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, 6 (9), e1001133

Identification of Early Requirements for Preplacodal Ectoderm and Sensory Organ Development


Identification of Early Requirements for Preplacodal Ectoderm and Sensory Organ Development

Hye-Joo Kwon et al. PLoS Genet.


Preplacodal ectoderm arises near the end of gastrulation as a narrow band of cells surrounding the anterior neural plate. This domain later resolves into discrete cranial placodes that, together with neural crest, produce paired sensory structures of the head. Unlike the better-characterized neural crest, little is known about early regulation of preplacodal development. Classical models of ectodermal patterning posit that preplacodal identity is specified by readout of a discrete level of Bmp signaling along a DV gradient. More recent studies indicate that Bmp-antagonists are critical for promoting preplacodal development. However, it is unclear whether Bmp-antagonists establish the proper level of Bmp signaling within a morphogen gradient or, alternatively, block Bmp altogether. To begin addressing these issues, we treated zebrafish embryos with a pharmacological inhibitor of Bmp, sometimes combined with heat shock-induction of Chordin and dominant-negative Bmp receptor, to fully block Bmp signaling at various developmental stages. We find that preplacodal development occurs in two phases with opposing Bmp requirements. Initially, Bmp is required before gastrulation to co-induce four transcription factors, Tfap2a, Tfap2c, Foxi1, and Gata3, which establish preplacodal competence throughout the nonneural ectoderm. Subsequently, Bmp must be fully blocked in late gastrulation by dorsally expressed Bmp-antagonists, together with dorsally expressed Fgf and Pdgf, to specify preplacodal identity within competent cells abutting the neural plate. Localized ventral misexpression of Fgf8 and Chordin can activate ectopic preplacodal development anywhere within the zone of competence, whereas dorsal misexpression of one or more competence factors can activate ectopic preplacodal development in the neural plate. Conversely, morpholino-knockdown of competence factors specifically ablates preplacodal development. Our work supports a relatively simple two-step model that traces regulation of preplacodal development to late blastula stage, resolves two distinct phases of Bmp dependence, and identifies the main factors required for preplacodal competence and specification.

Conflict of interest statement

The authors have declared that no competing interests exist.


Figure 1
Figure 1. Models for the role of Bmp in preplacodal specification.
(A) Classical model in which a Bmp morphogen gradient directly specifies multiple fates, including epidermal ectoderm, preplacodal ectoderm (PPE), neural crest (NC) and neural plate, at discrete threshold concentrations. (B) Bmp-attenuation model in which Bmp-antagonists, secreted from the dorsal tissue of the embryo, promotes preplacodal fate in nonneural ectoderm abutting the anterior neural plate. In this model, Bmp must be fully blocked to permit preplacodal specification.
Figure 2
Figure 2. Distinct responses of neural crest and preplacodal ectoderm to graded impairment of Bmp.
(A) Lateral views of foxd3 expression at 11 hpf with anterior up and dorsal to the right. Embryos were treated with indicated concentrations of DM added at 4 hpf. Where indicated the DM concentration was increased to 200 µM (complete Bmp-inhibition) at 5 hpf, 6 hpf or 7 hpf. (B) Lateral views of six4.1 expression at 11 hpf in embryos treated with indicated concentrations of DM beginning at 4 hpf. Treatment with 25 µM DM yields two discrete responses, one in which six4.1 remains confined to two bilateral stripes flanking the neural plate and the other in which six4.1 expression is lost. (C) Lateral views showing expression of preplacodal competence factors tfap2a, tfap2c, foxi1 and gata3 in embryos were treated with 50 µM DM beginning at 4 hpf. Note that tfap2a/c remain broadly expressed in ventral ectoderm whereas foxi1 and gata3 are nearly eliminated.
Figure 3
Figure 3. Stage-dependent requirements for Bmp.
(A–E, G–L) Analysis of indicated gene expression patterns in control embryos and embryos treated with 100 µM dorsomorphin (DM) at 5 hpf or 7 hpf. Lateral views with dorsal to the right and anterior up. Expression of six4.1, eya1 and dlx3b (A–C) in PPE, krox20 in hindbrain(D) and p63 in epidermal ectoderm (E). Expression of competence factor genes tfap2a, tfap2c, foxi1 and gata3 (G–J). Reporters of Bmp-signaling, Phospho-Smad1/5/8 antibody staining (K) and sizzled in situ hybridization (L). Note the complete loss of Bmp signaling by 100 µM DM-treatment either at 5 hpf or 7 hpf. (F) Lateral views of live embryos at 30 hpf. Embryos treated with DM at 7 hpf show a partially dorsalized C3 phenotype . (M, N) Tg(hs:chd) and/or Tg(hs:dnBmpr) embryos heat-shocked and treated with 100 µM DM at 7.5 hpf. eya1 expression (M) and C3 phenotypes (N) are comparable to embryos treated with 100 µM DM alone.
Figure 4
Figure 4. Formation of cranial placodes requires competence factors but not Bmp during gastrulation.
Analysis of various cranial placode markers in control embryos, embryos treated with 100 µM DM at 7 hpf, or foxi1/gata3/tfap2a/c quadruple morphants (4-MO). Arrows indicate relevant expression domains in placodal tissues. (A–C) Dorsal views (anterior up) of pitx3 expression in anterior pituitary and lens placode. (D–F) Lateral views (anterior to left) of foxe3 expression in the lens placode. (G–I) Frontal views of cxcr4b expression in olfactory placode. (J–L) Lateral views (anterior to left) showing the lens and nasal pits in live specimens at 30 hpf. Asterisks in (L) depict the absence of morphologically discernable structures. (M–O) Lateral views (anterior up) of isl1 expression in the trigeminal placode. (P–R) Lateral views (anterior up) of sox3 expression in the epibranchial placode. (S–U) Dorsal views (anterior up) of pax2a expression in the otic placode. (V–X) Dorsal views (anterior up) of cldna expression in the otic vesicle. All placodal markers are expressed normally in DM-treated embryos. Expression of cldna is severely deficient in quadruple morphants (X, n  =  13/21) or ablated altogether (8/21, not shown). All other placodal markers are ablated in quadruple morphants (n≥10 for each marker).
Figure 5
Figure 5. Knockdown of competence factors impairs preplacodal specification.
(A–C) Expression of preplacodal markers at 10.5 hpf in (A) foxi1/gata3 double morphants, (B) tfap2a/c double morphants, (C) foxi1/gata3/tfap2a/c quadruple morphants (4-MO). Note the complete loss of preplacodal markers in C. (D) Expression of krox20, p63, P-smad and sizzled during gastrulation in foxi1/gata3/tfap2a/c quadruple morphants. Morphology of a live quadruple morphant at 30 hpf is also shown. (E) Expression of six4.1 and eya1 in p63 morphants alone or in combination with tfap2a/c-MO or foxi1/gata3-MO. All images show lateral views with dorsal to the right and anterior up, except for the live specimen in (D), which shows a lateral view with anterior to the left.
Figure 6
Figure 6. Misexpression of competence factors induces ectopic expression of preplacodal markers.
(A–C) Analysis of indicated gene expression patterns in control embryos and embryos carrying hs:gata3, hs:foxi1 and/or hs:tfap2a heat-shocked at 30% epiboly (4.5 hpf). Dorsal views with anterior up except bottom row in A, inset in B, which are lateral views with dorsal to the right. Note the ectopic expression of PPE markers, six4.1 (A), eya1 (B) and dlx3b (C) in neuroectoderm of embryos misexpressing one or more competence factors. (D–H) Dorsolateral views (anterior up) of mosaic embryos showing ectopic expression of dlx3b and six4.1 at 10.5 hpf. Donor cells obtained from Tg(hs:foxi1) injected with hs:gata3 and hs:tfap2a plasmid were transplanted into wild type hosts and heat shocked at 4.5 hpf at 39°C. Transplanted cells were identified with Strepavidin-FITC (arrows F, H). Mosaic embryos shows cell autonomous expression of dlx3b and six4.1 in the neural plate (compare E, F and G, H).
Figure 7
Figure 7. The entire nonneural ectoderm is competent to form preplacodal tissue.
(A–H) Expression of preplacodal markers in (A, B, E, F) Tg(hs:fgf8) embryos incubated at 35°C from 7.5–10.5 hpf, or (C, D, G, H) Tg(hs:fgf3) embryos incubated at 36°C from 7–10.5 hpf. 100 µM DM was added as indicated. (I–K) Expression of erm in (I) Tg(hs:fgf8) embryo incubated at 35°C without DM, (J) a non-transgenic embryos incubated at 35°C with 100 µM DM, and (K) a Tg(hs:fgf8) embryo incubated at 35°C with 100 µM DM. (L–S) Mosaic misexpression of Fgf8 and/or Chordin. (L–O) Brightfield images (top row) and fluorescent images (bottom row) of host embryos with cells transplanted from Tg(hs:fgf8) (L), Tg(hs:chd) (M) or Tg(hs:fgf8); Tg(hs:chd) donor embryos (N, O). Donor embryos were injected with lineage tracer (biotin-dextran) and transplanted at mid-blastula (L, M, N) or early gastrula stage (O) into unlabeled host embryos. Embryos were heat-shocked at 39°C for 30 minutes at 7 hpf and examined for six4.1 expression at 10.5 hpf. Transplanted transgenic cells were identified by Strepavidin-FITC staining after in situ hybridization. All panels show lateral views of host embryos with anterior up. Mosaic embryos with Tg(hs:fgf8);Tg(hs:chd) double transgenic cells showed ectopic six4.1 expression in surrounding ventral ectoderm (N, O), whereas no ectopic six4.1 expression was detected following activation of hs:fgf8 alone (L) or hs:chd alone (M). (P–S) Brightfield images (top row) and fluorescent images (bottom row) of host embryos with cells transplanted during early gastrula stage from double heterozygous Tg(hs:fgf8); Tg(hs:chd) embryos. Embryos were heat shocked for 30 minutes at 39°C beginning at 8.5 hpf and examined for expression of six4.1 (preplacodal ectoderm), foxd3 (neural crest), sox19b (neural plate) or p63(epidermal ectoderm) at 10.5 hpf. All panels show lateral views except (P) which shows a ventral view (lateral view in inset) and (S) which shows ventro-lateral view. Heat shock activation at 8.5 hpf (P) leads to stronger ectopic expression of six4.1 than heat shock at 7 hpf (O). No ectopic expression of foxd3 or sox19b is detected (Q, R) whereas p63 expression appears downregulated in and around transgenic cells (arrows in S).
Figure 8
Figure 8. A model for sequential phases of preplacodal development.
During late blastula stage, Bmp acts as a morphogen that specifies neural crest (NC) within a narrow but low range of signaling, whereas higher levels of Bmp signaling establish the nonneural ectoderm as a broad zone of uncommitted cells with potential to form epidermal or preplacodal ectoderm (PPE). Within the nonneural ectoderm, changing levels of Bmp do not distinguish preplacodal from epidermal potential, and preplacodal competence factors are uniformly induced throughout this domain. However, expression of tfap2a/c overlaps with the lateral edges of the neural plate where, perhaps in combination with neural markers, they cell-autonomously specify NC fate. During late gastrula stage (9–10 hpf), PPE fate is specified in competent cells near the neural-nonneural border by dorsally expressed Bmp antagonists, Fgf and Pdgf. Complete attenuation of Bmp is required for PPE specification. Relevant markers for each ectodermal domain are shown.

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