Identification and characterization of HMX3/Hmx3a binding partners

Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)




Lewis, Katharine

Subject Categories

Biology | Developmental Biology | Neurosciences


Mammalian HMX3 and its zebrafish ortholog Hmx3a are homeodomain proteins with essential roles in CNS and ear development. In particular, our lab has demonstrated that Hmx3a is required for dI2 interneurons in the spinal cord to utilize excitatory neurotransmitters, which is essential for their correct function in neuronal circuitry. While classically thought of as a transcription factor, our recent data suggest that Hmx3a may not require its DNA-binding domain to function, raising the important question of what molecular interactions mediate its effects and prompting a search for proteins that interact with it. Prior to this work, no protein binding partners were known for HMX3/Hmx3a in any species. Our lab performed a yeast two-hybrid screen with mouse HMX3 that generated 3200 clones containing plasmid inserts encoding putative binding partners. I isolated and sequenced the vast majority of these, yielding 539 unique putative binding partners of mouse HMX3. Using co-immunoprecipitation experiments with zebrafish Hmx3a and zebrafish orthologs of a subset of proteins identified through this screening, I tested whether a prioritized subset of these interactions are conserved in zebrafish and found that Tle3b, Azin1b, Prmt2, Hmgb1a, and Hmgn3 bind Hmx3a. I tested whether these proteins bind the products of four distinct homeodomain-lacking hmx3a mutant alleles, only two of which cause altered embryonic phenotypes, in order to correlate interaction profiles to phenotypes. I used both acute knockdown with CRISPRi and created stable mutant lines using conventional CRISPR in zebrafish to study the roles of these binding partners and related genes in vivo. 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.


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