In Vivo Neuronal Subtype-Specific Targets of Atoh1 (Math1) in Dorsal Spinal Cord

Department of Neuroscience, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
The Journal of Neuroscience : The Official Journal of the Society for Neuroscience (Impact Factor: 6.34). 07/2011; 31(30):10859-71. DOI: 10.1523/JNEUROSCI.0445-11.2011
Source: PubMed


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-sequencing experiments followed by enhancer reporter analyses identified five direct neuronal subtype-specific targets of Atoh1 in vivo along with their Atoh1-responsive enhancers. These targets, Klf7, Rab15, Rassf4, Selm, and Smad7, have diverse functions that range from transcription factors to regulators of endocytosis and signaling pathways. Only Rab15 and 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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Available from: Tiemo Klisch, Nov 20, 2015
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    • "For decades, researchers have tried to understand how bHLH factors can select and regulate their specific gene targets, given that they bind similar degenerate E-box motifs in vitro [reviewed by Bertrand et al. (2002); Lai et al. (2013)]. With advances in chromatin immunoprecipitation (ChIP) and the increasing number of identified cis-regulatory elements that are under the control of bHLH factors, a preference for binding certain E-boxes in vivo has been shown, thus explaining some of the functional specificity (Fong et al., 2012; Klisch et al., 2011; Lai et al., 2011; Meredith et al., 2013; Seo et al., 2007). Additional influences on specificity probably involve the epigenetic landscape (Fong et al., 2012; Meredith et al., 2013) and the pool of transcriptional co-factors that are available in a given population of progenitors (Lai et al., 2013). "
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    ABSTRACT: The proper balance of excitatory and inhibitory neurons is crucial for normal processing of somatosensory information in the dorsal spinal cord. Two neural basic helix-loop-helix transcription factors (TFs), Ascl1 and Ptf1a, have contrasting functions in specifying these neurons. To understand how Ascl1 and Ptf1a function in this process, we identified their direct transcriptional targets genome-wide in the embryonic mouse neural tube using ChIP-Seq and RNA-Seq. We show that Ascl1 and Ptf1a directly regulate distinct homeodomain TFs that specify excitatory or inhibitory neuronal fates. In addition, Ascl1 directly regulates genes with roles in several steps of the neurogenic program, including Notch signaling, neuronal differentiation, axon guidance and synapse formation. By contrast, Ptf1a directly regulates genes encoding components of the neurotransmitter machinery in inhibitory neurons, and other later aspects of neural development distinct from those regulated by Ascl1. Moreover, Ptf1a represses the excitatory neuronal fate by directly repressing several targets of Ascl1. Ascl1 and Ptf1a bind sequences primarily enriched for a specific E-Box motif (CAGCTG) and for secondary motifs used by Sox, Rfx, Pou and homeodomain factors. Ptf1a also binds sequences uniquely enriched in the CAGATG E-box and in the binding motif for its co-factor Rbpj, providing two factors that influence the specificity of Ptf1a binding. The direct transcriptional targets identified for Ascl1 and Ptf1a provide a molecular understanding of how these DNA-binding proteins function in neuronal development, particularly as key regulators of homeodomain TFs required for neuronal subtype specification.
    Development 06/2014; 141(14). DOI:10.1242/dev.105866 · 6.46 Impact Factor
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    • "The trhlmodel is the result of anEMS induced hypomorphic allele of the Atoh1 geneon the C57BL/6J (B6) mouse strain[13]. The phenotype of these hypomorphic mutant mice includes a trembling gait and hearing loss (symbol: trhl). "
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    ABSTRACT: Atoh1 is a transcription factor that regulates neural development in multiple tissues and is conserved among species. Prior mouse models of Atoh1, though effective and important in the evolution of our understanding of the gene, have been limited by perinatal lethality. Here we describe a novel point mutation of Atoh1 (designated Atoh1(trhl) ) underlying a phenotype of trembling gait and hearing loss. Histology revealed inner ear hair cell loss and cerebellar atrophy. Auditory Brainstem Response (ABR) and Distortion Product Otoacoustic Emission (DPOAE) showed functional abnormalities in the ear. Normal lifespan and fecundity of Atoh1(trhl) mice provide a complementary model to facilitate elucidation of ATOH1 function in hearing,central nervous system and cancer biology.
    PLoS ONE 11/2013; 8(11):e79791. DOI:10.1371/journal.pone.0079791 · 3.23 Impact Factor
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    • "Together, our observations support the view that Npn2 regulates commissural axon guidance in a population-specific manner and raise the possibility that Atoh1 or another transcription factor expressed by post-mitotic dI1 neurons directly regulates the expression of Npn2. However, our preliminary studies indicate that Npn2 expression is unaltered in mice lacking Atoh1 (EC and ZK, unpublished observation) and Npn2 was not identified in a systematic screen for direct lineage-specific in vivo targets of Atoh1[43]. "
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    ABSTRACT: Spinal commissural axons represent a model system for deciphering the molecular logic that regulates the guidance of midline-crossing axons in the developing central nervous system (CNS). Whether the same or specific sets of guidance signals control the navigation of molecularly distinct subtypes of these axons remains an open and largely unexplored question. Although it is well established that post-crossing commissural axons alter their responsiveness to midline-associated guidance cues, our understanding of the repulsive mechanisms that drive the post-crossing segments of these axons away from the midline and whether the underlying guidance systems operate in a commissural axon subtype-specific manner, remains fragmentary at best. Here, we utilize axonally targeted transgenic reporter mice to visualize genetically distinct dorsal interneuron (dI)1 and dI4 commissural axons and show that the repulsive class 3 semaphorin (Sema3) guidance receptor Neuropilin 2 (Npn2), is selectively expressed on the dI1 population and is required for the guidance of post-crossing dI1, but not dI4, axons. Consistent with these observations, the midline-associated Npn2 ligands, Sema3F and Sema3B, promote the collapse of dI1, but not dI4, axon-associated growth cones in vitro. We also identify, for the first time, a discrete GABAergic population of ventral commissural neurons/axons in the embryonic mouse spinal cord that expresses Npn2, and show that Npn2 is required for the proper guidance of their post-crossing axons. Together, our findings indicate that Npn2 is selectively expressed in distinct populations of commissural neurons in both the dorsal and ventral spinal cord, and suggest that Sema3-Npn2 signaling regulates the guidance of post-crossing commissural axons in a population-specific manner.
    Neural Development 07/2013; 8(1):15. DOI:10.1186/1749-8104-8-15 · 3.45 Impact Factor
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