A Novel Protein-Level Interaction Between Ebf3 and Six1 during Development in Xenopus

Abstract

Branchio-Oto-Renal (BOR) syndrome arises from disruptions in transcriptional networks that govern craniofacial and sensory development. Although pathogenic variants in SIX1 and EYA1 explain many cases, a substantial proportion of clinically diagnosed individuals have an unknown genetic cause, indicating that additional regulatory factors remain unidentified. Early B-Cell Factor 3 (Ebf3) is a transcription factor required for neurogenic and mesodermal lineages, but its role in craniofacial development remains incompletely defined. This study examined whether Ebf3 interacts with the Six1–Eya1 bipartite complex. Whole-mount in situ hybridization (WISH) of Xenopus laevis embryos revealed overlapping expression of ebf3 and six1 in the placodes at neurula stages and in the otic vesicle and branchial arches in larval stages. Immunofluorescence analysis demonstrated nuclear co-localization, and biochemical assays established a physical interaction between Ebf3 and Six1. Functional assays showed that Ebf3 represses Six1 Eya1 transcriptional activity and appear to disrupt the Six1–Eya1 complex formation in vitro. In vivo knockdown of ebf3 produced limited changes in dlx5 expression and no detectable alterations in six1 expression or craniofacial cartilage. However, interpretation was limited likely because of morpholino toxicity. These findings support a model in which Ebf3 modulates Six1-dependent transcription by regulating Six1-Eya1 interaction. Additional in vivo studies with optimized loss of function approaches are required to clarify its developmental role in the craniofacial structures disrupted in BOR patients.