August 1, 2010;
Conserved expression of mouse Six1 in the pre-placodal region (PPR) and identification of an enhancer for the rostral PPR.
All cranial sensory organs and sensory neurons of vertebrates develop from cranial placodes
. In chick, amphibians and zebrafish, all placodes originate from a common precursor domain, the pre-placodal region (PPR), marked by the expression of Six1
/4 and Eya1
/2. However, the PPR has never been described in mammals and the mechanism involved in the formation of PPR is poorly defined. Here, we report the expression of Six1
in the horseshoe-shaped mouse ectoderm
surrounding the anterior neural plate
in a pattern broadly similar to that of non-mammalian vertebrates. To elucidate the identity of Six1
-positive mouse ectoderm
, we searched for enhancers responsible for Six1
expression by in vivo enhancer assays. One conserved non-coding sequence, Six1
-14, showed specific enhancer activity in the rostral PPR of chick and Xenopus and in the mouse ectoderm
. These results strongly suggest the presence of PPR in mouse and that it is conserved in vertebrates. Moreover, we show the importance of the homeodomain protein-binding sites of Six1
-14, the Six1
rostral PPR enhancer, for enhancer activity, and that Dlx5
are candidate binding factors that regulate the level and area of Six1
expression, and thereby the location of the PPR. Our findings provide critical information and tools to elucidate the molecular mechanism of early sensory development and have implications for the development of sensory precursor/stem cells.
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The conserved non-coding sequence Six1-14 activates gene expression in the PPR of chick and Xenopus. (A–E) Enhancer activity of Six1-14 in chick. Enhancer activity was identified by electroporation (A). Whole mount embryo at 6 h.p.e. with ptkEGFP-mSix1-14×4 showing EGFP expression (arrowheads) in the region surrounding the anterior neural plate (B, dorsal view). Green channel (EGFP) is superimposed on bright-field image. Inset in B shows expression of HcRed (magenta, pseudocolor) from co-electroporated pCAG-HcRed, which drives ubiquitous expression of HcRed, in a broad area of the embryo. Transverse section showing uniform expression of HcRed (magenta) in the ectoderm (Ca) and specific EGFP expression (arrowheads) in the non-neural ectoderm (Cb). Transverse section of another embryo at 6 h.p.e. with ptkEGFP-mSix1-14×2 showing EGFP expression (arrowheads) in a similar pattern (D). Green channel (EGFP) is superimposed on DIC image. The majority of EGFP-positive cells are located outside the neural plate labeled with anti-Sox2 antibody (magenta, pseudocolor) (E). (F–H) Enhancer activity of Six1-14 in Xenopus. Enhancer activity was identified by transgenesis (F). Frontal view of a stage 15 embryo transgenic for ISpBSIISK + betaGFP mSix1-14 (G). EGFP mRNA distribution (arrowheads) is detected in the region surrounding the anterior neural plate by in situ hybridization. Sagittal section (100-μm) of another embryo showing EGFP expression in the anterior non-neural ectoderm or the PPR (arrowheads) (H). (I–P) Distribution of cells expressing mRFP1 driven by Six1-14 in chick. ptkmRFP1-cSix1-14PCRx4 and ptkEGFP-mSix1-21×2 activate gene expression in the PPR and otic/epibranchial placodes, respectively (I). Both reporters were co-electroporated into the entire epiblast, examined at 6 (J) and 24 h.p.e. (K,L) in whole mount. Ventral (J,K) and dorsal (L) views. Merged images of cSix1-14PCR-labeled cells (red, pseudocolor) and mSix1-21-labeled cells (green, pseudocolor) superimposed on bright-field image. At 6 h.p.e., mRFP1-positive cells are found in the rostral PPR, while at 24 h.p.e, mRFP1-positive cells are present in the olfactory (K,L), lens (K,L) and adenohypophyseal (K) placode areas and ventral head ectoderm (K) but are largely absent from the otic/epibranchial placodes (L) labeled with EGFP. (M–P) Transverse sections of the embryo at 24 h.p.e. shown in K,L (the approximate positions of sections are indicated in L). Merged confocal images confirmed the presence of mRFP1-positive cells in the olfactory placode area (M), lens placode stained by anti-Pax6 antibody (green, pseudocolor, N) and ventral head ectoderm (yellow arrows in N). In contrast, positive cells were mostly absent in the trigeminal placode stained by anti-Pax3 antibody (green, pseudocolor, O) and otic/epibranchial placodes labeled by mSix1-21 (P). There are multiple mRFP1-positive cells stained by anti-Pax6 antibody in the lens placode (white arrowheads in N). In contrast, no mRFP-positive cell is labeled with anti-Pax3 antibody in the trigeminal placode (O) and only a single mRFP1-positive cell is labeled by mSix1-21 in the otic/epibranchial placodes (a white arrowhead in P). DAPI is used for nuclear staining (blue in M–P). All confocal images were taken at the same magnification and the mRFP1 and DAPI channels were taken at the same exposure time. In B,H,J–L, anterior is to the left. In C–E,G,H,M–P, dorsal side is to the top. ad: adenohypophyseal placode, ep: epibranchial placodes, hn: Hensen's node, le: lens placode, np: neural plate, ol: olfactory placode, ot: otic placode, ppr: pre-placodal region. Scale bars: 100 μm (C–E,M–P), 200 μm (H), 400 μm (B,G,J,K).