Linkages between BMP, canonical WNT/β-catenin, and FGF20 signaling during placode formation in the chicken anterior eye segment

The mechanisms that regulate the even spacing of placodes during embryonic development remain intriguing. These mechanisms typically involve complex interactions between signaling pathways, which can be further influenced by mechanical forces as the embryo grows. Here, we investigate the patterning of the ring of conjunctival placodes in the anterior eye of chicken embryos by functionally manipulating the BMP signaling pathway. Specifically, we electroporated a TWSG1 plasmid at HH27 to modulate BMP signaling during the pre-patterning phase and examined the effects on placode formation and key developmental pathways. Our results show that modulation of BMP signaling at HH27 influences placode development, morphology and spacing three days later, at HH34. qPCR data confirm an initial and statistically significant upregulation of FGF20 and WNT2B 6 h after electroporation. However, one day later (at HH29), only β-catenin is significantly elevated. Multiplex fluorescent in situ hybridization shows WNT2 expression in the conjunctival placodes and papillae for the first time. This WNT2 expression is colocalized with β-catenin in controls and remains spatially colocalized after electroporation. Together, these results provide functional evidence that BMP signaling regulates both canonical WNT/β-catenin and FGF pathways during early placode formation and support a model in which BMP may act as the inhibitor in a Turing-like reaction-diffusion mechanism underlying conjunctival placode patterning in the anterior eye.