Neural basic helix-loop-helix (bHLH) transcription factors are crucial in regulating the differentiation and neuronal subtype specification of neurons. Precisely how these transcription factors direct such processes is largely unknown due to the lack of bona fide targets in vivo. Genetic evidence suggests that bHLH factors have shared targets in their common differentiation role, but unique targets with respect to their distinct roles in neuronal subtype specification. However, whether neuronal subtype-specific targets exist remains an unsolved question. To address this question, we focused on Atoh1 (Math1), a bHLH transcription factor that specifies distinct neuronal subtypes of the proprioceptive pathway in mammals including the dI1 (dorsal interneuron 1) population of the developing spinal cord. We identified transcripts unique to the Atoh1-derived lineage using microarray analyses of specific bHLH-sorted populations from mouse.Chromatin immunoprecipitation-sequencingexperimentsfollowedbyenhancer reporter analyses identified five directneuronal subtype-specific targets of Atoh1 in vivo along with their Atoh1-responsive enhancers. These targets, Klf7, Rab15, Rassf4, Selm, and Smad7, havediverse functionsthat range from transcription factorstoregulatorsofendocytosisand signalingpathways. OnlyRab15and Selm are expressed across several different Atoh1-specified neuronal subtypes including external granule cells (external granule cell layer) in the developing cerebellum, hair cells of the inner ear, and Merkel cells. Our work establishes on a molecular level that neuronal differentiation bHLH transcription factors have distinct lineage-specific targets.
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