Regulation of Six1 and Eya1 during the formation of the preplacodal ectoderm in xenopus - towards a gene regulatory network of the developing neural plate border

Cranial placodes are ectodermal thickenings in vertebrate embryos that give rise to many sense organs and ganglia. All placodes arise from a common precursor, the pre-placodal ectoderm (PPE). The latter develops during gastrulation at the neural plate border (NPB) just lateral to the neural crest – another embryonic tissue that contributes to cranial ganglia and skull formation. The transcription factor Six1 and its cofactor Eya1 play a central role in the early induction, formation and differentiation of PPE and later in placode development but it is still unknown, how these two transcriptional regulators become established at the NPB during the transition from early gastrulation to neural plate stage. In the first part of this study, I used gain and loss of function experiments in Xenopus laevis to elucidate the role of transcription factors (TFs)with dorsally/neurally (Pax3, Hairy2b, Zic1) and ventrally/non-neurally (AP2, Vent2, Msx1, Foxi1a) restricted expression domains for early expression of Six1 and Eya1 in the PPE. I also analyzed how Six1 and Eya1 cross-regulate the expression of these TFs. Taken together, this allowed me to describe an early gene regulatory network up- and downstream of Six1 and Eya1 in the PPE. In the second part of this study, I have tried to further elucidate the regulation of Eya1 by identifying Eya1 cis-regulatory elements using two different approaches. First, reporter constructs of candidate enhancers identified by phylogenetic footprinting were tested in transgenic Xenopus embryos. Second, regulatory regions surrounding the Eya1 locus were characterized by deletion analysis of reporter constructs of bacterial artificial chromosomes (BACs). The latter approach allowed the identification of several new cis-regulatory regions of Eya1 including regions that control its expression in the PPE and several placodes. Taken together, this study provides novel insights into the earliest processes underlying development of cranial sense organs in vertebrate embryos.2018-01-0