Six1 and Irx1 have reciprocal interactions during cranial placode and otic vesicle formation

The specialized sensory organs of the vertebrate head are derived from thickened patches of cells in the ectoderm called cranial sensory placodes. The developmental program that generates these placodes and the genes that are expressed during the process have been studied extensively in a number of animals, yet very little is known about how these genes regulate one another. We previously found via a microarray screen that Six1, a known transcriptional regulator of cranial placode fate, up-regulates Irx1 in ectodermal explants. In this study, we investigated the transcriptional relationship between Six1 and Irx1 and found that they reciprocally regulate each other throughout cranial placode and otic vesicle formation. Although Irx1 expression precedes that of Six1 in the neural border zone, its continued and appropriately patterned expression in the pre-placodal region (PPR) and otic vesicle requires Six1. At early PPR stages, Six1 expands the Irx1 domain, but this activity subsides over time and changes to a predominantly repressive effect. Likewise, Irx1 initially expands Six1 expression in the PPR, but later represses it. We also found that Irx1 and Sox11, a known direct target of Six1, reciprocally affect each other. This work demonstrates that the interactions between Six1 and Irx1 are continuous during PPR and placode development and their transcriptional effects on one another change over developmental time.