Anteroposterior Patterning Of The Zebrafish Ear Through Fgf- And Hh-Dependent Regulation Of Hmx3a Expression

In the zebrafish, Fgf and Hh signalling assign anterior and posterior identity, respectively, to the poles of the developing ear. Mis-expression of fgf3 or inhibition of Hh signalling results in double-anterior ears, including ectopic expression of hmx3a. To understand how this double-anterior pattern is established, we characterised transcriptional responses in Fgf gain-of-signalling or Hh loss-of-signalling backgrounds. Mis-expression of fgf3 resulted in rapid expansion of anterior otic markers, refining over time to give the duplicated pattern. Response to Hh inhibition was very different: initial anteroposterior asymmetry was retained, with de novo duplicate expression domains appearing later. We show that Hmx3a is required for normal anterior otic patterning, and that otic patterning defects in hmx3a(-/-) mutants are a close phenocopy to those seen in fgf3(-/-) mutants. However, neither loss nor gain of hmx3a function was sufficient to generate full ear duplications. Using our data to infer a transcriptional regulatory network required for acquisition of otic anterior identity, we can recapitulate both the wild-type and the double-anterior pattern in a mathematical model.Author summary Understanding how signalling molecules impart information to developing organ systems, and how this is interpreted through networks of gene activity, is a key goal of developmental genetic analysis. In the developing zebrafish inner ear, differences in gene expression arise between the anterior and posterior poles of the ear placode, ensuring that sensory structures in the ear develop in their correct positions. If signalling pathways are disrupted, a mirror-image ear can result, developing with two anterior poles. We have used genetic, pharmacological and mathematical modelling approaches to decipher the pathway of gene action required to specify anterior structures in the zebrafish ear. Patterns of gene expression are dynamic and plastic, with two different routes leading to the formation of duplicate anterior structures. Expression of the hmx3a gene is an early response to the anterior signalling molecule Fgf3, but is not sufficient to drive the formation of ectopic anterior structures at the posterior of the ear. The hmx3a gene codes for a protein that regulates other genes, and in humans, mutation of HMX genes results in diseases affecting inner ear function. Our work provides a framework for understanding the dynamics of early patterning events in the developing inner ear.