Hmx3a does not require its homeodomain for its essential functions in spinal cord, ear and lateral line development

Homeodomain-containing transcription factors have crucial functions in most aspects of cellular function and embryonic development in both animals and plants. Hmx proteins are a sub-family of NK homeodomain proteins and previous research has shown that they have crucial functions in the development of sensory structures such as the eye and the ear. However, the functions of Hmx proteins in spinal cord development have not been analyzed. Here we show that zebrafish hmx2 and hmx3a are co-expressed in spinal dI2 and V1 neurons, whereas hmx3b, hmx1 and hmx4 are not expressed in the spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously demonstrated role in ear development, Hmx3a is required for survival to adulthood, lateral line progression and correct specification of spinal interneuron neurotransmitter fates. However, despite the fact that hxm2 is also expressed in the developing ear, lateral line and spinal cord, we have not detected any requirement for Hmx2 in the development of these structures, or for embryo viability, even when almost all of the hmx2 locus is deleted. It is possible that there is a small amount of partial redundancy between hmx2 and hmx3a in the specification of spinal interneuron neurotransmitter fates, as double mutants have slightly more severe phenotypes than hmx3a single mutants at some stages of development, but hmx2 single mutants have normal otolith development, lateral line progression and numbers of glutamatergic spinal cord neurons. Even more surprisingly, we have found that Hmx3a does not require its homeodomain for its roles in viability or embryonic development. This is, to our knowledge, the first time that such a prototypical homeodomain protein has been shown not to require its homeodomain.