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

Abstract

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 dorsal interneuron 1 (dI1) 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 (ChIP-seq) 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 (EGL) 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.

Making sense out of spinal cord somatosensory development

“…Thus, terminal neuronal phenotypes can be directly regulated by sustained expression of HD factors in mature neurons. Furthermore, transiently expressed TFs, such as the bHLH TFs ATOH1, PTF1A and ASCL1, have been shown to directly regulate genes that control terminal neuronal phenotypes in addition to their role in regulating the expression of HD TFs (Borromeo et al, 2014;Lai et al, 2011;Russ et al, 2015;Wildner et al, 2013). For example, PTF1A directly regulates genes encoding GABA synthesizing enzymes and GABA and glycine transporters required for inhibitory neuronal functions, but it also regulates the expression of PAX2 (Borromeo et al, 2014).…”

Atoh1 in sensory hair cell development: constraints and cofactors

“…Despite this, the Atoh1 binding sites found in intestinal secretory cells show only a small overlap with those found in cerebellum granule neurons (951 out of 8729 detected in the gut) (23). So far, there are no data on Atoh1 binding sites in hair cells or dorsal interneurons: Atoh1 ChIP-seq experiments are problematic due to the very small numbers of these cell types (25,26 (27). Below, we review the possible mechanisms that could explain context-dependent activity of Atoh1.…”

Axonal Patterns and Targets of dA1 Interneurons in the Chick Hindbrain

“…The tested enhancers were the mouse Atoh1 (Helms et al, 2000;Ebert et al, 2003;Lai et al, 2011) and edI1 enhancer element (Avraham et al, 2009). These elements were cloned upstream to Cre recombinase and coelectroporated at E2.5 along with a Credependent nuclear Green Fluorescent Protein (nGFP) reporter plasmid (pCAGG-LoxP-Stop-LoxP-nGFP; Fig.…”