted subsequent neurogenesis (Dude et al. 2009). To test if Pax3 transfection would also repress Pax6, a chick Pax3 expression vector (see Matsunaga et al. 2001) was co-transfected with EGFP into the PPR of the prospective ophthalmic trigeminal placode. Ten hours after transfection, Pax6 expression in the transfected PPR was normally observed compared with the contra-lateral side (n = 6/6; Fig. 6B), despite the transfected Pax3 expression having been confirmed by immuno-staining (Fig. 6A). Twenty-four hours after transfection, however, a downregulation of Pax6 was observed in the Pax3-transfected area (n = 4/5; Fig. 6C). However, expression of co-transfected EGFP and endogenous Pax6 expression could not be correlated at a single cell level, along with the relatively slow response of Pax6 expression to Pax3 transfection, suggesting a possible indirect effect of Pax3 misexpression.

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Figure 6. Pax3 transfection affects Pax6 expression in the pre-placodal region (PPR). Pax3 is co-transfected with EGFP. (A–A″) Ectopic expression of Pax3 protein is confirmed in the transfected area 10 h after transfection (arrowheads). (B–B″) A nearby section of (A). Pax6 protein expression does not appear to be affected by Pax3 misexpression at this stage. (C–C″) Pax6 expression is reduced in the Pax3-transfected side (arrowheads) 24 h after transfection. Indicated area corresponds to the Pax6-positive domain partially overlapping with EGFP-expressing transfected area. (C″′–C″′″′) Higher magnification of transfected area indicated in (C–C″). Despite the overall reduction of Pax6 expression in the Pax3/EGFP-co-transfected area, some EGFP-positive cells still express high levels of Pax6 (arrowheads).

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Potential mutual repression between Pax3 and Pax6 in the embryonic ectoderm lineages

Reciprocal expression of Pax3 and Pax6 proteins did not seem to be restricted to the ophthalmic trigeminal placode region. As indicated in Figure 7, Pax6 expression is high in the nasal epithelium (Fig. 7A), lens placode (Fig. 7C), and ventral non-neural ectoderm (Fig. 7E) of stage 14 embryo. Expression of Pax3 was low or absent in these tissues, but nearby ectoderm expressed Pax3 (Fig. 7B, D, F). These observations suggested that the mutual repression between Pax3 and Pax6 occurred in various locations, potentially contributing to the regional specifications of the developing ectoderm.

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Figure 7.  Reciprocal expression of Pax6 and Pax3 proteins in the developmental ectoderm of stage 14 embryo. (A, A′, B, B′) While nasal epithelium (ne) express Pax6, Pax3 expression is high in nearby ectoderm (arrowheads). (C, C′, D, D′) While Pax6 is expressed in the lens placode (lp), retina (re), and a part of diencephalon (di), Pax3 expression is restricted in the dorsal ectoderm (arrowheads). (E, E′, F, F′) Pax6 and Pax3 are differentially expressed in the hindbrain (hb) with some overlap. Pax3 is also expressed in the ophthalmic trigeminal placode (otg) and migrating placode neurons (arrowheads).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

While the importance of Pax6 function in formation of optic and olfactory placodes has been well studied (e.g. Fujiwara et al. 1994), the role of Pax6 in the development of other placodes has not been considered. In this paper, I reveal the importance of the mutual repression of Pax3 and Pax6 in formation of the ophthalmic trigeminal placode of avian embryo, although the importance of this regulation will have to be tested in other animals. While several studies showed instructive signals involved in the ophthalmic trigeminal placode specification (Lassiter et al. 2007; Canning et al. 2008; McCabe & Bronner-Fraser 2008), how the island-like ophthalmic trigeminal placode is positioned in PPR has not been understood. Pax6 now seems to play a role to determine the ventro-lateral edge of the ophthalmic trigeminal placode. Pax3 expression comes on in the very dorsal part of the midbrain PPR and expands laterally, likely by the inductive signals, but the dorsal most of the PPR eventually become occupied by Pax3-negative cells. It remains to be shown, however, how the island of the Pax3-positive ophthalmic trigeminal placode is separated from the dorsal midline and further shifts posteriorly, as shown before (Canning et al. 2008). This can be achieved by a loss of Pax3 expression in the dorsal PPR cells. Alternatively, Pax3-negative dorsal PPR cells nearby may translocate/migrate anteriorly and/or posteriorly to separate Pax3-positive cells from the dorsal midline. To test these hypotheses, extensive cell-labeling studies will be needed.

In this study, I also show (i) that both wild type and constitutively activator-form of Pax6 repress Pax3 expression, (ii) that the constitutively repressor-form of Pax6 has little effect on Pax3 expression in the midbrain level PPR, and (iii) that constitutively activator-form, but not the wild type, Pax6 induced an ectopic expression of a lens placode marker. Thus, in this context, Pax6 protein would likely act as a transcriptional activator as in many previous examples (e.g. Tang et al. 1998; Muta et al. 2002) to repress Pax3 expression, and therefore this regulation should be indirect. While the activator-form of Pax6 could theoretically activate the lens/olfactory target genes in ectopic regions, only a lens marker was induced. Thus, the functional difference of Pax6 in distinct placodal regions is likely caused by the epigenetic context, at least in part. It has been known that Pax6 forms heterodimers to activate certain target genes. The well-known example as one of the target gene is δ-crystallin. In this case, Pax6 binds the lens-specific enhancer of δ-crystallin gene along with Sox2 and activates the transcription (Kamachi et al. 2001). Thus, the functional difference of Pax6 in the ophthalmic trigeminal placode-forming PPR and in the more anterior placodal regions could rather be attributed to the composition of co-factor expression.

It is also shown that transfection of Pax3 repressed Pax6 protein expression in the midbrain level PPR. Considering that this repression occurs slowly, and that the correlation between expression levels of the transgene and the endogenous Pax6 expression was weak, the Pax3-mediated repression of Pax6 expression seems to be indirect and likely be non-cell autonomous. While it remains to be shown how Pax3 represses Pax6, because the expression of Pax3 and Pax6 in the embryonic ectoderm often appears reciprocal (Fig. 7), the mutual repression seems to be important in establishing the expression pattern.

As shown above, transfection of Pax6-En into PPR results in the posterior expansion of Pax3 expression domain. However, because the posterior PPR expresses Pax2, but not Pax6 (see Fig. 2), the observed expansion of Pax3 domain cannot be explained by blocking the endogenous Pax6 protein function. Pax6-En may promote proliferation of Pax3-positive cells. However, as a repression of Pax2 protein expression occurs in Pax6-En-transfected posterior PPR cells (data not shown), this may secondarily permit Pax3 expression. In a previous study, it has been shown that an ectopic expression of Pax3 repressed Pax2 in the posterior PPR (Dude et al. 2009). Differential Pax family gene expressions, along with other homeobox transcription factors, have been known to specify neural progenitors to distinct neuronal subtypes in the developing central nervous system (see Takahashi & Osumi 2002; and references therein. See also Fig. 7). Thus, I suggest that Pax2, Pax3, and Pax6 in an analogous way repress each other to specify the identities of each placode, as a “Pax code”.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Drs Koji Tamura and Hiroshi Yajima for comments on the manuscript. This work was supported by a grant to YW from the Japan Society for promotion of Science (21570215).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References