Transcription factors that contain a homeodomain DNA-binding domain 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-containing proteins that have fundamental roles in development of sensory structures such as the eye and the ear. However, Hmx functions in spinal cord development have not been analyzed. Here we show that zebrafish (Danio rerio) hmx2 and hmx3a are co-expressed in spinal dI2 and V1 interneurons, whereas hmx3b, hmx1 and hmx4 are not expressed in spinal cord. Using mutational analyses, we demonstrate that, in addition to its previously reported role in ear development, hmx3a is required for correct specification of a subset of spinal interneuron neurotransmitter phenotypes, as well as correct lateral line progression and survival to adulthood. Surprisingly, despite similar expression patterns of hmx2 and hmx3a during embryonic development, zebrafish hmx2 mutants are viable and have no obviously abnormal phenotypes in sensory structures or neurons that require hmx3a. In addition, embryos homozygous for deletions of both hmx2 and hmx3a have identical phenotypes to severe hmx3a single mutants. However, mutating hmx2 in hypomorphic hmx3a mutants that usually develop normally, results in abnormal ear and lateral line phenotypes. This suggests that while hmx2 cannot compensate for loss of hmx3a, it does function in these developmental processes, although to a much lesser extent than hmx3a. More surprisingly, our mutational analyses suggest that Hmx3a may not require its homeodomain DNA-binding domain for its roles in viability or embryonic development.

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Ladybird homeobox (Lbx) transcription factors have crucial functions in muscle and nervous system development in many animals. Amniotes have two Lbx genes, but only Lbx1 is expressed in spinal cord. In contrast, teleosts have three lbx genes and we show here that zebrafish lbx1a, lbx1b, and lbx2 are expressed by distinct spinal cell types, and that lbx1a is expressed in dI4, dI5, and dI6 interneurons, as in amniotes. Our data examining lbx expression in Scyliorhinus canicula and Xenopus tropicalis suggest that the spinal interneuron expression of zebrafish lbx1a is ancestral, whereas lbx1b has acquired a new expression pattern in spinal cord progenitor cells. lbx2 spinal expression was probably acquired in the ray‐finned lineage, as this gene is not expressed in the spinal cords of either amniotes or S. canicula. We also show that the spinal function of zebrafish lbx1a is conserved with mouse Lbx1. In zebrafish lbx1a mutants, there is a reduction in the number of inhibitory spinal interneurons and an increase in the number of excitatory spinal interneurons, similar to mouse Lbx1 mutants. Interestingly, the number of inhibitory spinal interneurons is also reduced in lbx1b mutants, although in this case the number of excitatory interneurons is not increased. lbx1a;lbx1b double mutants have a similar spinal interneuron phenotype to lbx1a single mutants. Taken together these data suggest that lbx1b and lbx1a may be required in succession for correct specification of dI4 and dI6 spinal interneurons, although only lbx1a is required for suppression of excitatory fates in these cells.

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 hmx2 and hmx3a have similar expression patterns during embryonic development, we have not detected any requirement for Hmx2 in these developmental processes, even when almost all of the hmx2 locus is deleted. We have also not detected any redundancy between hmx2 and hmx3a: double mutants have identical phenotypes to hmx3a single mutants. 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.Hmx2 in viability, or otolith, lateral line or specification of spinal interneuron neurotransmitter fates, even when almost all of the hmx2 locus is deleted (in our most severe mutant allele, hmx2 SU39 , only 84 nucleotides of 5' sequence and 57 nucleotides of 3' coding sequence remain). We have also not detected any redundancy between hmx2 and hmx3a: hmx2;hmx3a double mutants have identical phenotypes to severe hmx3a single mutants. 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.

HMX3 is a homeodomain protein with essential roles in CNS and ear development. Homeodomains are DNA-binding domains and hence homeodomain-containing proteins are usually assumed to be transcription factors. However, intriguingly, our recent data suggest that zebrafish Hmx3a may not require its homeodomain to function, raising the important question of what molecular interactions mediate its effects. To investigate this, we performed a yeast two-hybrid screen and identified 539 potential binding partners of mouse HMX3. Using co-immunoprecipitation, we tested whether a prioritized subset of these interactions are conserved in zebrafish and found that Tle3b, Azin1b, Prmt2, Hmgb1a, and Hmgn3 bind Hmx3a. Next, we tested whether these proteins bind the products of four distinct hmx3a mutant alleles that all lack the homeodomain. Embryos homozygous for two of these alleles develop abnormally and die, whereas zebrafish homozygous for the other two alleles are viable. We found that all four mutations abrogate binding to Prmt2 and Tle3b, whereas Azin1b binding was preserved in all cases. Interestingly, Hmgb1a and Hmgn3 had more affinity for products of the viable mutant alleles. These data shed light on how HMX3/Hmx3a might function at a molecular level and identify new targets for future study in these vital developmental processes.