We analyzed the spatiotemporal pattern of expression of 15 transcription factors (Six1, Six4, Eya1, Sox3, Sox2, Pax6, Pax3, Pax2, Pax8, Dlx3, Msx1, FoxI1c, Tbx2, Tbx3, Xiro1) during placode development in Xenopus laevis from neural plate to late tail bud stages. Out of all genes investigated, only the expression of Eya1, Six1, and Six4 is maintained in all types of placode (except the lens) throughout embryonic development, suggesting that they may promote generic placodal properties and that their crescent-shaped expression domain surrounding the neural plate defines a panplacodal primordium from which all types of placode originate. Double-labeling procedures were employed to reveal the precise position of this panplacodal primordium relative to neural plate, neural crest, and other placodal markers. Already at neural plate stages, the panplacodal primordium is subdivided into several subregions defined by particular combinations of transcription factors allowing us to identify the approximate regions of origin of various types of placode. Whereas some types of placode were already prefigured by molecularly distinct areas at neural plate stages, the epibranchial, otic, and lateral line placodes arise from a common posterior placodal area (characterized by Pax8 and Pax2 expression) and acquire differential molecular signatures only after neural tube closure. Our findings argue for a multistep mechanism of placode induction, support a combinatorial model of placode specification, and suggest that different placodes evolved from a common placodal primordium by successive recruitment of new inducers and target genes.

Ectodermal placodes, from which many cranial sense organs and ganglia develop, arise from a common placodal primordium defined by Six1 expression. Here, we analyse placodal Six1 induction in Xenopus using microinjections and tissue grafts. We show that placodal Six1 induction occurs during neural plate and neural fold stages. Grafts of anterior neural plate but not grafts of cranial dorsolateral endomesoderm induce Six1 ectopically in belly ectoderm, suggesting that only the neural plate is sufficient for inducing Six1 in ectoderm. However, extirpation of either anterior neural plate or of cranial dorsolateral endomesoderm abolishes placodal Six1 expression indicating that both tissues are required for its induction. Elevating BMP-levels blocks placodal Six1 induction, whereas ectopic sources of BMP inhibitors expand placodal Six1 expression without inducing Six1 ectopically. This suggests that BMP inhibition is necessary but needs to cooperate with additional factors for Six1 induction. We show that FGF8, which is expressed in the anterior neural plate, can strongly induce ectopic Six1 in ventral ectoderm when combined with BMP inhibitors. In contrast, FGF8 knockdown abolishes placodal Six1 expression. This suggests that FGF8 is necessary and together with BMP inhibitors sufficient to induce placodal Six1 expression in cranial ectoderm, implicating FGF8 as a central component in generic placode induction.

SUMMARYIt is still controversial whether cranial placodes and neural crest cells arise from a common precursor at the neural plate border or whether placodes arise from non-neural ectoderm and neural crest from neural ectoderm. Using tissue grafting in embryos of Xenopus laevis, we show here that the competence for induction of neural plate, neural plate border and neural crest markers is confined to neural ectoderm, whereas competence for induction of panplacodal markers is confined to non-neural ectoderm. This differential distribution of competence is established during gastrulation paralleling the dorsal restriction of neural competence. We further show that Dlx3 and GATA2 are required cell-autonomously for panplacodal and epidermal marker expression in the non-neural ectoderm, while ectopic expression of Dlx3 or GATA2 in the neural plate suppresses neural plate, border and crest markers. Overexpression of Dlx3 (but not GATA2) in the neural plate is sufficient to induce different non-neural markers in a signaling-dependent manner, with epidermal markers being induced in the presence, and panplacodal markers in the absence, of BMP signaling. Taken together, these findings demonstrate a non-neural versus neural origin of placodes and neural crest, respectively, strongly implicate Dlx3 in the regulation of non-neural competence, and show that GATA2 contributes to non-neural competence but is not sufficient to promote it ectopically.

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